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Preclinical Evaluation of Synergistic Drug Combinations in Acute Myeloid Leukemia
by
Lianne Emily Rotin
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Institute of Medical Science
University of Toronto
© Copyright by Lianne E. Rotin (2016)
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Preclinical Evaluation of Synergistic Drug Combinations in
Acute Myeloid Leukemia
Lianne E. Rotin
Doctor of Philosophy
Institute of Medical Science University of Toronto
2016
Abstract
The FDA-approved Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has
significantly improved patient outcomes in B-cell malignancies, where BTK
signaling is implicated in disease progression. Documented expression of
constitutively active BTK in AML cells has generated interest in evaluating the
potential therapeutic role of ibrutinib in the treatment of AML. We investigated the
role of ibrutinib in AML by screening this drug against libraries of approved drugs
in AML cell lines, identifying the poly(ADP-ribose) glycohydrolase inhibitor
ethacridine lactate as a profoundly synergistic hit. This drug combination was
preferentially cytotoxic to patient-derived AML cells over normal controls, and
synergistic cell death was preceded by reactive oxygen species (ROS)
production. Ibrutinib similarly synergized with current first-line AML chemotherapy
agent daunorubicin. Interestingly, neither ethacridine, nor daunorubicin’s synergy
with ibrutinib appeared to be BTK-dependent. Further study with the epidermal
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growth factor receptor (EGFR) inhibitor erlotinib—which also has preclinical anti-
AML activity—revealed equally profound EGFR-independent synergy with
ethacridine, with an increase in ROS that paralleled that induced by the ibrutinib-
ethacridine combination. We determined that erlotinib-mediated potentiation of
ethacridine accumulation was responsible for this combination’s synergistic
cytotoxicity, and hypothesize that ibrutinib and ethacridine likely synergize via the
same mechanism. In summary, we have identified a novel BTK-independent role
for ibrutinib in AML, and for the first time, report a potential role for PARG
inhibition as a combination candidate for AML therapy.
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Acknowledgements
I extend my deepest gratitude to my supervisor, Dr. Aaron Schimmer, for his
guidance and encouragement throughout my PhD studies. Aaron is a wonderful
mentor and a role model for the clinician-scientist I hope to one day become.
I would also like to thank my program advisory committee members, Drs. Mark
Minden and Meredith Irwin, for their helpful advice and constructive feedback
during and in between committee meetings. Furthermore, I would like to thank
Dr. Minden for providing me with the opportunity to attend his weekly leukemia
clinic for the better part of two years; observing these patient visits gave me a
better understanding of this disease and its impact on patients and their families,
as well as a sincere appreciation for the need for new ways to tackle it.
Working in the Schimmer Lab has been a truly incredible experience; it has been
a privilege to train alongside a team of such bright, creative, and enthusiastic
scientists. I would especially like to thank Marcela Gronda, Rose Hurren, and
Neil MacLean: you have taught me countless lab techniques, assisted with many
important experiments, and you have helped me become a better researcher.
Finally, this acknowledgements section would be incomplete without mention of
my wonderful support team outside of the lab. I’m grateful to my parents, Daniela
and Robbie, for instilling in me the importance of education, hard work, and
putting your heart into everything you do. I am also thankful for their continued
and unwavering support throughout this long educational journey. Lastly, I would
like to thank my fiancé Zach, who has patiently sat through every single one of
my practice talks, asked some impressively pertinent questions, and who has
always had something positive and encouraging to say. Thank you!
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Contributions
This thesis consists of 3 data chapters. Chapter 3 was published in the journal
Oncotarget (Rotin et al., 2016b) and Chapter 4 was published in the journal
Leukemia and Lymphoma (Rotin et al., 2016a). Chapter 5 has yet to be
published.
The author performed all experiments and analyses outlined in the thesis, except
as indicated below:
Mr. Neil MacLean – performed combination high-throughput drug screens
against ibrutinib and ethacridine, provided assistance with combination
drug screens against erlotinib, analyzed screen data, and prepared
lentiviral stocks
Ms. Marcela Gronda – performed immunoblots (BTK, phospho-BTK, BMX,
RLK, TEC, ITK, EGFR), olaparib assays, reactive oxygen species
measurements depicted in figures 4-10 and 4-11, and provided assistance
with the PARG inhibitor assay
Ms. Rose Hurren – conducted in vivo combination ibrutinib-ethacridine
studies, radiolabeled daunorubicin uptake studies, and Z-VAD-FMK
experiments
Ms. XiaoMing Wang – carried out in vivo combination ibrutinib-ethacridine
studies
Dr. Ahmed Aman – mass spectrometry analysis of ethacridine
accumulation in AML cell lines
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Dr. Feng-Hsu Lin – designed software program for excess-over-Bliss
analysis of drug screen data
Dr. Alessandro Datti – guidance in planning high-throughput drug
screening procedures
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Table of Contents ACKNOWLEDGEMENTS……….………………………………..……………….IV
CONTRIBUTIONS………….………………………………………..…….............V
TABLE OF CONTENTS……………………………………………..……………VII
LIST OF TABLES………………………………………………………………….XI
LIST OF FIGURES………………………………………………….…….……….XII
LIST OF ABBREVIATIONS………………………………………………………XIV
Table of Contents Preface ................................................................................................................. 1 Chapter 1: Literature Review ............................................................................. 2 1.1 Acute Myeloid Leukemia .............................................................................. 3
1.1.1 Normal Hematopoiesis ...................................................................................... 3 1.1.2 Acute Myeloid Leukemia ................................................................................... 5
1.1.2.1 AML Pathogenesis ........................................................................................ 5 1.1.2.2 Epidemiology of AML .................................................................................... 6 1.1.2.3 AML Classification and Prognostication ........................................................ 6 1.1.2.4 AML Management ......................................................................................... 7
1.2 Tyrosine Kinase Inhibitor Therapy in AML ............................................... 10 1.2.1 Targeted Cancer Therapies ............................................................................. 10
1.2.1.1 Tyrosine Kinase Inhibitors ........................................................................... 10 1.2.2 Oncogenic Tyrosine Kinases in AML ............................................................. 11
1.2.2.1 FMS-Related Tyrosine Kinase 3 ................................................................. 11 1.3 Ibrutinib ........................................................................................................ 14
1.3.1 Bruton’s tyrosine kinase: background & role in signal transduction from the B-cell receptor .................................................................................................... 14
1.3.1.1 BTK domains ............................................................................................... 14 1.3.1.2 BTK expression ........................................................................................... 15 1.3.1.3 BTK: Role in B-cell Maturation .................................................................... 15 1.3.1.4 BTK Signaling in B-Cells ............................................................................. 15
1.3.2 A Rationale for Targeting BTK in B-cell Malignancies ................................. 16 1.3.2.1 Chronic Lymphocytic Leukemia .................................................................. 17 1.3.2.2 Mantle-Cell Lymphoma ............................................................................... 17 1.3.2.3 Waldenström Macroglobulinemia ................................................................ 17
1.3.3 Development of Ibrutinib as a Selective and Irreversible BTK Inhibitor with In Vivo Activity .......................................................................................................... 18 1.3.4 Preclinical and clinical activities of ibrutinib in B-cell cancers ................... 19
1.3.4.1 Chronic Lymphocytic Leukemia .................................................................. 19
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1.3.4.2 Mantle Cell Lymphoma ............................................................................... 20 1.3.4.3 Waldenström Macroglobulinemia ................................................................ 21
1.3.5 B-cell independent BTK signaling: myeloid-lineage cells ........................... 22 1.3.5.1 Mast Cells ................................................................................................... 23 1.3.5.2 Macrophages .............................................................................................. 24 1.3.5.3 Erythroid Cells ............................................................................................. 25 1.3.5.4 Platelets ...................................................................................................... 26 1.3.5.5 Neutrophils .................................................................................................. 27
1.3.6 A Role for Targeting BTK Beyond B-Cell Cancers ....................................... 29 1.3.6.1 Rheumatoid Arthritis .................................................................................... 29 1.3.6.2 Multiple Myeloma ........................................................................................ 30 1.3.6.3 Acute Myeloid Leukemia ............................................................................. 31 1.3.6.4 Prostate Cancer .......................................................................................... 32
1.4 EGFR inhibitors in AML: Anti-Leukemic Mechanisms of Action and Preclinical and Clinical Activity ....................................................................... 34
1.4.1 Development of Small Molecule EGFR Tyrosine Kinase Inhibitors ............ 34 1.4.2 Expression of EGFR in AML Cells .................................................................. 35 1.4.3 Preclinical EGFR-TKI activity against AML ................................................... 36
1.4.3.1 Differentiation .............................................................................................. 36 1.4.3.2 Cell Cycle Arrest and Cell Death ................................................................. 37
1.4.4 Proposed Anti-Leukemic Targets of EGFR-TKIs .......................................... 37 1.4.4.1 JAK2 Inhibition ............................................................................................ 37 1.4.4.2 SRC Family Kinase Inhibition ...................................................................... 38 1.4.4.3 SYK Inhibition .............................................................................................. 39 1.4.4.4 Bruton’s Tyrosine Kinase Inhibition ............................................................. 39 1.4.4.5 Inhibition of ATP-Binding Cassette Transporter Efflux Activity ................... 40
1.4.6 Clinical EGFR-TKI Activity Against AML ....................................................... 41 1.4.7 Summary ........................................................................................................... 42
1.5 Ethacridine Lactate ..................................................................................... 44 1.5.1 Ethacridine Lactate Indications ...................................................................... 44 1.5.2 Ethacridine Lactate Mechanisms of Action ................................................... 45
1.5.2.1 Poly(ADP-ribose) Glycohydrolase Inhibition ............................................... 45 1.5.2.2 Non-Genotoxic Activation of p53 ................................................................ 46
Chapter 2: Project Rationale and Aims ........................................................... 48 2.1 Thesis Aims ................................................................................................. 49
2.1.1 Aim I: Identify compounds that synergize with ibrutinib in AML ................ 49 2.1.2 Aim II: Evaluate the mechanism of synergy between ibrutinib and daunorubicin in AML ................................................................................................ 49 2.1.3 Aim III: Identify compounds that synergize with erlotinib in AML .............. 50
Chapter 3: Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism ........................................................................................................ 51 3.1 Abstract ....................................................................................................... 52 3.2 Introduction ................................................................................................. 53 3.3 Methods ....................................................................................................... 54
3.3.1 Materials ........................................................................................................... 54
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3.3.2 Cell Culture ....................................................................................................... 54 3.3.3 Primary cells ..................................................................................................... 55 3.3.4 In vivo Combination Treatment ...................................................................... 55 3.3.5 Immunoblotting ................................................................................................ 56 3.3.6 Cell Growth and Viability Assays ................................................................... 56 3.3.7 Combination High-Throughput Screen .......................................................... 57 3.3.8 Excess-over-Bliss Additivism for Calculating Synergy ............................... 57 3.3.9 Intracellular and Mitochondrial Reactive Oxygen Species Measurement .. 58 3.3.10 shRNA Knockdown Experiments ................................................................. 58 3.3.11 PARG Activity Assay ..................................................................................... 59 3.3.12 Statistical Analysis ........................................................................................ 59
3.4 Results ......................................................................................................... 60 3.4.1 BTK is overexpressed and constitutively active in AML cells .................... 60 3.4.2 AML cell lines are insensitive to chemical BTK inhibition with ibrutinib ... 62 3.4.3 A combination chemical screen with ibrutinib in AML cell lines identifies the PARG inhibitor, ethacridine lactate, as an ibrutinib sensitizer ...................... 64 3.4.4 The ibrutinib-ethacridine combination is preferentially cytotoxic to a subset of primary AML cells compared to normal hematopoietic cells .............. 70 3.4.5 The combination of ibrutinib and ethacridine delays the growth of AML cells in vivo ................................................................................................................ 74 3.4.6 Ethacridine synergizes with other small molecule BTK inhibitors, but not inhibitors of unrelated kinases ................................................................................ 76 3.4.7 Ibrutinib and ethacridine synergize to induce cell death via a ROS-dependent mechanism ............................................................................................. 80 3.4.8 The chemical PARG inhibitor gallotannin also synergizes with ibrutinib to induce cell death by excessive ROS production ................................................... 82 3.4.9 The synergy of ibrutinib with ethacridine is independent of the inhibitory effect on BTK ............................................................................................................. 85
3.5 Discussion ................................................................................................... 88 Chapter 4: Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells. .............................................. 91 4.1 Abstract ....................................................................................................... 92 4.2 Introduction ................................................................................................. 93 4.3 Methods ....................................................................................................... 94
4.3.1 Radiolabelled daunorubicin accumulation assay ......................................... 94 4.4 Results & Discussion ................................................................................. 94 Chapter 5: Erlotinib synergizes with the poly(ADP-ribose) glycohydrolase inhibitor ethacridine in acute myeloid leukemia cells ................................. 106 5.1 Abstract ..................................................................................................... 107 5.2 Introduction ............................................................................................... 108 5.3 Materials and Methods ............................................................................. 110
5.3.1 Reagents ......................................................................................................... 110 5.3.2 Cell culture ..................................................................................................... 110 5.3.3 Primary cells ................................................................................................... 111
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5.3.4 Immunoblotting .............................................................................................. 111 5.3.5 Cell viability assays ....................................................................................... 111 5.3.6 High-throughput combination drug screening & excess-over-Bliss additivism synergy calculations ............................................................................ 112 5.3.7 Reactive oxygen species measurement ...................................................... 112 5.3.8 Mass spectrometry ........................................................................................ 113
5.4 Results ....................................................................................................... 114 5.4.1 TEX and OCI-AML2 cell line sensitivity to erlotinib .................................... 114 5.4.2 A high-throughput combination chemical screen identifies erlotinib sensitizers in TEX and OCI-AML2 cells ................................................................ 116 5.4.3 The erlotinib-ethacridine combination synergizes in primary AML cells and other AML cell lines ................................................................................................ 119 5.4.4 Combining erlotinib and ethacridine generates lethal levels of reactive oxygen species ....................................................................................................... 122 5.4.5 Ethacridine synergizes with EGFR-targeting kinase inhibitors ................ 125 5.4.6 TEX and OCI-AML2 cell lines do not express EGFR .................................. 125 5.4.7 Erlotinib potentiates ethacridine accumulation in TEX and OCI-AML2 cells. .................................................................................................................................. 126 5.4.8 High-dose ethacridine treatment mimics ROS production observed from the erlotinib-ethacridine combination. .................................................................. 126
5.5 Discussion ................................................................................................. 131 Chapter 6: General Discussion & Conclusion .............................................. 134 6.1 Discussion ................................................................................................. 135
6.1.1 BTK-independent anti-leukemic activity of ibrutinib .................................. 135 6.1.1.1 Ibrutinib potentiates ethacridine accumulation .......................................... 135 6.1.1.2 Synergy between ibrutinib and daunorubicin is mediated by a mechanism unrelated to that of ibrutinib/erlotinib and ethacridine ........................................... 137
6.1.2 Anti-leukemic activity of ethacridine lactate ............................................... 138 6.1.3 Clinical relevance of BTK-independent effects of ibrutinib ....................... 139
6.2 Conclusion ................................................................................................. 142 Chapter 7: Future Directions .......................................................................... 143 7.1 Future Directions ...................................................................................... 144
7.1.1 Determining the mechanism of ethacridine accumulation by erlotinib and ibrutinib .................................................................................................................... 144
7.1.1.1 ABC transporters ....................................................................................... 144 7.1.2 Determining the relevant target of ethacridine ........................................... 145
7.1.2.1 PARG inhibition ......................................................................................... 145 7.1.2.2 p53 induction and the ribosomal stress pathway ...................................... 146
References ....................................................................................................... 148 Appendix 1 ....................................................................................................... 167
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List of Tables
Table 3-1: Patient demographics………………………………….…………………71
Table 5-1: Patient demographics…………………………...………………………121
List of Figures
Figure 1-1: Hematopoiesis: original and revised models. ..................................... 4
Figure 1-2: Structure of Ethacridine lactate ........................................................ 44
Figure 1-3: Mechanism of PARG activity ............................................................ 47
Figure 3-1: BTK mRNA levels in AML cell lines are similar to those of B-cell
malignancies. ............................................................................................... 61
Figure 3-2: AML cell lines express constitutively active BTK, but are insensitive to
ibrutinib. ....................................................................................................... 63
Figure 3-3: The PARG inhibitor ethacridine lactate sensitizes AML cell lines to
ibrutinib. ....................................................................................................... 65
Figure 3-4: Combination chemical screen validation for pentamidine. .............. 67
Figure 3-5: Cell death caused by ibrutinib-ethacridine combination is caspase
independent. ................................................................................................ 68
Figure 3-6: The ibrutinib-ethacridine combination is strongly synergistic in HL60,
U937, and K562, but not KG1a AML cell lines. ........................................... 69
Figure 3-7: The ibrutinib-ethacridine combination is preferentially cytotoxic to
primary AML cells over normal hematopoietic cells. .................................... 73
Figure 3-8: Ibrutinib-ethacridine combination displays anti-AML activity in mice. 75
Figure 3-9: Ethacridine synergizes with other small-molecule BTK inhibitors. ... 77
Figure 3-10: Ethacridine does not synergize with inhibitors of unrelated kinases.
..................................................................................................................... 78
Figure 3-11: Dasatinib and imatinib do not synergize with ethacridine in OCI-
AML2 cells. .................................................................................................. 79
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Figure 3-12: The ibrutinib-ethacridine combination induces cytotoxic levels of
intracellular ROS. ......................................................................................... 81
Figure 3-13: The PARG inhibitor gallotannin synergizes with ibrutinib ............... 83
Figure 3-14: Treatment of TEX and OCI-AML2 cells with olaparib in combination
with ibrutinib and ethacridine. ...................................................................... 84
Figure 3-15: Ibrutinib’s synergy with ethacridine is independent of BTK. ........... 86
Figure 3-16: Expression of TEC family kinases in AML cell lines. ...................... 87
Figure 4-1: Ibrutinib and daunorubicin synergize in TEX and OCI-AML2 cells. .. 95
Figure 4-2: Combination ibrutinib-cytarabine treatment of TEX and OCI-AML2
cells. ............................................................................................................. 96
Figure 4-3: Ibrutinib inhibits BTK phosphorylation. ............................................. 96
Figure 4-4: BTK knockdown confirmation. .......................................................... 98
Figure 4-5: Daunorubicin treatment of BTK-knockdown cells. ............................ 98
Figure 4-6: Ibrutinib treatment of BTK-knockdown TEX cells. .......................... 100
Figure 4-7: Daunorubicin accumulation in the presence or absence of ibrutinib.
................................................................................................................... 100
Figure 4-8: α-tocopherol rescue of combination-treated TEX and OCI-AML2 cells.
................................................................................................................... 102
Figure 4-9: Combination ibrutinib-daunorubicin treatment increases intracellular
ROS ........................................................................................................... 102
Figure 4-10: Intracellular ROS production following combination ibrutinib-
daunorubicin treatment in the presence or absence of α-tocopherol. ........ 103
Figure 4-11: Mitochondrial ROS production following combination ibrutinib-
daunorubicin treatment in TEX and OCI-AML2 cells. ................................ 104
Figure 5-1: AML cell line sensitivity to erlotinib. ................................................ 115
Figure 5-2: Erlotinib sensitizers in TEX and OCI-AML2 cells. ........................... 117
Figure 5-3: Validation of synergistic hits. .......................................................... 118
Figure 5-4: Erlotinib and ethacridine synergize in additional AML cell lines and
primary AML blasts. ................................................................................... 120
Figure 5-5: Combination erlotinib-ethacridine treatment induces lethal
intracellular ROS production. ..................................................................... 123
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Figure 5-6: Erlotinib enhances ethacridine accumulation in TEX and OCI-AML2
cells. ........................................................................................................... 128
Figure 5-7: Imatinib does not synergize with ethacridine in TEX and OCI-AML2
cells. ........................................................................................................... 130
List of Appendices
Appendix 1: Clinically achievable concentrations of kinase inhibitors………….167
xiv
List of Abbreviations ABC ATP-binding cassette ABC-DLBCL Activated B-cell-like subtype of diffuse large B-cell lymphoma Abl Abelson kinase ADP Adenosine diphosphate ADP-HPD Adenosine diphosphate (hydroxymethyl)pyrrolidinediol AIF Apoptosis inducing factor AML Acute myeloid leukemia APL Acute promyelocytic leukemia ATP Adenosine triphosphate ATRA All-trans retinoic acid Bcl-2 B-cell lymphoma 2 BCR B-cell receptor BCR Breakpoint cluster region BCRP Breast cancer resistance protein BMSC Bone marrow stromal cell BMX Bone marrow kinase in chromosome X BTK Bruton’s tyrosine kinase CAIA Anti-collagen antibody-induced arthritis CFU-L Colony forming unit-leukemia CIA Collagen induced arthritis CLL Chronic lymphocytic leukemia CLP Common lymphoid progenitor CML Chronic myeloid leukemia CMP Common myeloid progenitor CR Complete response/remission CRP C-reactive protein CXCR4 Chemokine (C-X-C Motif) Receptor 4 CXCR5 Chemokine (C-X-C Motif) Receptor 5 DAG Diacylglycerol DiOC2(3) 3,3’-diethyloxacarbocyanine DMSO dimethyl sulfoxide DNA Deoxyribonucleic acid EGFR Epidermal growth factor receptor EOBA Excess-over-Bliss additivism Epo Erythropoietin EpoR Erythropoietin receptor ER Endoplasmic reticulum ERK Extracellular signal related kinase FAB French American British classification FcεRI High-affinity IgE receptor FDA Food and Drug Administration FITC Fluorescein isothiocyanate FL FLT ligand FLT3 FMS-like tyrosine kinase 3
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FLT3-ITD FMS-like tyrosine kinase 3 with internal tandem duplication FLT3-TKD FMS-like tyrosine kinase 3 with tyrosine kinase domain mutation fMLP N-Formyl-methionyl-leucyl-phenylalanine GAPDH Glyceraldehyde-3-phosphate dehydrogenase G-CSF Granulocyte-colony stimulating factor GM-CSF Granulocyte macrophage colony-stimulating factor GMP Granulocyte-monocyte progenitor GPCR G-protein coupled receptor GPVI Glycoprotein VI HRP Horseradish peroxidase HSC Hematopoietic stem cell HSCT Hematopoietic stem cell transplant IFN Interferon IgE Immunoglobulin E IgM Immunoglobulin M IκBα Nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, alpha IL Interleukin iNOS Inducible nitric oxide synthase IP3 Inositol triphosphate IRAK Interleukin-1 receptor-associated kinase ITAM Immunoreceptor tyrosine-based activation motif ITK Interleukin-2-inducible T-cell kinase JAK Janus kinase LCK Lymphocyte-specific protein tyrosine kinase LPS Lipopolysaccharide LSC Leukemic stem cell M-CSF Macrophage colony-stimulating factor MAPK Mitogen-activated protein kinase MCL Mantle cell lymphoma MDM2 Murine double minute 2 MDS Myelodysplastic syndrome MEP Megakaryocyte erythroid progenitor MM Multiple myeloma MPP Multipotent progenitor mRNA Messenger ribonucleic acid MRP Multidrug resistance-associated protein MRR Major response rate mTOR Mammalian target of rapamycin MYD88 Myeloid differentiation primary response gene 88 NAD Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate NFAT Nuclear facor of activated T-cells NFκB Nuclear factor kappa-light-chain-enhancer of activated B-cells NSCLC Non small-cell lung cancer NO Nitric oxide
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OB Osteoblast OC Osteoclast ORR Overall response rate P-gp P-glycoprotein P53/TP53 Tumor protein 53 PAR Poly(ADP-ribose) PARG Poly(ADP-ribose) glycohydrolase PARP Poly(ADP-ribose) polymerase pBTK Phosphorylated BTK PDAC Pancreatic ductal adenocarcinoma PDGFRA Platelet-derived growth factor receptor alpha PDGFRB Platelet-derived growth factor receptor beta PH Pleckstrin homology PI Propidium iodide PI3K Phosphoinositide 3-kinase PIP2 Phosphatidylinositol 4,5-bisphosphate PIP3 Phosphatidylinositol 3,4,5-triphosphate PKC Protein kinase C PLCγ2 Phospholipase C gamma 2 qPCR Quantitative polymerase chain reaction RA Rheumatoid arthritis RAEB-2 Refractory anemia with excess blasts-2 RANKL Receptor activator of nuclear factor-kappaB ligand RIPA Radioimmunoprecipitation assay RLK/TXK Resting lymphocyte kinase ROS Reactive oxygen species rRNA Ribosomal RNA RT-PCR Reverse-transcriptase polymerase chain reaction SCF Stem cell factor SCID Severe combined immunodeficiency SDF1 Stromal cell-derived factor 1 SFK SRC-family kinase SH2 Src homology domain 2 SH3 Src homology domain 3 shRNA short hairpin RNA siRNA Short-interfering RNA SRB Sulforhodamine-B STAT Signal transducer and activator of transcription SYK Spleen tyrosine kinase T-ALL T-cell acute lymphoblastic leukemia TH TEC homology TK Tyrosine kinase TKI Tyrosine kinase inhibitor TLR Toll-like receptor TNFα Tumor necrosis factor alpha TRAIL TNF-related apoptosis-inducing ligand
xvii
VEGFR Vascular endothelial growth factor receptor WHO World Health Organization WM Waldenström macroglobulinemia WT Wild type Xid X-lined immunodeficiency XLA X-linked agammaglobulinemia
1
Preface
Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the
accumulation of improperly differentiated – and thus nonfunctional – myeloid
lineage cells. The mainstay of first-line therapy for this disease is aggressive
treatment with chemotherapy, which aims to eradicate leukemic cells and to
restore normal hematopoiesis. Unfortunately, this approach is inadequate for the
majority of patients: treatment-related toxicities and drug resistance have
translated to five-year survival rates of 25%. Thus, there is a great need for novel
approaches to AML treatment. One potential strategy for reducing therapy-
associated toxicity and improving efficacy is to combine anti-leukemic drugs with
synergizing agents in order to enhance AML cell sensitivity to these drugs. We
therefore screened drugs with documented preclinical anti-AML activity against
chemical libraries in AML cell lines to identify synergistic drug combination
candidates.
2
Chapter 1: Literature Review
3
1.1 Acute Myeloid Leukemia
1.1.1 Normal Hematopoiesis
In the traditional model of normal hematopoiesis in humans, maturation of
hematopoietic cells follows a hierarchy in which stem cells differentiate to give
rise to the lineages that produce mature blood cells: hematopoietic stem cells
(HSCs) self-renew or differentiate to multipotent progenitor cells (MPPs), which in
turn give rise to the oligopotent common myeloid (CMP) and common lymphoid
(CLP) progenitor cells. CLPs differentiate to T- and B-lymphocytes and natural
killer cells, while CMPs differentiate into megakaryocyte erythroid progenitors
(MEP) and granulocyte monocyte progenitors (GMP). MEPs ultimately give rise
to erythrocytes and megakaryocytes (from which platelets form), while GMPs
differentiate to infection and pathogen-fighting granulocytes (neutrophils,
eosinophils, and basophils) and monocytes. This model is illustrated in Figure 1-1 (top panel).
Recent work has contested this original model: (Notta et al., 2015) provided
evidence to support a two-tier model of hematopoiesis in the adult bone marrow
wherein multipotent HSCs give rise to unipotent progenitor cells that mature to
form monocytes, granulocytes, erythrocytes, and lymphocytes. Interestingly,
megakaryocytes were found to originate from the multipotent tier, and thus do not
arise from the same progenitors (CMPs) as the rest of the myeloid lineage, as
had previously been thought (Figure 1-1, bottom-right).
4
Figure reproduced from Notta et al. (2015), license #3817030184707 Figure 1-1: Hematopoiesis: original and revised models. Top panel: classical model of hematopoiesis. Bottom panel: revised model of hematopoiesis in the adult bone marrow (right) and fetal liver bone marrow (left). Abbreviations: HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte erythroid progenitor; GMP, granulocyte-monocyte progenitor; Ly, lymphoid cell; Er, erythroid cell; Mk, megakaryocyte; Gran, granulocyte; Mono, monocyte; My, granulocyte/monocyte
5
1.1.2 Acute Myeloid Leukemia
Acute myeloid leukemia (AML) comprises many different hematologic neoplasms
with one unifying feature: the presence of proliferative, clonal, myeloid-lineage
cells that are improperly differentiated and the ensuing absence of one or more
mature myeloid-lineage cell types (reviewed in Döhner et al. (2015)). These
improperly differentiated cells—termed blasts—accumulate in the bone marrow
and peripheral blood. Generally, when the blast percentage in the blood and
bone marrow reaches or exceeds 20%, a diagnosis of AML is made (Vardiman et
al., 2002).
1.1.2.1 AML Pathogenesis
AML results from a series of mutations that cooperate to confer a proliferative
and survival advantage to the leukemic clone. The cells of origin in AML are
leukemic stem cells (LSCs), which develop from alterations in normal
hematopoietic stem cells (HSCs) (Bonnet & Dick, 1997; Hope et al., 2004;
Lapidot et al., 1994). LSCs, like HSCs, are highly primitive (often possessing the
CD34+CD38- immunophenotype) and have the capacity to recapitulate the entire
AML cell hierarchy (Bonnet & Dick, 1997; Lapidot et al., 1994). LSCs have the
ability to self-renew or give rise to non self-renewing leukemic progenitor cells
(also known as CFU-L, or colony-forming unit-leukemia cells) (Bonnet & Dick,
1997; Hope et al., 2004). CFU-L cells actively proliferate and undergo incomplete
differentiation to leukemic blasts (reviewed by (Griffin & Löwenberg, 1986). Left
untreated, blast accumulation ultimately prevents the formation of mature
myeloid cells, such as neutrophils, erythrocytes, and platelets, leading to
infection susceptibility, anemia, and hemorrhage, respectively.
6
1.1.2.2 Epidemiology of AML
AML is the most common form of acute leukemia in adults, and the risk of
developing the disease increases significantly with age. The incidence in those
under the age of 65 is approximately one per 100,000, while this figure climbs to
12 per 100,000 in those over the age of 65. AML is also slightly more prevalent in
males than females, with a 5:3 ratio (Siegel et al., 2012).
1.1.2.3 AML Classification and Prognostication
The French American British (FAB) Cooperative Group classification system was
an early system used to divide AML into subtypes based on morphology (Bennett
et al., 1976). Organized from M0-M7, each subtype reflects the cell type from
which the leukemia originated and its degree of differentiation, with M0
representing the most primitive (“undifferentiated acute myeloid leukemia”), and
M6 and M7 representing the most mature AML subtypes (“acute erythroid
leukemia” and acute megakaryoblastic leukemia”, respectively). Several FAB
subtypes have associated somatic cytogenetic abnormalities, and identifying
such abnormalities in newly diagnosed AML patients may at times offer useful
insights into individualized disease management. For instance, FAB M3 (acute
promyelocytic leukemia, APL) is most commonly associated with a t(15;17)
translocation, producing the oncogenic PML-RARα fusion protein, which can be
successfully targeted through addition of all trans-retinoic acid differentiation
therapy in conjunction with an anthracycline-containing chemotherapy regimen or
arsenic trioxide (Tallman & Altman, 2009).
For most other AML patients, however, the FAB classification is of limited clinical
utility, as it does not take into consideration other, non-morphologic abnormalities
that also provide prognostic insight and thus inform AML treatment approaches.
These limitations prompted the development of the World Health Organization
(WHO) classification of myeloid neoplasms, which in addition to AML blast
7
morphology, utilizes genetic, immunophenotypic, biologic, and clinical information
to categorize AML subtypes (Vardiman et al., 2002). The WHO system classifies
AML into the following four major subgroups: AML with recurrent genetic
abnormalities (which include mutations such as t(8:21)(q22;q22),
inv(16)(p13.1q22), t(16;16)(p13.1;q22), and t(15;17)(q22;q12)), AML with
myelodysplasia-related changes, therapy-related AML (caused by previous
treatment with alkylating agents, radiation, or topoisomerase II inhibitors), and
AML, not otherwise specified (Vardiman et al., 2009). The majority of identified
recurrent genetic abnormalities associated with AML have been stratified into
favourable, intermediate-I, intermediate-II, and adverse prognostic categories
(Döhner et al., 2010). Three-year overall survival rates for each category, in both
younger and older AML patients, have been reported (Mrózek et al., 2012):
patients with alterations such as t(8;21)(q22;q22), inv(16)(p13.1q22) and
t(16;16)(p13.1;q22) have “favourable” prognoses, with a 3-year overall survival of
66% and 33% in the under- and over-60 age groups, respectively. Patients with
karyotypically normal AML harbouring the FLT3-ITD mutation with or without
NPM1 mutation, or karyotypically normal AML without the NPM1 or FLT3-ITD
mutations fall under the intermediate-I prognostic classification, with 3-year
survival rates of 28% and 11% in the under- and over-60 age group, respectively.
The t(9;11)(p22;q23) translocation is classified prognostically as intermediate-II
and is associated with 3-year survival rates of 45% in patients under the age of
60, and 16% in patients over the age of 60. Finally, adverse-risk alterations such
as inv(3)(q21q26.2), t(3;3)(q21;q26.2), t(6;9)(p23;q34), -5, del(5q), -7, and
complex karyotype AML are associated with 3-year survival rates of 12% and 3%
in adults under- and over the age of 60, respectively.
1.1.2.4 AML Management
Standard pharmacologic AML therapy aims to eradicate leukemic blasts and
restore normal multilineage hematopoietic cell growth. It often consists of two
8
phases: induction and consolidation. During the induction phase, a combination
of the chemotherapy drugs, daunorubicin and cytarabine (known as the 3+7
regimen) is administered over a seven-day period. Daunorubicin, an
anthracycline antibiotic, likely induces death of AML cells via several proposed
mechanisms. Daunorubicin binds to and inhibits topoisomerase II, preventing
replication fork progression and creating single- and double-stranded DNA
breaks and inducing subsequent cell death. In addition to topoisomerase II
inhibition, daunorubicin blocks DNA synthesis via DNA intercalation, induces the
generation of free radicals, and may disrupt DNA helicase activity (Gewirtz,
1999).
Cytarabine is a nucleoside analogue. It inhibits DNA polymerases and competes
with deoxycitidine for incorporation into newly synthesized DNA. Cytarabine
incorporation halts DNA synthesis, inducing subsequent cell death (Grant, 1998;
Inagaki et al., 1969).
Induction therapy produces complete remissions (defined as <5% bone marrow
blasts, and recovery of absolute neutrophil and platelet counts to >1.0x109/L and
>10x109/L, respectively (Döhner et al., 2010) in 60-85% of AML patients under
the age of 60, and in 40-60% of those over the age of 60 (Döhner et al., 2015).
However, induction therapy alone is inadequate to produce lasting remissions in
patients: without further treatment, AML patients relapse within several months
(Cassileth et al., 1988). Additional therapy is therefore necessary in order to
reduce AML relapse risk and prolong remissions.
The post-remission treatment approach to AML is dependent upon the prognostic
classification of the patient’s cytogenetic and/or molecular abnormalities, as well
as the patient’s age and presence or absence of comorbidities. In general,
pharmacologic consolidation therapy with cytarabine is recommended in patients
with favourable-risk AML, with this approach leading to cure in 60-70% of
patients aged <60 (Döhner et al., 2015). Allogeneic hematopoietic stem cell
9
transplantation (HSCT) is recommended in patients with intermediate or adverse-
risk disease, as these individuals are unlikely to be cured with cytarabine
consolidation; however, HSCT carries significant risk of mortality, lifelong
morbidity, and requires identification of an appropriately matched donor (Döhner
et al., 2015).
10
1.2 Tyrosine Kinase Inhibitor Therapy in AML
1.2.1 Targeted Cancer Therapies
The significant toxicity associated with standard chemotherapy treatment for AML
is related to the lack of specificity of these drugs: DNA synthesis and replication
are ubiquitous processes and daunorubicin and cytarabine are therefore also
lethal to many non-cancerous cell types. Therapies with targets unique—or at
least more specific—to cancer cells have thus been investigated as a less toxic
therapeutic strategy compared to standard chemotherapies.
1.2.1.1 Tyrosine Kinase Inhibitors
Tyrosine kinase inhibitors (TKIs) are one class of targeted therapy used to treat
malignancies. Although the expression of protein tyrosine kinases (TKs) is not
exclusive to cancer cells, their critical role in many cell growth, proliferation, and
survival signaling pathways (which are frequently aberrantly activated in cancer)
make them ideal candidates for therapeutic targeting. Tyrosine kinases catalyze
the transfer of a phosphoryl group from ATP to a tyrosine residue on protein
substrates. Tyrosine phosphorylation of protein targets may serve as activation
or regulatory signals, and the majority of TKIs intercept TK activity by competing
with ATP for binding to the ATP-binding site of the TK (reviewed by Roskoski
(2015)).
A classic example of the success of TKIs in cancer therapy is that of imatinib.
This small-molecule inhibitor of Abelson tyrosine kinase (Abl) has vastly
improved the survival of patients with chronic myelogenous leukemia (CML).
CML is characterized by the presence of the Philadelphia translocation
(t(9;22)(q34;q11.2)), which results in expression of the oncogenic breakpoint
cluster region (BCR)-Abl fusion protein. This fusion of BCR and Abl renders Abl
11
constitutively active, which causes leukemic transformation. The small molecule
imatinib binds to the inactive conformation of Abl (Capdeville et al., 2002) and
produces long-term complete remissions in CML patients: in a long term follow
up study of first-line imatinib therapy for this disease, six-year progression-free
survival was found to be 87% (with overall survival at 89%) (Castagnetti et al.,
2015), compared to a median survival of five to seven years with interferon α
therapy (Apperley, 2015).
1.2.2 Oncogenic Tyrosine Kinases in AML
The striking success of imatinib in CML prompted the evaluation of TKs as
therapeutic targets in other malignancies, including AML. Given the
comparatively heterogeneous nature of AML, however, this approach has proven
to be far more challenging for this disease: no one TK target is consistently
deregulated in the majority of AML cases. In reality, many different kinases
implicated in proliferation and survival pathways may be deregulated in AML and
may thus contribute to the pathogenesis of this malignancy (Kelly & Gilliland,
2002). While there are currently no TKIs approved for clinical use in AML, several
inhibitors are under clinical investigation, and other kinases have been proposed
as potential therapeutic targets.
1.2.2.1 FMS-Related Tyrosine Kinase 3
FMS-related tyrosine kinase 3 (FLT3) is an extensively studied receptor TK that
is expressed in normal hematopoietic stem and progenitor cells (Rosnet et al.,
1996; Small et al., 1994). FLT3 signaling plays an important role in
hematopoiesis: stimulation of this receptor with its ligand, FL, induces
proliferation and contributes to differentiation of hematopoietic progenitor cells
(Gabbianelli et al., 1995; Lyman et al., 1994). FLT3 is also expressed in primary
12
AML cells (Birg et al., 1992; Carow et al., 1996). FL-mediated FLT3 stimulation
was found to induce proliferation and clonogenic growth in a subset of AML cell
lines and primary AML blasts, respectively (Dehmel et al., 1996; Stacchini et al.,
1996).
Two common oncogenic FLT3 mutations have been identified in AML: an internal
tandem duplication (FLT3-ITD), present in approximately 20% of cases (Nakao et
al., 1996; Yokota et al., 1997), and a point mutation in the second tyrosine kinase
domain (FLT3-TKD), which is present in 7% of AML cases (Yamamoto et al.,
2001). Both mutations induce constitutive (FL-independent) FLT3 activity in AML
(Hayakawa et al., 2000; Kiyoi et al., 1998; Yamamoto et al., 2001), and FLT3-ITD
mutations have been associated with particularly poor AML patient prognoses
(Whitman et al., 2001; Yamamoto et al., 2001). Thus, there has been significant
interest in investigating the therapeutic potential of FLT3 inhibitors in patients
with AML.
Clinical trials for some FLT3 inhibitors have reported some benefit to patients
with FLT3-ITD positive AML. The first-generation FLT3 inhibitors sunitinib,
sorafenib, midostaurin and lestaurtinib—all multi-receptor TK inhibitors—caused
reductions in peripheral blast count, with sunitinib producing complete remissions
as a single-agent, and midostaurin inducing remissions when administered in
combination with chemotherapy agents (Wander et al., 2014). However, these
studies reported transient reductions of peripheral or marrow blasts (sunitinib,
midostaurin, lestaurtinib), and lestaurtinib in combination with chemotherapy
failed to improve complete remission rates or overall survival in
relapsed/refractory AML patients in a Phase III trial (Wander et al., 2014).
Subsequent generations of FLT3 inhibitors have produced more promising
results in the clinical trial setting. Quizartinib (AC220), which has been shown to
target FLT3, KIT, PDGFRA, PDGFRB, and RET kinases in preclinical studies,
produced responses in nine of 17 AML patients harboring the FLT3-ITD
mutation, five of 37 FLT3-ITD negative patients, and nine of 22 patients with
13
undetermined FLT3-ITD status in a Phase I trial (Cortes et al., 2013). In a Phase
II trial of single-agent quizartinib in patients over the age of 60 with
relapsed/refractory AML, 54% of FLT3-ITD positive and 32% of FLT3-ITD
negative patients achieved a composite complete remission (Cortes et al., 2012).
Crenolanib and PLX3397 are newer FLT3 inhibitors with impressive preclinical
activity against FLT3-mutant AML cell lines and are currently undergoing
evaluation in clinical trial (Wander et al., 2014).
14
1.3 Ibrutinib
Ibrutinib (PCI-32765) is a Bruton’s tyrosine kinase (BTK) inhibitor that is currently
clinically approved for the treatment of chronic lymphocytic leukemia (CLL),
mantle cell lymphoma (MCL), and Waldenström’s macroglobulinemia (WM)
(FDA, 2015a). BTK is an important therapeutic target in these B-cell
malignancies, and ibrutinib was designed to inhibit this cytoplasmic kinase with
high specificity. In the clinic, ibrutinib is well tolerated by patients and clinical
trials for ibrutinib use in these cancers have demonstrated improved patient
outcomes. As preclinical research into novel roles for BTK in different diseases
continues, the list of FDA-approved ibrutinib indications is likely to rapidly
expand.
1.3.1 Bruton’s tyrosine kinase: background & role in signal transduction from the B-cell receptor
1.3.1.1 BTK domains
The BTK gene is located at the Xq21.3-Xq22 locus (Kwan et al., 1986; Malcolm
et al., 1987) and encodes a 659 amino acid protein (Vetrie et al., 1993). BTK
belongs to the TEC family of cytoplasmic protein kinases, which also includes
TEC, ITK, RLK/TXK and BMX. BTK, like all TEC family kinases, is similar in
sequence to SRC family kinases (SFKs): it contains Src homology (SH) 2 and
SH3 domains, as well as a C-terminal kinase domain (Vetrie et al., 1993).
However, BTK and the majority of the other TEC family members are
distinguishable from SFKs by the presence of a Tec homology (TH) domain, an
N-terminal plasma membrane-targeting pleckstrin homology (PH) domain (as
opposed to a myristoylation sequence found in SFKs), as well as the absence of
a negative regulatory tyrosine analogous to the C-terminal Y527 of SFKs (Vetrie
et al., 1993).
15
1.3.1.2 BTK expression BTK is a cytoplasmic protein and its expression is restricted to hematopoietic
cells. This kinase is expressed throughout B-cell maturation, with BTK mRNA
and/or protein detection in pro-B cell, early pre-B cell, late pre-B cell, and mature
B cell lines (de Weers et al., 1993; Genevier et al., 1994). BTK is also expressed
in many myeloid cell lines, but its expression is downregulated in plasma cells
and T-cells (de Weers et al., 1993; Genevier et al., 1994; Smith et al., 1994).
1.3.1.3 BTK: Role in B-cell Maturation
The importance of BTK function during B-cell maturation is illustrated clinically in
X-linked agammaglobulinemia (XLA), a human primary immunodeficiency
caused by germline BTK mutations (Tsukada et al., 1993; Vetrie et al., 1993).
Patients with XLA often have normal pre-B cell levels, but severely reduced or
absent levels of B-cells and plasma cells (and thus immunoglobulins), implying
that BTK is critical for maturation beyond the pre-B cell stage (de Weers et al.,
1993). This disease manifests itself clinically as an increased susceptibility to
recurrent bacterial infections in infant males, and is treated with donor-derived
immunoglobulin therapy, and antibiotics in the presence of confirmed or
suspected infections (Bruton, 1952; Timmers et al., 1991).
1.3.1.4 BTK Signaling in B-Cells
In B-cells, BTK is required for appropriate signal transduction from the B-cell
receptor (BCR) upon receptor crosslinking (reviewed in-depth by Dal Porto et al.
(2004)). When an antigen binds to the immunoglobulin (IgM) portion of the BCR,
the CD79A (Igα) and CD79B (Igβ) components of the BCR are tyrosine-
16
phosphorylated on their immunoreceptor tyrosine-based activation motifs
(ITAMs) by LYN and other SRC family kinase (SFK) members. Tyrosine
phosphorylation of ITAMs attracts spleen tyrosine kinase (SYK) via SYK’s SRC
homology 2 (SH2) domains, and SYK is subsequently phosphorylated and
activated by vicinal SFKs. Antigen binding to the BCR also triggers simultaneous
activation of phosphoinositide 3-kinase (PI3K), which phosphorylates the
membrane phospholipid, PIP2. Phosphorylated PIP2 (also known as PIP3)
recruits cytoplasmic BTK via its pleckstrin homology domain to the plasma
membrane. Here, proximal SFKs and SYK phosphorylate BTK at Y551 of its
kinase domain and BTK undergoes subsequent autophosphorylation at Y223 in
its SH3 domain, which stabilizes its active conformation (Rawlings et al., 1996;
Wahl et al., 1997). Activated BTK and SYK then phosphorylate and activate
phospholipase Cγ2 (PLCγ2) (Takata & Kurosaki, 1996), which cleaves the
membrane-associated PIP2 to form IP3 and DAG. IP3 generation leads to Ca2+
mobilization from intra- and extracellular Ca2+ stores, ultimately triggering NFAT
activation. Ca2+ and DAG, together, activate protein kinase Cβ, which causes
NFKB pathway activation. BCR signaling also triggers the activation of MAPK
and RAS signaling pathways. The end result of BCR signaling, mediated by BTK,
is B-cell survival and proliferation.
1.3.2 A Rationale for Targeting BTK in B-cell Malignancies BTK emerged as an attractive therapeutic target in B-cell malignancies due to its
relatively restricted expression pattern and its role as a signal transducer in
several pathways implicated in the pathogenesis and progression of these
diseases.
17
1.3.2.1 Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of
CD5-expressing mature B cells within the bone marrow and secondary lymphoid
tissues. Proliferation and survival of CLL cells is dependent upon stimulatory
signals from their respective tissue microenvironments, as well as increased
antigen-dependent or independent (“tonic”) BCR signaling (Burger, 2013). Given
that BTK is implicated in signal transduction downstream of both the BCR and
chemokine receptor (CXCR4 and CXCR5) pathways (Hendriks et al., 2014), BTK
inhibition would thus act as a two-pronged approach to interfering with CLL cell
proliferation and survival.
1.3.2.2 Mantle-Cell Lymphoma
Mantle-cell lymphoma (MCL) is a type of non-Hodgkin lymphoma that is
associated with aberrant cyclin D1 overexpression in mantle-zone B-cells. Cyclin
D1 overexpression drives proliferation of these cells. BCR signaling pathway
proteins, including BTK, are overexpressed in MCL and this pathway is highly
active in MCL cell lines (Cinar et al., 2013; Pighi et al., 2011). BCR signaling
contributes to MCL cell proliferation and survival, and inhibiting SYK within this
pathway (which is upstream of BTK) has been shown to induce apoptosis and
decrease cyclin D1 expression (Pighi et al., 2011; Rinaldi et al., 2006).
1.3.2.3 Waldenström Macroglobulinemia
Waldenström macroglobulinemia (WM) is an indolent lymphoma characterized by
the overproduction of IgM-producing lymphoplasmacytic cells in the bone
marrow. One commonly identified feature of this disease is the presence of the
myeloid differentiating factor 88 (MYD88) L265P mutation, which has been
detected in over 90% of WM patients (Treon et al., 2012; Varettoni et al., 2013;
18
Xu et al., 2013). This mutation promotes WM cell growth and survival by
increased activation of NF-KB, which is mediated in part by BTK signaling (Yang
et al., 2013). In contrast, wild-type MYD88 does not associate with, nor signal
through, BTK (Yang et al., 2013). Constitutively active BTK has also been
detected in two WM cell lines harbouring the MYD88 L265P mutation (Tai et al.,
2012).
1.3.3 Development of Ibrutinib as a Selective and Irreversible BTK Inhibitor with In Vivo Activity
Prior to the development of ibrutinib, there were no existing small molecule BTK
inhibitors with in vivo activity. Pan et al. (2007) used a scaffold screening
approach to identify compounds with inhibitory activity against BTK, noting that
one of these compounds inhibited BTK at a Ki of 8.2nM in enzymatic assays.
This compound, however, was not BTK-specific, as it also potently inhibited other
TEC and SRC family kinases. To design a compound with enhanced potency
and selectivity, this group mapped the predicted binding site of the original
compound to within the kinase domain of BTK and LCK, a SRC family kinase.
They reported the presence of a nucleophilic residue within the kinase domain of
BTK (Cys481) that was absent in LCK, and noting that other kinases with
equivalent Cys residues were potently and irreversibly inhibited by small
molecules with electrophilic centres, they designed several compounds to
analogously inhibit BTK. The most potent of these compounds, “Compound 4”
(later designated PCI-32765, or ibrutinib), inhibited BTK activity at an IC50 of
0.72nM and the activity of the BTK substrate PLCγ1 at an IC50 of 14nM and was
more than 500 times more selective for BTK than SYK or the SRC family kinase,
LYN. Ibrutinib treatment prevented arthritis development in an anticollagen
antibody and LPS-induced murine arthritis model.
19
Further investigation of the activity of ibrutinib by Honigberg et al. (2010)
confirmed its selectivity against BTK and provided additional in vivo evidence to
support its use as a clinical BTK inhibitor in several B-cell disease models. Oral
ibrutinib administration improved renal function in the MRL-Fas(lpr) murine model
of lupus (which induces glomerulonephritis), and improved clinical arthritis scores
in mouse models of rheumatoid arthritis. Ibrutinib also produced partial
responses in a canine model of non-Hodgkin lymphoma.
1.3.4 Preclinical and clinical activities of ibrutinib in B-cell cancers
1.3.4.1 Chronic Lymphocytic Leukemia
BTK inhibition by ibrutinib was found to only modestly induce patient-derived CLL
cell apoptosis, but significantly reduced CLL cell proliferation at clinically
achievable concentrations (Cheng et al., 2014; Herman et al., 2011; Ponader et
al., 2012) and delayed CLL progression in an adoptive transfer TCL1 murine
model of CLL (Ponader et al., 2012). Perhaps the most striking effect of ibrutinib
treatment CLL cells was the impact of this drug on the interactions between
these cells and their microenvironment: ibrutinib counteracted IL-6, IL-10, and
TNFα production by T-cells (Herman et al., 2011), abrogated the pro-survival and
proliferative effects imparted by Hs5 stromal cell (Herman et al., 2011) and
nurse-like cell (Ponader et al., 2012) co-culture, reduced CLL cell migration
toward the tissue homing chemokines CXCL12 and CXCL13 (Ponader et al.,
2012), and blocked IgM-stimulated CLL cell adhesion to fibronectin and VCAM-1
(de Rooij et al., 2012). The ibrutinib-mediated disruption of CLL cell homing to
microenvironment-produced chemokines has been hypothesized as the
explanation for the observed transient peripheral blood lymphocytosis following
ibrutinib treatment in vivo (Ponader et al., 2012) and in clinical trial participants
(Byrd et al., 2013).
20
Clinical trials of ibrutinib in both treatment-naïve and pre-treated CLL patients
have yielded impressive responses: in a Phase 1b/2 trial assessing single-agent
ibrutinib treatment in relapsed/refractory CLL, the overall response rate was 71%
and 26-month progression-free survival was 75% (Byrd et al., 2013). Plasma
ibrutinib concentrations in these patients reached ~450nM (see Appendix 1). A
three-year follow-up of this study demonstrated the long-term efficacy of ibrutinib,
with overall response rates of 90% and 84% in patients with relapsed/refractory
and treatment-naïve CLL, respectively (Byrd et al., 2015). In a Phase 1b/2 trial
assessing ibrutinib in newly diagnosed CLL in patients over the age of 65,
ibrutinib was generally well tolerated and efficacious, with 71% of patients
achieving an objective response, and 4/22 responders achieving complete
responses (O'Brien et al., 2014). In a Phase 3 trial in relapsed/refractory CLL,
ibrutinib was superior to the anti-CD20 monoclonal antibody ofatumumab in
extending progression-free and overall survival and overall survival (Byrd et al.,
2014). Ibrutinib is now FDA-approved for CLL with 17p deletion and for
previously treated CLL (FDA, 2015a).
1.3.4.2 Mantle Cell Lymphoma
In preclinical studies, ibrutinib was found to block BTK activity in primary MCL
cells stimulated by IgM or co-culture with stromal cells, and in the MCL cell lines
Mino, Jeko, and HBL2 (Chang et al., 2013). This drug only modestly reduced
MCL cell line growth and viability at clinically achievable concentrations, and
induced apoptosis at supraclinical (10-20 µM) concentrations (Cinar et al., 2013).
Ibrutinib has been shown to synergize with the proteasome inhibitor bortezomib,
with the combination inducing ER stress, AKT and NFkB inhibition,
downregulation of Bcl-2 family proteins, and apoptotic cell death in Granta519
MCL cells (Dasmahapatra et al., 2013). Ibrutinib-mediated downregulation of Bcl-
21
2 family anti-apoptotic proteins was confirmed in Mino, an MCL cell line, by Cinar
et al. (2013).
In the context of MCL-microenvironment signaling, ibrutinib reduced chemokine
production by MCL cell lines and blocked their adhesion and migration in
response to BCR-, CXCL12- and CXCL13-mediated stimulation. Ibrutinib
treatment of C57BI/6 mice reduced MCL cell migration toward lymphoid tissues,
and reduced MCL cell infiltration of lymph nodes and bone marrow in a murine
model of MCL lymphadenopathy (Chang et al., 2013).
A Phase II clinical trial in relapsed/refractory MCL patients demonstrated an
overall response rate (ORR) of 68%, with 21% achieving complete responses
(CR) (Wang et al., 2013) and led to the drug’s accelerated approval for
previously-treated MCL by the FDA. A longer-term follow-up of this trial (median
follow-up of 26.7 months) demonstrated similarly impressive outcomes, with an
ORR of 67%, CR of 23%, and 31% progression-free survival at 24 months
(Wang et al., 2015).
1.3.4.3 Waldenström Macroglobulinemia
In preclinical studies, ibrutinib treatment of the MYD88 L265P BCWM.1 and
MWCL1 WM cell lines reduced IKBα phosphorylation (IKBα phosphorylation
permits nuclear translocation and activation of NFKB). In addition, these cell lines
were more sensitive to killing by ibrutinib compared to MYD88 WT WM cell lines
(Yang et al., 2013). Combining ibrutinib with an inhibitor of interleukin – receptor-
associated kinase (IRAK) 1 and 4—the other reported pathway by which MYD88
signalling activates NFKB—profoundly enhanced NFKB inhibition and induced
synergistic cell death (Yang et al., 2013).
22
Somatic activating C-terminus CXCR4 mutations are present in approximately
30% of WM patients (Roccaro et al., 2014). WM cells engineered to express
CXCR4 C-terminus mutations commonly found in WM patients, exhibited
constitutive receptor activity due to impaired CXCR4 internalization following
ligand (SDF-1a) binding, as well as sustained ERK and AKT activation (Cao et
al., 2015a; Cao et al., 2015b). Mutant CXCR4-mediated ERK and AKT activation
were shown to contribute to ibrutinib resistance in WM, which was reversible by
CXCR4 inhibitor (AMD3100, plerixafor) administration (Cao et al., 2015a; Cao et
al., 2015b).
A clinical trial evaluating the efficacy of ibrutinib in WM provided strong evidence
to support targeting BTK as a therapeutic strategy for this disease. In this study,
ibrutinib treatment of 63 pre-treated WM patients resulted in an overall response
rate of 90.5%, with major responses in 73% of participants. Ibrutinib efficacy was
greatest in WM patients harboring MYD88 L265P and wild-type CXCR4 (91.2%
major response rate (MRR), 100% overall response rate (ORR)), but still highly
effective in patients with both the MYD88 and CXCR4 mutations (61.9% MRR,
85.7% ORR). Ibrutinib was the least effective in patients harbouring both wild
type MYD88 and CXCR4 (28.6% MRR, 71.4% ORR) (Treon et al., 2015). The
findings of this study led to the FDA-approval of ibrutinib for WM.
1.3.5 B-cell independent BTK signaling: myeloid-lineage cells
BTK is expressed in both primitive and mature myeloid-lineage cells (Schmidt et
al., 2004a). Given the observation that individuals with BTK mutations do not
appear to have abnormal myeloid cell numbers or defective myeloid cell activity,
it was long assumed that BTK played an insignificant or redundant role in the
development and function of these cells. However, several groups have
23
demonstrated evidence of the important role of this kinase in both normal and
malignant myeloid cells.
1.3.5.1 Mast Cells
In mast cells, BTK is activated upon high-affinity IgE receptor (FcεRI) cross-
linking (Kawakami et al., 1994). The mechanism of BTK activation in these cells
resembles that of mature B-cells: FcεRI cross-linking in response to antigen
binding leads to phosphorylation of receptor-associated ITAMs by LYN. ITAM
phosphorylation triggers the recruitment and activation of SYK and additional
LYN, which in turn phosphorylate and activate BTK. Btk itself does not associate
with FcεRI in murine mast cells (Kawakami et al., 1994), however it is
constitutively bound to protein kinase C (PKC) via its PH domain (Yao et al.,
1994). PKC phosphorylates Btk and this event is inhibitory: Btk
autophosphorylation is decreased and pharmacologic PKC inhibitors enhance
tyrosine phosphorylation of Btk upon FcεRI stimulation (Yao et al., 1994).
In contrast to B-cells, BTK does not appear to be required for mast cell
development. In two murine models of defective Btk (btk null and xid), mast cell
numbers, morphology and expression of important signaling proteins were not
different compared to wild type controls (Hata et al., 1998).
BTK does appear to be important, however, for normal mast cell signaling and
function: Btk-defective mice exhibited diminished anaphylactic responses relative
to wild type mice. Upon FcεRI cross-linking of mast cells cultured from xid and/or
btk-null mice, these cells exhibited reduced Ca2+ mobilization, impaired
degranulation (as indicated by reduced histamine release), and demonstrated
severely compromised TNF-α transcription, and TNF-α, IL-2, IL-6, and GM-CSF
secretion (Hata et al., 1998; Setoguchi et al., 1998). Consistent with these
findings, transfection of multiple siRNAs directed against Btk in RBL-2H3 rat
24
mast cells induced a 20-25% decrease in histamine release upon FcεRI
stimulation (Heinonen et al., 2002). This observation was further corroborated
with pharmacologic Btk inhibitors: the leflunomide metabolite LFM-A13 also
moderately reduced histamine release in FcεRI-stimulated RBL-2H3 cells
(Heinonen et al., 2002) and the quinone epoxide terreic acid decreased TNF-α
and IL-2 secretion in antigen-stimulate mouse bone marrow-derived mast cells
(Kawakami et al., 1999).
In a more recent study by Soucek et al. (2011), oral administration of ibrutinib in
a Myc-driven murine insulinoma model (in which mast cell recruitment is critical
to tumor expansion and angiogenesis) was found to block mast cell
degranulation and induce tumor regression. Furthermore, in addition to blocking
mast cell degranulation and tumor cell proliferation in murine models of
pancreatic ductal adenocarcinoma (PDAC), ibrutinib was found to block mast
cell-mediated fibrosis of stromal tissue, which is commonly associated with
therapy resistance in this tumor type (Massó-Vallés et al., 2015).
1.3.5.2 Macrophages
BTK expression does not appear essential for macrophage development, as
bone marrow-derived and splenic macrophages from Btk-deficient mice were
found to be phenotypically similar to those of wild-type mice (Schmidt et al.,
2006). In addition, macrophages derived from Xid mice exhibited no differences
in phagocytic activity (Mangla et al., 2004).
BTK does however appear to be important for other macrophage functions.
Macrophages derived from Xid mice demonstrated impaired bactericidal activity
(Mukhopadhyay et al., 2002), and cytokine production by Btk-deficient
macrophages was altered: TLR stimulation of Btk-deficient macrophages
resulted in reduced production of the anti-inflammatory cytokine IL-10 relative to
25
wild-type controls (Schmidt et al., 2006). Moreover, Btk-deficient
monocyte/macrophage production of pro-inflammatory cytokines TNFα and IL-1β
following LPS stimulation was impaired relative to control macrophages
(Horwood et al., 2003; Mukhopadhyay et al., 2002).
BTK also appears to be implicated in reactive oxygen species (ROS) and nitric
oxide (NO) generation in response to macrophage stimulation: relative to
macrophages derived from wild-type mice, LPS stimulation of macrophages from
Xid mice resulted in reduced ROS generation (Mangla et al., 2004). Xid mouse-
derived stimulated macrophages were also found to have profoundly reduced NO
production, as a result of impaired induction of the STAT1/IFN regulatory factor-
1/iNOS pathway relative to control mice (Mukhopadhyay et al., 1999). Reduced
NO production was associated with increased production of IL-12 in these Xid
macrophages (Mukhopadhyay et al., 1999), a cytokine that drives T-cells to
activate macrophages via IFNγ production.
1.3.5.3 Erythroid Cells
In mouse erythroid progenitor cells, erythropoietin (Epo) and stem cell factor
(SCF) stimulation of the erythropoietin receptor (EpoR) and cKit, respectively,
results in progenitor cell proliferation, and EpoR stimulation by Epo induces cell
differentiation into erythrocytes. Btk expression in erythroblastoid cell lines
derived from chickens was first reported by Robinson et al. (1998). Building on
this work, Schmidt et al. (2004b) investigated the role of Btk in erythroid
progenitor cell signaling. They noted that Epo and/or SCF stimulation of mouse-
derived erythroid progenitor cells induced BTK phosphorylation at Y223. Btk-
deficient erythroid progenitor cells derived from mice demonstrated reduced
sensitivity to Epo and thus impaired phosphorylation of EpoR, Jak2, Stat5, and
Plcγ1 following Epo stimulation, relative to wild type-derived controls. In addition,
SCF treatment was found to induce Btk association with TNF-related apoptosis-
26
inducing ligand (TRAIL) receptor 1, and Btk-deficient cells were more sensitive to
TRAIL-induced apoptosis in the presence of SCF. On a functional and
morphologic level, exposure of Btk-deficient cells to physiologic concentrations of
Epo and SCF resulted in a block in proliferation and premature terminal
differentiation of these cells, relative to cells derived from wild-type littermate
controls.
While Btk evidently plays an important role in erythroid progenitor signaling and
proliferation in mouse-derived cells, the clinical significance of BTK disruption in
human erythroid progenitors and erythrocytes is not known: to our knowledge,
there have been no reports of erythroid progenitor or erythrocyte abnormalities in
patients with XLA.
1.3.5.4 Platelets
BTK is expressed in platelets (Futatani et al., 2001) and is implicated in platelet
activation. Upon collagen (or collagen-related peptide) binding to the FcRγ-
associated collagen receptor glycoprotein VI (GPVI), FcRγ chain ITAMs are
phosphorylated by the GPVI-bound SFKs LYN and FYN (Watson et al., 2005).
These phosphorylated ITAMs serve as docking sites for SYK, which is recruited
and tyrosine-phosphorylated, resulting in its activation. BTK is activated
downstream of SYK and in turn phosphorylates and activates neighbouring
PLCγ2 (Quek et al., 1998). BTK is also activated downstream of platelet
stimulation by thrombin; integrin αIIb/β3 and PI3K mediate this activation
(Laffargue et al., 1999).
The functional importance of BTK in platelet activation was first reported by Quek
et al. (1998), who found that platelets isolated from XLA patients demonstrated
significantly reduced aggregation, calcium mobilization, dense granule secretion,
and PLCγ2 phosphorylation in response to collagen or CRP, relative to platelets
27
derived from healthy controls. In line with these observations, Rushworth et al.
(2013) noted that ex vivo ibrutinib treatment of platelets from CLL and MCL
patients impaired platelet aggregation in response to collagen or adenosine
diphosphate (ADP) stimulation. Kamel et al. (2015) later confirmed the capacity
of ibrutinib to reduce collagen-mediated platelet aggregation in patients
undergoing ibrutinib treatment, however they observed no effect of ibrutinib on
platelet aggregation in response to ADP stimulation. Kamel et al. (2015) and
Levade et al. (2014) also found an association between disruption of collagen-
mediated platelet aggregation and likelihood of adverse bleeding events in
patients with CLL or MCL.
There have been no clinical reports of platelet dysfunction in patients with XLA,
possibly due to the fact that TEC kinase—which is also expressed in platelets—
has some functional redundancy with BTK in these cells (Atkinson et al., 2003).
However, given that ibrutinib inhibits TEC kinase in addition to BTK (Honigberg et
al., 2010), this drug may contribute to adverse bleeding events experienced by
patients undergoing ibrutinib therapy. Adverse bleeding events have been
reported in ibrutinib clinical trials: in a Phase Ib-II CLL trial carried out by Byrd et
al. (Byrd et al., 2013), 16% of patients experienced Grade 1 or 2 bruising, and
5% of patients experienced bleeding that was Grade 3 or higher. Similarly, in a
Phase II trial for MCL, 17% of patients experienced Grade 1 or 2 bruising and 5%
of patients experienced Grade 3 bleeding (Wang et al., 2013). While CLL itself is
associated with diminished collagen-mediated platelet aggregation, ibrutinib was
shown to transiently worsen collagen-mediated platelet aggregation in these
patients (Lipsky et al., 2015). These findings therefore have implications for CLL
patients undergoing concurrent ibrutinib and antiplatelet or anticoagulant therapy
(Lipsky et al., 2015).
1.3.5.5 Neutrophils
28
Neutrophils are a major component of the innate immune response. In the
presence of invading pathogens, these cells are recruited to the site of infection
by chemokines and kill microbes via oxidative burst or phagocytosis.
BTK appears to be important for normal neutrophil development: GM-CSF- and
TLR- stimulation of granulocyte-monocyte progenitors (GMPs) isolated from a
Btk-deficient murine model of XLA preferentially induced granulopoiesis at the
expense of monocytes or undifferentiated myeloid cells (Fiedler et al., 2011).
However, resultant neutrophils exhibited maturation defects, as evidenced by a
reduced number of granules and expression of granule contents (Fiedler et al.,
2011).
In human peripheral blood neutrophils, TEC family kinases (including BTK) are
activated downstream of G-protein coupled receptor (GPCR) stimulation and
PI3K activation: the chemotactic factor (and GPCR ligand) fMet-Leu-Phe (fMLP)
induced tyrosine phosphorylation and membrane translocation of these kinases,
and GPCR and PI3K inhibition with pertussis toxin and wortmannin, respectively,
disrupted this activation response (Lachance et al., 2002).
Several studies support an important role for BTK in normal neutrophil function.
Mangla et al. (2004) and Fiedler et al. (2011) both noted defective neutrophil
migration into tissues and reduced tissue edema following inflammatory stimuli in
two murine models of XLA. While no significant differences in the phagocytic
activities of neutrophils between Xid and wild-type mice were noted, reduced
generation of reactive oxygen intermediates and nitric oxide following LPS
stimulation were apparent (Mangla et al., 2004). This finding is in contrast to that
of Honda et al. (2012), who demonstrated that stimulation of neutrophils isolated
from human XLA patients induced exaggerated levels of NADPH oxidase-
mediated reactive oxygen species (ROS) production compared to neutrophils
from healthy controls, resulting in higher levels of apoptotic cell death. This
finding was not reproduced, however, by Broides et al. (2014): this group did not
29
observe increased ROS production following fMLP stimulation of XLA patient-
derived neutrophils relative to stimulated neutrophils from healthy volunteers. It is
important to note, however, that both groups used differing methods of neutrophil
stimulation and ROS quantification for their respective studies.
Clinically, a link between BTK mutations and neutropenia has been reported: in a
chart review of 50 XLA patients, Farrar et al. (1996) noted that 26% had
experienced severe episodes of neutropenia. Similarly, an XLA database of 201
patients reported neutropenia as the initial clinical presentation in 11% of XLA
patients (Winkelstein et al., 2006). The cause of neutropenia in XLA patients has
been attributed to abnormally elevated ROS production by neutrophils in
response to pathogens (Farrar et al., 1996; Honda et al., 2012) and defective
neutrophil function as a result of abnormal maturation (Fiedler et al., 2011).
1.3.6 A Role for Targeting BTK Beyond B-Cell Cancers
1.3.6.1 Rheumatoid Arthritis
Rheumatoid arthritis (RA) is an autoimmune disease initiated by B-cell-mediated
production of autoantibodies targeting self-antigens within joints. Autoantibody-
autoantigen interactions within the joint trigger immune complex formation, and
these immune complexes subsequently activate innate immune cells
(predominantly macrophages, but also neutrophils and mast cells) via Fc
receptor (FcR) crosslinking. FcR crosslinking of these cells triggers processes
such as phagocytosis and inflammatory cytokine production, leading to the
cartilage and bone destruction that is characteristic of this disease (Whang &
Chang, 2014).
BTK emerged as a promising therapeutic target in RA because of its role as a
node in two independent signaling pathways implicated in RA pathogenesis: the
30
BCR signaling pathway in B-cells, and the FcR pathway in myeloid cells (Di
Paolo et al., 2011). Blocking BCR signaling with the small molecule BTK inhibitor
CGI1746 prevented arthritis development and decreased autoantibody formation
in a murine collagen induced arthritis (CIA) model, which are B-cell-dependent
(Di Paolo et al., 2011). Similarly, ibrutinib reversed joint inflammation, decreased
infiltration of granulocytes and macrophages (and correspondingly decreased
synovial fluid proinflammatory cytokines), and was protective against joint
destruction in a murine CIA model (Chang et al., 2011; Honigberg et al., 2010).
Meanwhile, blocking FcR (specifically, FcγRIII) signaling with CGI1746
significantly diminished proinflammatory cytokine production by murine
macrophages, and prevented arthritis development in a murine anti-collagen
antibody-induced arthritis (CAIA) model, which is an FcγR-dependent (and thus
myeloid cell-dependent) model of autoantibody-mediated arthritis (Di Paolo et al.,
2011). Ibrutinib likewise prevented inflammation, blocked myeloid cell infiltration,
and was protective against cartilage and bone destruction in a murine CAIA
model (Chang et al., 2011).
Given the impressive preclinical activity of small molecule BTK inhibitors in
murine models of RA, several BTK inhibitor compounds are currently being
investigated in Phase I (GDC-0834, HM-71224) and II (CC-292) clinical trials for
this disease (Whang & Chang, 2014).
1.3.6.2 Multiple Myeloma
Multiple myeloma (MM) is characterized by the clonal proliferation of plasma cells
in the bone marrow, as well as the presence of osteolytic bone lesions. MM
patients exhibit excessive osteoclast (OC) activity, with inadequate opposing
osteoblast (OB) activity and thus significant bone resorption. Interactions
between MM cells and the cells of their microenvironment (bone marrow stromal
31
cells (BMSCs), OBs, and OCs) are critical for MM cell proliferation and survival,
and BTK signaling mediates some of these interactions.
BTK, which is expressed in primary MM cells and in a subset of MM cell lines
(Bam et al., 2013; Tai et al., 2012), appears to be important for MM cell growth,
survival, migration, and adhesion to BMSCs: ibrutinib treatment reduced
proliferation and induced cytotoxicity in the BTK-expressing MM cell line INA6
stimulated with IL-6, or when cocultured with patient-derived BMSCs or OCs (Tai
et al., 2012). Ibrutinib similarly reduced MM burden in SCID-hu mice. BTK in MM
cells was activated in response to the chemokine SDF-1 (Bam et al., 2013; Tai et
al., 2012), and ibrutinib treatment of SDF-1-stimulated MM cells (both cell lines
and primary MM samples) blocked cell migration and adherence to BMSCs (Tai
et al., 2012).
BTK and TEC kinase are strongly expressed in OCs (but not OBs) and are
required for osteoclast differentiation (osteoclastogenesis), as evidenced by the
observation that Tec-/-Btk-/- mice exhibit decreased bone resorption and
consequent osteopetrosis (Shinohara et al., 2008). BTK inhibition with ibrutinib
blocked osteoclastogenesis and reduced bone resorption in healthy donor-
derived precursor OC cells stimulated with M-CSF and RANKL and in SCID-hu
mice (Tai et al., 2012).
Ibrutinib is currently under investigation for the treatment of relapsed/refractory
multiple myeloma, both as a single agent and as a combination candidate with
dexamethasone, or carfilzomib and dexamethasone, in two Phase II clinical trials
(NCT01478581 and NCT01962792).
1.3.6.3 Acute Myeloid Leukemia
32
Acute myeloid leukemia (AML) is characterized by excessive proliferation of
aberrantly differentiated hematopoietic cells of the myeloid lineage. BTK
expression in AML cell lines and primary blasts was first reported in 1993 by de
Weers et al. and has been extensively corroborated by other groups in the years
since. While the presence of non-mutated BTK expression (Ritis et al., 1998) in
AML cells is well established, the role of this kinase in this disease has only
recently been investigated. Rushworth et al. (2014) were the first to identify the
presence of constitutively active BTK (by detection of Tyr223 phosphorylation) in
AML cell lines and primary blasts, noting a positive correlation between the pBTK
to total BTK ratio and ibrutinib sensitivity. They also noted that BTK knockdown
impaired colony formation by AML progenitor cells in a subset of patients. In a
separate study, this group demonstrated the impact of BTK inhibition on AML cell
migration, demonstrating that at clinically relevant concentrations, ibrutinib can
block SDF1-induced CXCR4-mediated migration of AML cell lines and primary
patient blasts (Zaitseva et al., 2014). BTK knockdown yielded a similar, though
less dramatic, inhibitory effect on migration, suggesting that BTK is involved in
signal transduction from the CXCR4 receptor (Zaitseva et al., 2014).
Another important study investigated the impact of BTK signaling in AML cell
survival and proliferation in both FLT3-ITD positive and FLT3-ITD negative AML
cell lines. Oellerich et al. (2015) demonstrated that in AML cells harboring the
FLT3-ITD mutation, BTK couples FLT3-ITD signaling to STAT5 and MYC
activation. Interestingly, in AML cells expressing the FLT3 wild type receptor,
BTK does not interact with FLT3, but instead couples TLR9 signaling to STAT5
and NFkB activation.
1.3.6.4 Prostate Cancer
BTK has been reported as a potential therapeutic target in prostate cancer. Guo
et al. (2014) were the first to report overexpression of BTK in this disease and
33
observed that the degree of BTK overexpression correlated with tumor grade, a
finding that was more recently corroborated by Kokabee et al. (2015). BTK
silencing in the prostate cancer cell line PC3 was found to reduce cell
proliferation, an effect which was not observed following BTK knockdown in a
normal prostate cell line (RWPE1) (Guo et al., 2014). Moreover, treatment of
prostate cancer cell lines LNCaP and DU145 with ibrutinib and other small
molecule BTK inhibitors was found to reduce cell growth and viability, albeit only
modestly at clinically achievable concentrations (Kokabee et al., 2015).
Combined knockdown of BTK and the TEC family member BMX (which has
known oncogenic activity in prostate cancer) in PC3 cells additively reduced cell
proliferation. A small molecule dual BTK/BMX inhibitor, CTN06, demonstrated
potent autophagic and apoptotic activity against several prostate cancer cell lines
and in a PC3 xenograft model (Guo et al., 2014).
Interestingly, Kokabee et al. (2015) found that two different BTK isoforms are
commonly expressed in prostate cancer tissue samples and cell lines: BTK-A
(the isoform expressed in B-cell cancers) and BTK-C, with the latter isoform
having higher expression in cell lines of this tumor type.
34
1.4 EGFR inhibitors in AML: Anti-Leukemic Mechanisms of Action and Preclinical and Clinical Activity
There is an unmet need for novel approaches to acute myeloid leukemia (AML)
treatment, where five-year patient survival is 25.9% (NIH, 2012) despite
aggressive chemotherapy treatment regimens. The need for new therapies is
especially great for those older than the age of 65 for two reasons: first-line
chemotherapy is less tolerable for these individuals (Döhner et al., 2015), and
AML prevalence rises sharply in this age group (NIH, 2012).
Small molecule epidermal growth factor receptor (EGFR) inhibitors are clinically
approved for use in non small-cell lung cancer (NSCLC) and pancreatic
adenocarcinoma. They are well tolerated in elderly patients, prolonging patient
survival often without compromising quality of life. Within the last ten years,
several groups have described the activity of EGFR inhibitors—specifically
erlotinib and gefitinib—against AML, despite the absence of EGFR expression in
these tumor cells. This review will summarize the mechanisms of action that
likely account for these desirable off-target effects, and will summarize the
current preclinical and clinical evidence for their indicated use in the treatment of
AML.
1.4.1 Development of Small Molecule EGFR Tyrosine Kinase Inhibitors
Small-molecule inhibitors of EGFR were developed as a strategy to block
signaling from this receptor in solid tumors exhibiting increased activation of this
pathway. Activation of EGFR by ligand-receptor binding and subsequent receptor
homo- or heterodimerization promotes signal transduction through the
35
Ras/Raf/MAPK and PI3K/Akt/mTOR pathways, which in turn stimulate cell
proliferation and survival, respectively (Sharma et al., 2007; Siegelin & Borczuk,
2014). Given the pro-survival and proliferative downstream effects of this
pathway, it is not surprising that aberrant EGFR signaling—due to mutations
conferring constitutive receptor activation, receptor overexpression, ligand
upregulation, or reduced receptor turnover (Ciardiello & Tortora, 2001)—can
strongly favor tumorigenesis.
The EGFR tyrosine kinase inhibitors (EGFR-TKIs) erlotinib and gefitinib compete
with ATP for binding to the cytoplasmic kinase domain of this kinase, resulting in
the inhibition of C-terminal autophosphorylation (Moyer et al., 1997; Wakeling et
al., 2002). These EGFR-TKIs therefore prevent the formation of docking sites for
downstream effectors of EGFR signaling.
Gefitinib was the first oral EGFR-TKI to receive FDA approval. It is currently
approved for first-line use in NSCLC patients with EGFR exon 19 deletions or
exon 21 L858R mutations (FDA, 2015b). Erlotinib is also currently indicated for
first-line use in EGFR-mutant (exon 19 deletions or exon 21 L858R) NSCLC and
in combination with gemcitabine in the treatment of locally advanced, metastatic,
or unresectable pancreatic cancer. Erlotinib is also approved for use as
maintenance therapy in locally advanced or metastatic NSCLC following first-line
treatment with platinum-based chemotherapy, and in NSCLC patients who have
failed at least one chemotherapy regimen (FDA, 2013).
1.4.2 Expression of EGFR in AML Cells
To investigate EGFR inhibitor targets in AML, several groups have examined the
expression of this receptor in AML cell lines and primary blasts. Sun et al. (2012)
noted EGFR mRNA expression by RT-PCR in 48 of 143 AML samples of various
36
French American British subtypes, however the majority of studies are in conflict
with these findings, having reported no EGFR expression in this tumor type.
Walz et al. (1993) were the first to report the absence of EGFR expression in the
HL60 cell line. Stegmaier et al. (2005) and Boehrer et al. (2008a) confirmed the
lack of EGFR expression in HL60, in addition to determining that EGFR
expression was absent in Kasumi-1, KG1, and P39 cells. In line with these
observations, EGFR was undetectable in primary AML blasts from eight patients
(DeAngelo et al., 2014). Finally, in agreement with the above observations,
querying EGFR expression using the Cancer Cell Line Encyclopedia revealed
lower EGFR mRNA levels in AML cell lines relative to cell lines of other tumor
types, except for T-ALL (Barretina et al., 2012).
1.4.3 Preclinical EGFR-TKI activity against AML
1.4.3.1 Differentiation
The differentiating capacities of erlotinib and gefitinib have been examined in
several AML cell lines as well as primary AML blasts. Gefitinib treatment was
found to induce functional, morphologic, and gene expression changes
(Stegmaier et al., 2005) in the AML cell lines HL60, Kasumi-1, and U937, which
were consistent with their maturation (Stegmaier et al., 2005). Erlotinib induced
morphologic maturation of KG1, HL60, and P39 cell lines, with P39 and HL60
expressing the myeloid differentiation marker CD11b in response to this drug
(Boehrer et al., 2008a; Boehrer et al., 2008b). Erlotinib also induced CD11b
expression in CD34+ primary AML cells (Boehrer et al., 2008a).
Lainey et al. (2013b) found that as single agents, gefitinib and erlotinib had
negligible effects on HL60 and primary AML blast differentiation, however they
noted that both drugs potentiated the differentiation capacities of all-trans retinoic
37
acid (ATRA) and vitamin D3 in these cell lines (based on changes in CD11b and
CD14 marker levels), and that erlotinib potentiated ATRA and vitamin D3-
mediated differentiation in a subset of primary AML samples.
1.4.3.2 Cell Cycle Arrest and Cell Death
Erlotinib has been shown to induce G1 arrest (Boehrer et al., 2008a) and
subsequent apoptosis in KG1 cells (Boehrer et al., 2008a; Boehrer et al., 2008b).
Neither erlotinib, nor gefitinib induced apoptosis in HL60, P39, MV4-11, MOLM-
13, or U937 cells (Boehrer et al., 2008a; Boehrer et al., 2008b). Erlotinib induced
modest cell cycle arrest in HL60 and P39 cells (Boehrer et al., 2008a).
Erlotinib treatment of SCID mice prevented tumor formation following
intraperitoneal KG1 cell inoculation and significantly increased tumor-free
survival (Boehrer et al., 2008a). Primary CD34+ AML cells were also more
sensitive than normal CD34+ hematopoietic cells to apoptosis induction by
erlotinib and gefitinib (Boehrer et al., 2008a; Boehrer et al., 2008b). Likewise,
primary AML samples were preferentially sensitive to gefitinib treatment: gefitinib
IC50 in six out of eight primary AML samples was less than 5 µM, while the
average IC50 in five peripheral blood mononuclear control samples was greater
than 9 µM using the Cell Titer Glo assay (Stegmaier et al., 2005).
1.4.4 Proposed Anti-Leukemic Targets of EGFR-TKIs
1.4.4.1 JAK2 Inhibition JAK/STAT signaling is increased in AML blasts (Gouilleux-Gruart et al., 1996;
Ikezoe et al., 2011) and AML progenitor (CD34+) cells relative to normal
hematopoietic stem cells (Cook et al., 2014). JAK2 knockdown by siRNA and
38
treatment with JAK1/2 inhibitors blocked phosphorylation of STAT3/5 and
reduced colony formation and increased Annexin V staining in primary AML
CD34+ cells, implicating JAK2 as a promising therapeutic target in AML (Cook et
al., 2014).
Boehrer et al. (2008a) reported decreased phosphorylation of JAK2 at
Tyr1007/1008 and its substrate, STAT5 (Tyr694) in response to erlotinib
treatment of KG1 cells. This group also demonstrated the functional importance
of erlotinib-mediated inhibition of JAK2-STAT5 signaling using siRNA knockdown
of JAK2. KG1 cells transfected with JAK2 siRNA recapitulated the reduced
STAT5 phosphorylation and increased apoptosis induction seen with erlotinib
treatment.
1.4.4.2 SRC Family Kinase Inhibition
SRC family kinases (SFKs)—particularly the family member LYN—are reportedly
overexpressed and constitutively active in primary AML blasts and the primitive
CD34+CD38-CD123+ fraction, as evidenced by the presence of SFK tyrosine
phosphorylation at Y416 in these cells (Dos Santos et al., 2008). LYN was also
found to act as an upstream effector of mTOR signaling by this group. Inhibition
of SFKs has been shown to induce G1 arrest and inhibit colony formation in
primary AML cells, providing a rationale for targeting these kinases in the
treatment of AML (Dos Santos et al., 2008).
Boehrer et al. (2011) found that erlotinib decreased SFK phosphorylation in KG1
cells and AML blasts from one patient, and induced autophagy by inhibition of
mTORC1, as demonstrated by reduction of p70S6K phosphorylation. Weber et al.
(2012) validated the finding of decreased SFK phosphorylation following erlotinib
(and gefitinib) treatment of KG1 cells using quantitative phospho-mass
39
spectrometry. This group also demonstrated—using chemical proteomics and an
in vitro kinase assay—that these compounds directly bind SFKs.
1.4.4.3 SYK Inhibition Spleen tyrosine kinase (SYK) has been proposed as a therapeutic target in AML.
It is constitutively phosphorylated at Y525/526, a marker associated with its
activation, in multiple AML cell lines and primary blasts (Hahn et al., 2009), and is
an upstream effector of mTOR signaling in this disease (Carnevale et al., 2013).
Genetic knockdown of SYK has been shown to induce differentiation and reduce
proliferation of several AML cell lines, and the small molecule SYK inhibitor R406
reduced tumor burden in KG1- and primary AML-engrafted mice (Hahn et al.,
2009). SYK was identified as a target of gefitinib in AML in a phospho-mass
spectrometry study carried out by Hahn et al. (2009), in which SYK
phosphorylation (and the phosphorylation of SYK substrates) in HL60 cells was
reduced in response to gefitinib treatment. SYK was further confirmed as a target
of gefitinib (and erlotinib) in a study carried out by Weber et al. (2012). In this
phosphoproteomics study, erlotinib and gefitinib reduced SYK-Tyr352
phosphorylation in KG1 cells. Through chemical proteomics and in vitro kinase
activity assays, SYK was confirmed as an indirect target of both kinase inhibitors.
1.4.4.4 Bruton’s Tyrosine Kinase Inhibition
Bruton’s tyrosine kinase (BTK) is constitutively active in AML cell lines and
primary blasts (Rushworth et al., 2014). It has been proposed as a therapeutic
target in AML based on its reported capacity to promote AML cell proliferation
and survival by mediating signal transduction from the receptor tyrosine kinase
FLT3 in FLT3-mutant (FLT3-ITD) AML cells, and from TLR9 in FLT3-wild type
cells (Oellerich et al., 2015). BTK has also been shown to promote AML cell
40
migration (Zaitseva et al., 2014). In a study carried out by Weber et al. (2012),
erlotinib and gefitinib were found to reduce phosphorylation of BTK at Tyr551
(phosphorylation at this site is required for BTK activation). This work also
demonstrated that these kinase inhibitors target BTK directly.
1.4.4.5 Inhibition of ATP-Binding Cassette Transporter Efflux Activity
Expression and enhanced activity of ATP-binding cassette (ABC) transporter
family members such as P-glycoprotein (P-gp) in AML cells is a predictor of poor
patient prognosis (Campos et al., 1992; Pirker et al., 1991; Zöchbauer et al.,
1994). ABC transporters mediate the extrusion of cytotoxins, such as
chemotherapy drugs, from cells and their activity is thus associated with therapy
resistance in AML. Inhibiting the activity of one or more of these transporters has
been proposed as a strategy for the re-sensitization of AML cells to
chemotherapy agents. The addition of the P-gp inhibitor cyclosporine to an AML
chemotherapy regimen was found to prolong overall and relapse-free survival in
unfavourable-risk AML patients (List et al., 2001). Moreover, P-gp inhibitor
quinine addition to AML induction therapy was found to increase complete
remission rates in treatment-naïve AML patients with increased rhodamine-123
efflux (Solary et al., 2003). However, subsequent clinical trials investigating the
addition of the cyclosporine analog valspodar to AML regimens have not
demonstrated benefit to older or younger patient populations (Kolitz et al., 2010;
van der Holt et al., 2005)
Multiple small-molecule EGFR inhibitors are known to inhibit ABC transporters in
many tumor types (Dai et al., 2008; Kuang et al., 2010; Shi et al., 2007),
including AML. Erlotinib was found to inhibit the activities of P-glycoprotein (P-
gp), breast cancer resistance protein (BCRP), and multidrug resistance-
associated protein (MRP) in KG1 cells, as evidenced by increased retentions of
the P-gp substrate 3,3’-diethyloxacarbocyanine (DiOC2(3)), the MRP substrate
41
calcein, and the BCRP substrate Hoechst33342 in response to gefitinib and/or
erlotinib treatment (Lainey et al., 2012). Simultaneous inhibition of these
transporters with erlotinib and gefitinib led to the accumulation of the
chemotherapy agents doxorubicin, etoposide, and mitoxantrone, effectively
sensitizing KG1 cells to chemotherapy-induced cell death. Erlotinib and gefitinib
have also been found to potentiate the accumulation of the hypomethylating
agent azacytidine in AML cells (presumably a consequence of ABC-transporter
efflux inhibition). Combining these EGFR inhibitors with azacytidine was
synergistically cytotoxic (Lainey et al., 2013a).
1.4.6 Clinical EGFR-TKI Activity Against AML
Clinical evidence for EGFR inhibitor activity against AML was first described in
two case reports. In the former, Chan and Pilichowska (2007) reported complete
AML remission (less than 3% bone marrow blasts with normal hematopoietic
count recovery) in a 68 year-old male diagnosed with concomitant NSCLC and
AML and treated with single-agent erlotinib for three months. The patient’s
remission was maintained for six months following erlotinib discontinuation. The
second case report described a 64 year-old male, also diagnosed with
concomitant NSCLC and AML, who received daily erlotinib treatment for three
months (Pitini et al., 2008). The patient’s AML remission was maintained for at
least seven months.
To date, three clinical trials have investigated the activity of small molecule
EGFR inhibitors as single agents in AML, with very modest results. A Phase II
trial examining single-agent gefitinib treatment in 18 patients with intermediate-to-
poor-risk cytogenetics noted zero objective responses (DeAngelo et al., 2014). A
Phase I/II trial assessing response to erlotinib in 30 patients (12 with AML that
progressed from myelodysplastic syndrome (MDS) and 18 with high-risk MDS
(RAEB-2)) who had previously received azacytidine treatment reported
42
responses in six RAEB-2 patients (median duration of five months), of whom two
achieved complete remission (Thepot et al., 2014). Finally, a Phase I trial carried
out by Sayar et al. (2015) evaluated erlotinib use in 11 patients with AML,
including nine patients with treatment-naïve disease. Peripheral blast counts
were reduced greater than 50% in two patients, however both patients, as well as
the remaining nine patients, experienced disease progression.
1.4.7 Summary
Several groups have demonstrated convincing evidence of preclinical EGFR-
independent erlotinib and gefitinib activity in AML. Collectively, these groups
have identified the cellular targets of these EGFR-TKIs that may plausibly
account for their effects on differentiation, proliferation, and cell death: JAK2,
SFKs, SYK, BTK, mTOR and ABC transporters are all inhibited by erlotinib and
gefitinib, and have all been proposed as potential therapeutic targets in AML. To
date, the impressive preclinical activity of these drugs has not been observed in
the clinical trial setting, where responses to EGFR-TKIs have been modest at
best: erlotinib only mildly reduced bone marrow blasts in a small subset of AML
patients (Sayar et al., 2015), and produced complete remissions or hematologic
improvement in a subset of high-risk MDS patients (Thepot et al., 2014).
While it is difficult to pinpoint the exact reason for the failure of these TKIs in AML
clinical trials, it is important to note that none of these trials assessed the
pharmacodynamic effects of these TKIs on their reported targets in AML,
meaning that it is not known whether these drugs inhibited any of the targets that
may account for the anti-AML activity of erlotinib or gefitinib.
Finally, it is also important to note that these trials did not assess the clinical
activity of erlotinib or gefitinib in combination with other chemotherapy agents
approved for use in AML. Given that erlotinib and gefitinib exhibit profound
43
preclinical synergistic cytotoxicity with chemotherapy agents, further preclinical
and clinical investigation of these EGFR-TKIs in combination with AML
chemotherapies may be warranted.
44
1.5 Ethacridine Lactate
1.5.1 Ethacridine Lactate Indications
Ethacridine lactate (2-ethoxy-6,9-diaminoacridine monolactate, see Figure 1-2
for molecular structure) is an antiseptic ointment with modest activity in venous
leg ulcers (O’Meara et al., 2014). Ethacridine is also used as an extra-amniotic
second-trimester abortifacient in China and Cuba (Boza et al., 2008; Hou et al.,
2010). When administered in conjunction with tannin albuminate, ethacridine
lactate has moderate effectiveness as chemoprophylaxis for travellers’ diarrhea
(Ericsson, 2005).
Figure 1-2: Structure of Ethacridine lactate
Source: www.sigmaaldrich.com
45
1.5.2 Ethacridine Lactate Mechanisms of Action
1.5.2.1 Poly(ADP-ribose) Glycohydrolase Inhibition
Poly(ADP-ribose)ylation, also known as PARylation, is a post-translational
modification of nuclear and cytoplasmic acceptor proteins that contributes to
cellular processes such as DNA damage repair, transcriptional regulation, and
cell death (reviewed by Feng and Koh (2013)). In the presence of cellular
stressors such as DNA damage, activated poly(ADP-ribose) polymerase 1
(PARP1) catalyzes the transfer of PAR moieties to acceptor proteins such as
PARP1 itself, histones, DNA repair proteins, transcription factors, and chromatin
modulators. The activity of PARP1 is opposed by poly(ADP-ribose)
glycohydrolase (PARG), which hydrolyzes PAR polymers (Figure 1-3). Closely
coordinated activity between PARP1 and PARG is critical, as unopposed PARP1
activation (and thus PAR accumulation) can lead to necrotic (Feng & Koh, 2013)
or apoptosis-inducing factor-mediated cell death (Yu et al., 2006; Yu et al., 2002;
Zhou et al., 2011).
Ethacridine lactate is a PARG inhibitor (Bernardi et al., 1997; Boulikas, 1990;
Tavassoli et al., 1985). Ethacridine blocks PARG activity by binding directly to
PAR polymers, preventing PARG binding and thus PARG-mediated PAR
catabolism (Tavassoli et al., 1985).
1.5.2.1.1 Other Chemical PARG Inhibitors
Tannins, which are naturally occurring polyphenolic compounds derived from
plants, are also reported PARG inhibitors (Aoki et al., 1993; Tanuma et al., 1989;
Tsai et al., 1992). Gallotannin is the best studied of the tannins, and consists of
trigalloylglucose, tetragalloylglucose, and pentagalloylglucose compounds, which
are cell membrane permeable and inhibit PARG activity at an in vitro IC50 of
46
approximately 18-33 µM (Aoki et al., 1993; Tsai et al., 1992). Tsai et al. (1992)
and Aoki et al. (1993) also noted a positive relationship between the number of
galloyl groups and the degree of PARG inhibition in vitro.
Adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) is a more
potent inhibitor of PARG, relative to tannins. It is an ADP-ribose analogue that
inhibits PARG activity through partial noncompetitive inhibition, with an IC50 of
120nM (Slama et al., 1995a; Slama et al., 1995b). The use of this compound is
however limited to enzymatic assays, as this compound is not cell membrane
permeable (Feng & Koh, 2013).
1.5.2.2 Non-Genotoxic Activation of p53 Ethacridine lactate is a reported inducer of p53 activation via the ribosomal stress
pathway. In a recent study by Morgado-Palacin et al. (2014), ethacridine at a
concentration of 5 µM increased p53 expression and induced nucleolar
disruption—denoted by an increase in the ratio of rounded (disrupted) to irregular
(undisrupted) nucleoli—but failed to induce DNA damage, as indicated by the
absence of γH2A.X foci. The likely cause of nucleolar disruption-mediated p53
induction by ethacridine was inhibition of rRNA transcription by defective RNA
polymerase I: in this study, ethacridine treatment led to degradation of the
essential RNA polymerase I component, RP194. This group further
demonstrated that non-genotoxic p53 activation by acridine derivatives was
sufficient to induce cell cycle arrest and apoptosis in TP53-wild type, but not
TP53 knock-out HCT116 (human colon carcinoma) cells.
47
Figure 1-3: Mechanism of PARG activity PARP uses NAD+ as a substrate for PAR synthesis. PAR is assembled as polymers onto proteins. PARG hydrolyzes PAR polymers, releasing ADP-ribose. Reproduced from Feng and Koh (2013), license #3817021464698.
48
Chapter 2: Project Rationale and Aims
49
2.1 Thesis Aims
Given the limited effectiveness of currently approved therapy, novel approaches
to the treatment of AML are urgently needed for this disease. Identifying clinically
approved drugs that enhance the anti-AML activity of agents that are currently
approved for use in – or under investigation for – the treatment of AML would
provide a new therapeutic strategy that could be rapidly advanced to the clinic.
The central objective of this thesis was to identify synergistically cytotoxic
combinations of approved drugs that preferentially kill AML cells, and to uncover
the mechanisms by which these drug pairs synergize.
2.1.1 Aim I: Identify compounds that synergize with ibrutinib in AML
Several groups have described a potential role for the Bruton’s tyrosine kinase
inhibitor ibrutinib in the treatment of AML. With the goal of enhancing ibrutinib’s
anti-AML activity, we used a combination high-throughput screening approach to
identify ibrutinib-sensitizing agents. We subsequently investigated the synergistic
mechanism between ibrutinib and the top synergistic screen hit, hypothesizing
that this synergistic activity was dependent upon ibrutinib-mediated BTK
inhibition.
2.1.2 Aim II: Evaluate the mechanism of synergy between ibrutinib and daunorubicin in AML
Ibrutinib was previously reported to synergize with daunorubicin in AML cells. We
hypothesized that this synergy was dependent upon ibrutinib-mediated BTK
inhibition. We evaluated the role of BTK in ibrutinib-daunorubicin synergy by
treating BTK-knockdown AML cell lines with daunorubicin. We further evaluated
50
this synergistic mechanism by measuring combination-induced reactive oxygen
species production and ibrutinib-mediated intracellular daunorubicin
accumulation.
2.1.3 Aim III: Identify compounds that synergize with erlotinib in AML
The epidermal growth factor receptor (EGFR) inhibitor erlotinib has been
reported to exert modest EGFR-independent anti-AML activity in clinical trials.
We sought to identify erlotinib combination candidates by carrying out a
combination high-throughput chemical screen against this drug in erlotinib-
insensitive AML cell lines. We subsequently delineated the synergistic
mechanism of action between erlotinib and the top synergistically cytotoxic hit
using mass spectrometry.
51
Chapter 3: Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism
This chapter has been published:
Rotin LE, Gronda M, MacLean N, Hurren R, Wang X, Lin F, Wrana J, Datti A, Barber DL, Minden MD, Slassi M, Schimmer AD (2016). Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism. Oncotarget, 7(3): 2765-2779. doi: 10.18632/oncotarget.6409.
Open-Access License (Creative Commons Attribution License); no permissions required.
52
3.1 Abstract
Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor
ibrutinib has significantly improved patient outcomes in several B-cell
malignancies, with minimal toxicity. Given the reported expression and
constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has
been recent interest in investigating the anti-AML activity of ibrutinib. We noted
that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and
primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs.
Using a high-throughput combination chemical screen, we identified that the
poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized
with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of
ibrutinib and ethacridine induced a synergistic increase in reactive oxygen
species that was functionally important to explain the observed cell death.
Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent
of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not
sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings
indicate that ibrutinib may have a BTK-independent role in AML and that PARG
inhibitors may have utility as part of a combination therapy for this disease.
53
3.2 Introduction
Ibrutinib is a small-molecule Bruton’s tyrosine kinase (BTK) inhibitor approved for
clinical use in several B-cell malignancies, including chronic lymphocytic
leukemia (CLL). Inhibition of BTK induces cell death by blocking constitutive B-
cell receptor (BCR) signaling and impairing tumor-microenvironment interactions
in CLL cells (Herman et al., 2011; Ponader et al., 2012). BTK is expressed in
almost all B-hematopoietic malignancies, but is also expressed in myeloid cells
and myeloid malignancies where it can be activated through mechanisms distinct
from BCR signaling. Since BTK is expressed in myeloid cells, we evaluated
ibrutinib in acute myeloid leukemia (AML).
AML is a hematologic malignancy characterized by the overproduction of poorly
differentiated myeloid-lineage cells (Löwenberg et al., 1999). Previously, other
groups reported increased expression and constitutive activation of BTK in AML
cell lines and primary AML patient samples (Barretina et al., 2012; de Weers et
al., 1993; Oellerich et al., 2015; Rushworth et al., 2014; Wu et al., 2015). BTK
mediates signal transduction from the FLT3-ITD, TLR9 and CXCR4 receptors in
AML cell lines, thereby promoting leukemic cell survival, growth, and migration
(Oellerich et al., 2015; Zaitseva et al., 2014). We further characterized the anti-
AML activity of ibrutinib and identified drugs that sensitize AML cells to ibrutinib.
Through exploration of the synergistic activity of ibrutinib with other drugs, we
uncovered a BTK-independent role for ibrutinib with potential clinical utility in
AML.
54
3.3 Methods
3.3.1 Materials
BTK and GAPDH antibodies were purchased from Cell Signaling Technology
(Danvers, MA), BTK pTyr-223 antibody was obtained from Abcam (Cambridge,
MA), and β-actin antibody was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). HRP-conjugated goat anti-rabbit and goat anti-mouse secondary
antibodies were acquired from GE Healthcare (Buckinghamshire, UK). Kinase
inhibitors ibrutinib, CC-292, ONO–4059, PIM1/2, and STO-609 were provided by
the Ontario Institute for Cancer Research (Toronto, ON, Canada). Ibrutinib was
also obtained from Selleckchem (Houston, TX), as was olaparib. Z-VAD-FMK
was purchased from Enzo Life Sciences (Farmingdale, NY). Ethacridine lactate,
gallotannin, sulforhodamine-B, hydrogen peroxide, puromycin, and shRNA
plasmid-containing bacterial glycerol stocks were purchased from Sigma-Aldrich
(St. Louis, MO). The library of internationally prescribed drugs was purchased
from MicroSource Discovery Systems, Inc. (Gaylordsville, CT). Alamar Blue was
purchased from Life Technologies (Carlsbad, CA) and carboxy-H2DCFDA-FITC
and MitoSOX Red were obtained from Molecular Probes/Life Technologies
(Eugene, OR).
3.3.2 Cell Culture
TEX cells (Warner et al., 2005) were provided by Dr. John Dick (Ontario Cancer
Institute, Toronto, Canada) and grown in Iscove’s Modified Dulbecco’s Medium
(IMDM) supplemented with 15% fetal bovine serum (FBS) (Seradigm/VWR,
Radford, PA), 100 µg/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine
(Life Technologies, Carlsbad, CA), 20 ng/ml SCF (Miltenyi Biotec, San Diego,
CA), and 2 ng/ml IL-3 (R&D Systems, Minneapolis, MN). OCI-AML2, NB4, KG1a,
Daudi, Thp1, and U937 cells were provided by Dr. Mark Minden (Ontario Cancer
55
Institute, Toronto, Canada). K562 and HL60 cells were provided by Dr. Suzanne
Kamel-Reid (Ontario Cancer Institute, Toronto, Canada) and Jurkat D1.1 cells
were provided by Dr. Pamela Ohashi (Ontario Cancer Institute, Toronto,
Canada). OCI-AML2, K562, Thp1, and NB4 cells were grown in IMDM
supplemented with 10% FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin.
Daudi, Jurkat D1.1, KG1a, U937, and HL60 cells were grown in RPMI 1640
supplemented with 10% FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin.
All cell lines were maintained at 37°C and 5% CO2.
3.3.3 Primary cells
Bulk AML cells from AML patients and peripheral blood stem cells from healthy
G-CSF-treated stem cell donors were isolated by Ficoll density centrifugation and
apheresis, respectively. Isolated cells were maintained in IMDM supplemented
with 10% FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin, at 37°C and 5%
CO2. All samples were obtained from consenting patients. The collection and use
of human tissue for this study were approved by the University Health Network
(Toronto, Canada) institutional review board.
3.3.4 In vivo Combination Treatment
Animal studies were carried out with the approval of the Princess Margaret
Cancer Centre ethics review board, and in accordance with Canadian Council on
Animal Care regulations. SCID mice were subcutaneously injected with 1 × 106
OCI-AML2 cells. Once tumors were palpable (8 days following injection), mice
were treated with ibrutinib (300 mg/kg), ethacridine (20 mg/kg), both in
combination (300 mg/kg ibrutinib + 20 mg/kg ethacridine), alongside vehicle
control once per day, 5 days/week, for a total of 9 treatments. Mice were
subsequently sacrificed and tumor volumes were measured.
56
3.3.5 Immunoblotting
Cells were washed twice with 1xPBS and lysed in 1xLaemmli or
radioimmunoprecipitation assay (RIPA) buffer. Following quantification with the
DC Assay (1xLaemmli) or the Bradford assay (RIPA), protein lysates were
resolved by SDS-PAGE and transferred to a PVDF membrane. Membranes were
blocked for 1 hour at room temperature in 5% milk-TBST. Blotting with primary
antibody was carried out in 5% milk-TBST or 5% BSA-TBST overnight at 4°C.
Membranes were then incubated with HRP-conjugated secondary antibody (GE
Healthcare, Buckinghamshire, UK) for one hour at room temperature. Proteins
were detected by HRP chemiluminescence.
3.3.6 Cell Growth and Viability Assays
Cells were treated with drug(s) for 72 hours in a 96-well flat-bottomed, clear
microplate. Cell growth and viability was determined by the Alamar Blue assay as
per the manufacturers instructions or the sulforhodamine-B (SRB) assay as
previously described (LaPointe et al., 2005). Cell viability and apoptosis was
measured by staining cells with Annexin V-FITC (BioVision, Milpitas, CA) and
propidium iodide-PE (Sigma-Aldrich, St. Louis, MO) as per manufacturer’s
instructions. All flow cytometry experiments were carried out using Canto II 96w
or Fortessa LSR X20 cytometers (BD Biosciences, San Jose, CA). Flow
cytometry data were analyzed with FlowJo version 7.6.5 (TreeStar, Ashland,
OR).
57
3.3.7 Combination High-Throughput Screen
TEX and OCI-AML2 cells were treated with ibrutinib at its respective IC10 and
IC25 values, alongside vehicle (DMSO) controls. Ibrutinib- and vehicle-treated
cells were also treated with a library of known drugs at concentrations of 0.133
µM, 1.6 µM, 3.3 µM, 6.7 µM, and/or 13.3 µM. Combination-treated TEX and OCI-
AML2 cells were incubated for 72 h at 37°C and 5% CO2. The read-out for this
assay was percent growth and viability, measured with the sulforhodamine-B
(SRB) assay. These data were then used to calculate synergy according to
Excess-over-Bliss additivism criteria.
3.3.8 Excess-over-Bliss Additivism for Calculating Synergy
Excess-over-Bliss additivism (EOBA) (Borisy et al., 2003) provides an estimate of
resultant cytotoxicity when two drugs are combined. According to this model, any
cytotoxicity unaccounted for by the added effects of both drugs is due to synergy
between the two compounds. The formula for excess-over-Bliss is as follows:
EOBA = C − (A + B − (A × B))
where C is equal to the fractional inhibition of both drugs simultaneously, A is
equal to the fractional inhibition of drug A, and B is equal to the fractional
inhibition of drug B. Fractional inhibition is equal to 1.0 minus the viability
(expressed as a value from 0.0–1.0). Positive EOBA values reflect a synergistic
combination; the more positive the difference, the greater the synergy. Negative
EOBA values reflect an antagonistic drug combination, while near-zero EOBA
scores are indicative of an additive drug combination.
58
3.3.9 Intracellular and Mitochondrial Reactive Oxygen Species Measurement
Intracellular reactive oxygen species (ROS) in TEX and OCI-AML2 cells was
measured by carboxy-H2DCFDA staining on flow cytometry. Cells were stained
with 10 µM carboxy-H2DCFDA (dissolved in 100% ethanol) and incubated for 30
minutes at 37°C and 5% CO2. Dead cells were excluded by propidium iodide (PI)
staining. Fold change in intracellular reactive oxygen species production was
calculated by dividing the geometric mean of H2DCFDA+, PI−-staining treated
cells by the geometric mean of H2DCFDA+, PI−-staining untreated (vehicle-
treated) cells. Mitochondrial ROS was evaluated by the same procedure, using 5
µM MitoSOX (dissolved in DMSO) and Annexin V staining for dead cell
discrimination.
3.3.10 shRNA Knockdown Experiments
Stable knockdown of BTK in TEX and OCI-AML2 was achieved using lentiviral
transduction of short hairpin RNAs (shRNA) delivered by the PLKO.1 vector,
which contains a puromycin resistance gene. A 72-hour puromycin selection (2
µg/mL in TEX, and 1 µg/mL in OCI-AML2) was used to select for transduced
cells 24 hours after lentiviral infection. Following completion of puromycin
selection, knockdown was confirmed by immunoblot. The following shRNA
sequences directed against BTK (Accession No. NM_000061) were used:
shRNA-BTK_974: 5′-
GAAGCAGAAGACTCCATAGAACTCGAGTTCTATGGAGTCTTCTGCTTC-3′,
and shRNA-BTK_1066: 5′-
AGGAGGTTTCATTGTCAGAGACTCGAGTCTCTGACAATGAAACCTCCT-3′.
59
3.3.11 PARG Activity Assay
The HT Universal Colorimetric PARG Assay Kit (Trevigen, Gaithersburg, MD)
was used to measure the PARG inhibitor activity of ethacridine. The assay was
carried out as per manufacturer instructions.
3.3.12 Statistical Analysis
All graphed viability data are expressed as mean ± SD. Statistical significance
was determined by the unpaired Student’s t test with Holm-Sidak correction for
multiple comparisons or a one-way ANOVA with Tukey’s post-hoc test for
multiple comparisons. Statistical tests were performed using GraphPad Prism
6.03 software (La Jolla, CA).
60
3.4 Results
3.4.1 BTK is overexpressed and constitutively active in AML cells
To determine the relevance of BTK as a therapeutic target in AML, we examined
the protein and mRNA expression of BTK in a panel of AML cell lines. Analysis of
the Cancer Cell Line Encyclopedia (Barretina et al., 2012) demonstrated that
AML cells expressed levels of BTK mRNA similar to B-cell malignancies (Figure 3-1). Likewise, a subgroup of primary AML patient samples had increased BTK
expression (Figure 3-2A). Next, we evaluated the expression of BTK by
immunoblotting in a series of AML cell lines. OCI-AML2, THP1, U937, NB4,
K562, and the stem cell-like AML cell line TEX all expressed BTK, but this protein
was not detectable in KG1a AML cells or Jurkat D1.1 T-ALL cells (Figure 3-2B). Phosphorylation of BTK at Tyr223, a marker of BTK activation (Park et al., 1996;
Rawlings et al., 1996; Wahl et al., 1997), was detected in all cell lines expressing
BTK (Figure 3-2B), suggesting constitutive BTK activity
61
Figure 3-1: BTK mRNA levels in AML cell lines are similar to those of B-cell
malignancies. BTK mRNA expression AML relative to other cancer cell lines, as reported by the Cancer Cell Line Encyclopedia (Barretina et al., 2012).
62
3.4.2 AML cell lines are insensitive to chemical BTK inhibition with ibrutinib
To investigate the effects of BTK inhibition on AML viability and proliferation, we
treated AML cells with increasing concentrations of ibrutinib. Phospho-BTK was
reduced to undetectable levels at doses as low as 1 µM ibrutinib (Figure 3-2C). Compared to the sensitive B-lymphoblastic leukemia cell line, Daudi, the AML
cell lines TEX, OCI-AML2, HL60, and U937 were relatively insensitive to ibrutinib,
with IC50s ranging from 4- to 30-fold higher than Daudi cells in the Alamar Blue
Assay, which measures cell proliferation and viability (Figure 3-2D) and much
higher than the 1 µM concentration required to reduce levels of phospho-BTK.
Similar insensitivity to ibrutinib was seen when measuring cell viability with
Annexin V and PI staining (Figure 3-2E), which measures apoptosis.
Interestingly, KG1a cells lacking detectable expression of BTK were the most
sensitive to ibrutinib compared to other AML cell lines (KG1a IC50 = 2.87 µM by
Alamar Blue assay).
63
Figure 3-2: AML cell lines express constitutively active BTK, but are insensitive to
ibrutinib. (A) BTK mRNA expression in primary AML cells was determined by analysis of microarray gene expression (GEO accession code: GSE1159). BTK mRNA expression was examined in 267 patients with AML (Valk et al., 2004). (B) BTK and pBTK-Y223 expression in AML cell lines was determined by immunoblotting. (C) TEX and OCI-AML2 cells were treated with 1 µM ibrutinib for 1 h or 16 h. pBTK-Y223 and BTK expression in cell lysates was detected by immunoblotting. (D, E) AML cell lines were treated with increasing concentrations of ibrutinib over 72 h and cell growth and viability relative to untreated cells was determined by (D) Alamar Blue or (E) Annexin V and PI staining on flow cytometry. Data depict mean relative viability ± SD from a representative experiment performed in triplicate. Data are representative of three (D) or two (E) independent experiments.
64
3.4.3 A combination chemical screen with ibrutinib in AML cell lines identifies the PARG inhibitor, ethacridine lactate, as an ibrutinib sensitizer
Given the limited single-agent cytotoxicity in AML cell lines, we sought to
determine whether we could identify drugs that sensitize AML cells to ibrutinib.
To this end, we carried out a high-throughput combination chemical screen with
ibrutinib in both TEX and OCI-AML2 cells. The cell lines were co-treated with
ibrutinib and increasing concentrations of compounds from an in-house chemical
library containing 240 internationally prescribed drugs for a total of 5046 different
assays among the two cell lines in this screen. Following 72 hours of incubation,
cell growth and viability were measured by the sulforhodamine-B (SRB) assay.
Potential synergistic hits were identified using the excess-over-Bliss additivism
(EOBA) formula and average positive EOBA scores for each combination were
rank ordered (Figure 3-3A). Ethacridine lactate and pentamidine were top
synergistic hits common to both TEX and OCI-AML2 cells. We validated both
combinations in these cell lines, but pursued ethacridine lactate over pentamidine
because of its greater synergy with ibrutinib (EOBA scores of up to 0.58 and 0.47
by Alamar Blue in TEX and OCI-AML2, respectively) (Figure 3-3B & 3-3C, Figure 3-4). The ibrutinib-ethacridine combination induced cell death, as
determined by Annexin V and PI staining, but the mechanism of cell death was
caspase-independent (Figure 3-5). Ibrutinib and ethacridine also induced strong
synergistic cytotoxicity in U937, HL60, and K562 leukemia cells, but not in KG1a
cells (Figure 3-6).
Ethacridine lactate is used clinically as a topical antiseptic (O’Meara et al., 2014)
and intra-amniotic abortifacient (Mei et al., 2014). It is a DNA intercalator and
putative poly(ADP-ribose) glycohydrolase (PARG) inhibitor (Boulikas, 1990;
Tavassoli et al., 1985).
65
Figure 3-3: The PARG inhibitor ethacridine lactate sensitizes AML cell lines to
ibrutinib.
(A) Ibrutinib was co-treated with 240 drugs in TEX and OCI-AML2 cells for 72h. Growth and viability was determined with the SRB assay and synergy was calculated using the EOBA formula as described in the methods. Compounds were ranked in order of increasing average positive EOBA score. (B, C) TEX and OCI-AML2 cells were combination-treated with ibrutinib and ethacridine for 72 h and cell growth and viability relative to untreated cells was determined by Alamar Blue. (B) Data represent mean
66
growth and viability ± SD from a representative experiment performed in triplicate. (C) EOBA synergy scores (shown) were calculated for each of the combinations tested. EOBA values > 0.1 (lightest grey) denote a synergistic combination, while values > 0.5 (darkest grey) denote a strongly synergistic combination. Data represent mean EOBA scores from a representative experiment performed in triplicate.
67
Figure 3-4: Combination chemical screen validation for pentamidine. TEX and OCI-AML2 cells were subjected to 72h treatment with concentrations of ibrutinib and pentamidine similar to those tested during the combination chemical screen. Cell growth and viability was measured with the SRB assay, and calculated relative to untreated cells. Data represent mean percent growth and viability ± SD and mean EOBA scores from a single experiment performed in triplicate.
68
Figure 3-5: Cell death caused by ibrutinib-ethacridine combination is caspase
independent. Top: TEX and OCI-AML2 cells were subjected to combination ibrutinib (4 µM)-ethacridine (6 µM) treatment in the presence and absence of 50 µM Z-VAD-FMK (caspase inhibitor) for 48h. Viability was subsequently measured with Annexin V and PI staining on flow cytometry and calculated relative to vehicle-treated cells. Bottom: TEX and OCI-AML2 cells were treated at the indicated concentrations of ibrutinib and/or ethacridine for 48h, and induction of apoptosis was measured by Annexin V staining on flow cytometry.
69
Figure 3-6: The ibrutinib-ethacridine combination is strongly synergistic in HL60,
U937, and K562, but not KG1a AML cell lines. AML cell lines were treated with increasing concentrations of ibrutinib and ethacridine for 72h. Relative growth and viability was measured with the Alamar Blue assay. Data depict mean growth and viability ± SD and mean EOBA scores from a representative experiment performed in triplicate.
70
3.4.4 The ibrutinib-ethacridine combination is preferentially cytotoxic to a subset of primary AML cells compared to normal hematopoietic cells
Having identified the combination of ethacridine and ibrutinib as synergistic in
AML cell lines, we tested the combination in primary AML cells (n = 9) (see Table 3-1 for patient characteristics) and normal hematopoietic cells obtained from
consenting donors of G-CSF mobilized stem cells for allotransplantation (n = 9).
Primary cells were incubated with increasing concentrations of ethacridine and
ibrutinib for 48 hours in Iscove’s Modified Dulbecco’s Medium supplemented with
10% fetal bovine serum, without additional growth factors, and viability was
subsequently measured with Annexin V/PI staining and flow cytometry (Figure 3-7). Similar to the AML cell lines, ibrutinib had minimal single-agent cytotoxicity,
with IC50s exceeding 8 µM in all primary cells. We noted that primary AML cells,
on average, were more sensitive to single-agent ethacridine and combination
ibrutinib-ethacridine treatment compared to normals: a subset of 6 of 9 AMLs
demonstrated greater than 70% cell death from the combination, while only 1 of 9
normals (Normal 2) exhibited similar sensitivity. However, in some normal
samples, the drug combination induced ≥ 50% cell death, suggesting that the
ibrutinib-ethacridine combination may also have toxicity towards some normal
hematopoietic cells.
71
Table 3-1: Patient demographics.
Patient Characteristics Sample ID
Diagnosis
Age at Diagnosis Gender Cytogenetics Molecular
130794 AML 73 Male 46,XY[20] Not done 130819 AML, M5a 63 Female 46,XX[20] NPM1+, FLT3-ITD-, FLT3-TKD- 130826 AML, M4 58 Male 46,XY[20] NPM1+, FLT3-ITD+, FLT3-TKD- 130874 AML 69 Male 49,XY,+12,+16,+21[10] Not done 130877 AML 75 Male 48,XY,+9,+13[4]/46,XY[16] Not done 140994 AML 67 Male 45,XY,-7[10] NPM1-, FLT3-ITD-, FLT3-TKD- 141130 AML, M5b 80 Female Inconclusive CBFB-MYH11-
150177 AML 53 Female 42~46,XX,-2,der(3)add(3)(p21)?del(3)(q21q26),del(5)(q12), der(7)t(7;?11)(p13;q13),del(8)(p21),add(11)(q13),-18, add(20)(p13),+3mar[4]
Not done
150256 AML 24 Male 45,XY,der(6;7)t(6;7)(p21;q22)del(6)(q13q21)[17]/46,XY[3] Not done
72
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Figure 3-7: The ibrutinib-ethacridine combination is preferentially cytotoxic to
primary AML cells over normal hematopoietic cells. (Preceding page) Primary AML and normal hematopoietic cells (G-CSF mobilized peripheral blood stem cells) were treated with ibrutinib, ethacridine, or both in combination for 48 h. Viability was determined by Annexin V and PI staining. Data represent mean percent viability ± SD from a single experiment performed in triplicate. Ibru = ibrutinib, Ethac = ethacridine.
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3.4.5 The combination of ibrutinib and ethacridine delays the growth of AML cells in vivo
To assess the in vivo efficacy and toxicity of ibrutinib in combination with
ethacridine, we evaluated this combination in a mouse model of leukemia. SCID
mice were injected subcutaneously with OCI-AML2 cells. When tumors were
palpable, mice were treated with ibrutinib, ethacridine, or the combination of both
drugs. The combination of ibrutinib and ethacridine decreased the growth of OCI-
AML2 cells more than either drug alone (*P < 0.001 and **P < 0.0001). Of note,
no toxicity from combination treatment was detected as measured by changes in
body weight, behavior or gross examination of the organs at the end of the
experiment (Figure 3-8).
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Figure 3-8: Ibrutinib-ethacridine combination displays anti-AML activity in mice.
1 × 106 OCI-AML2 cells were subcutaneously injected in SCID mice. Eight days after injection, mice were treated with 300 mg/kg of ibrutinib by oral gavage, 20 mg/kg of ethacridine by i.p. injection, a combination of two drugs, or vehicle control (5% DMSO, 20% Cremophor, 0.9% NaCl) by oral gavage on the indicated days. Tumor volume (A) and body weight (B) were monitored over time. Mean ± SEM for tumor volume and mean ± SD for body weight, n = 7. *P < 0.001 and **P < 0.0001 from a two-way ANOVA with Tukey’s posttests comparing all treatment groups at day 18 and 20.
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3.4.6 Ethacridine synergizes with other small molecule BTK inhibitors, but not inhibitors of unrelated kinases
We sought to investigate whether the observed synergy with ethacridine was
specific to ibrutinib or a property common to other BTK inhibitors. We therefore
tested ethacridine in combination with two other BTK inhibitors currently in
clinical trials: CC-292 and ONO-4059. Cell growth and viability was measured 72
hours after incubation by the Alamar Blue assay and EOBA scores were
calculated. CC-292 and ONO-4059 synergized with ethacridine in TEX and OCI-
AML2 cells with efficacy similar to ibrutinib (Figure 3-9).
To further examine the specificity of the synergistic activity of ethacridine, we
sought to determine whether this compound generally sensitized AML cells to
kinase inhibitors. We therefore selected inhibitors of kinase targets bearing
minimal sequence similarity to BTK. Specifically, we tested PIM1/2 and STO-609,
inhibitors of Calcium/calmodulin-dependent protein kinase family members PIM
1/2 and CaMKK, respectively. TEX cells were treated with these compounds in
combination with ethacridine. Synergy was assessed by EOBA calculation
following viability determination at 72 hours with Annexin V and PI staining on
flow cytometry. Neither PIM1/2, nor STO-609 synergized with ethacridine in TEX
cells, with EOBA scores not exceeding 0.03 for either combination (Figure 3-10A).
We also tested the combination of ethacridine with the ABL kinase inhibitor
imatinib and the ABL and SRC family kinase inhibitor, dasatinib. Of note, ibrutinib
is reported to inhibit SRC family kinases (Honigberg et al., 2010) as they share
sequence homology to the TEC kinases. TEX and OCI-AML2 cells were
combination-treated with ethacridine and these kinase inhibitors. Following a 72-
hour incubation, cell growth and viability was determined by the Alamar Blue
assay. The combinations produced primarily additive effects as calculated by the
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EOBA formula (Figure 3-10B & Figure 3-11). Thus, the observed synergy with
ethacridine appears specific for TEC family kinase inhibitors.
Figure 3-9: Ethacridine synergizes with other small-molecule BTK inhibitors. TEX and OCI-AML2 cells were treated with increasing concentrations of ethacridine and (A) CC-292 or (B) ONO-4059 for 72 h. Growth and viability was measured by Alamar Blue and EOBA synergy scores were calculated. Data depict mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of three independent experiments.
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Figure 3-10: Ethacridine does not synergize with inhibitors of unrelated kinases. (A) TEX cells were combination-treated with ethacridine and PIM1/2 or STO-609 for 72 h. Viability was measured by Annexin V/PI staining and EOBA scores were generated. Combination ibrutinib-ethacridine treatment of TEX cells was included as a positive synergy control for this method of cell viability determination. (B) TEX cells were combination-treated with ethacridine and dasatinib or imatinib for 72 h. Growth and viability was measured by Alamar Blue and EOBA synergy scores were calculated. Data depict mean percent viability (A) or growth and viability (B) ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of three independent experiments.
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Figure 3-11: Dasatinib and imatinib do not synergize with ethacridine in OCI-
AML2 cells. OCI-AML2 cells were combination-treated with ethacridine and dasatinib (left) or imatinib (right) for 72 h. Cell growth and viability was measured with the Alamar Blue assay, and calculated relative to untreated cells. Synergy was calculated with the EOBA formula. Data represent mean percent growth and viability ± SD and mean EOBA scores from a single experiment performed in triplicate. Data are representative of three independent experiments.
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3.4.7 Ibrutinib and ethacridine synergize to induce cell death via a ROS-dependent mechanism
To investigate the mechanism of synergy between ethacridine and ibrutinib, we
examined ROS (reactive oxygen species) production in AML cells treated with
the drug combination. Using carboxy-H2DCFDA staining and flow cytometry, we
measured total intracellular ROS production after TEX and OCI-AML2 treatment
with ibrutinib, ethacridine or the drug combination. At concentrations that were
associated with synergistic cell death, neither drug alone markedly increased
intracellular ROS production. However, ROS production in live cells was
increased with the drug combination as early as two hours following treatment in
both TEX and OCI-AML2 cells (Figure 3-12A). Moreover, the increased ROS
production was functionally important for the observed cell death, as the addition
of the anti-oxidant α-tocopherol abrogated cytotoxicity from the combination in
both cell lines (Figure 3-12B). The observed increase in ROS following
combination treatment did not appear to be mitochondrial in origin, as MitoSOX
staining did not increase following combination treatment relative to single-agent
treatment (Figure 3-12C). Thus, these findings suggest that the synergistic
cytotoxicity caused by the ibrutinib-ethacridine combination is due to excessive
intracellular, but not mitochondrial, ROS production.
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Figure 3-12: The ibrutinib-ethacridine combination induces cytotoxic levels of
intracellular ROS. (A) TEX and OCI-AML2 cells were treated with ibrutinib, ethacridine, or both in combination for 2, 6 or 24 h. Intracellular ROS was measured by carboxy-H2DCFDA staining and dead cells were excluded by PI staining on flow cytometry. Fold increase in ROS was calculated relative to the geometric means of carboxy-H2DCFDA (FITC) staining in untreated cells. H2O2 treatment served as a positive control for intracellular ROS generation. (B) TEX and OCI-AML2 cells were pre-treated with α-tocopherol prior to a 48 h incubation with ibrutinib and/or ethacridine. Viability was measured by Annexin V and PI staining, and calculated relative to respective untreated controls (+ or − α-tocopherol). (C)TEX and OCI-AML2 cells were treated with ibrutinib, ethacridine, or both drugs in combination for 2, 6 or 24 h. Mitochondrial ROS was measured by MitoSOX Red staining, with dead cell exclusion by Annexin V staining on flow cytometry. Fold increase in mitochondrial ROS was calculated relative to the geometric means of carboxy-H2DCFDA (FITC) staining in untreated cells. Antimycin A (50 µM) treatment served as a positive control for mitochondrial ROS generation.
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Data represent mean fold increase in ROS production ± SD (A, C) or mean viability ± SD (B) from representative experiments performed in triplicate. Data are representative of two (A, C) or three (B) independent experiments. In all panels, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001 as determined by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons (A), or unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%) (B).
3.4.8 The chemical PARG inhibitor gallotannin also synergizes with ibrutinib to induce cell death by excessive ROS production
Ethacridine is a putative PARG inhibitor (Boulikas, 1990; Tavassoli et al., 1985)
and we demonstrated that ethacridine inhibited PARG (Figure 3-13A). Therefore, we evaluated the combination of ibrutinib and gallotannin, another
reported PARG inhibitor (Aoki et al., 1993; Formentini et al., 2008; Tsai et al.,
1992). We treated TEX and OCI-AML2 cells with increasing concentrations of
ibrutinib and gallotannin over 48 hours and then measured viability with Annexin
V and PI staining. The ibrutinib-gallotannin combination was also profoundly
synergistic, yielding EOBA values of up to 0.60 and 0.72 in TEX and OCI-AML2
cells, respectively (Figure 3-13B). Likewise, pre-treatment with α-tocopherol
abrogated ibrutinib-gallotannin cytotoxicity (Figure 3-13C). Pretreatment with the
poly(ADP-ribose) polymerase (PARP) inhibitor olaparib did not rescue
combination-induced cytotoxicity (Figure 3-14). However, olaparib was directly
toxic to the cells, thus potentially obscuring any protective effects.
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Figure 3-13: The PARG inhibitor gallotannin synergizes with ibrutinib (A) Ethacridine’s inhibitory activity against PARG was determined using a cell-free colorimetric assay that measures levels of biotinylated PAR attached to histones in the presence of PARG enzyme. A loss of absorbance at 450 nm correlates with increased PARG activity. Relative PARG activity was calculated by comparing the loss of absorbance at 450nm in the presence of ethacridine to that of no PARG control (maximal absorbance at 450 nm). Data represent mean PARG activity ± SD from a single experiment performed in triplicate. (B) TEX and OCI-AML2 cells were treated with increasing concentrations of ibrutinib and gallotannin for 48 h. Viability was measured by Annexin V and PI staining and EOBA scores were calculated. Data represent mean EOBA scores from a representative experiment performed in triplicate. Data are representative of two (OCI-AML2) or three (TEX) independent experiments. (C) TEX cells were pre-treated with α-tocopherol and subjected to 48 h treatment with ibrutinib and gallotannin. Viability was measured by Annexin V and PI staining and calculated relative to untreated controls. Data represent mean viability ± SD from a representative experiment performed in triplicate. Data are representative of three independent experiments. In all panels, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001 as determined by unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%) (C).
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Figure 3-14: Treatment of TEX and OCI-AML2 cells with olaparib in combination
with ibrutinib and ethacridine. TEX and OCI-AML2 cells were pre-treated with the PARP inhibitor olaparib 4 hours prior to a 72 h incubation with ibrutinib, ethacridine or both in combination at the indicated concentrations. Growth and viability was measured by the Alamar Blue assay and then calculated relative to untreated controls.
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3.4.9 The synergy of ibrutinib with ethacridine is independent of the inhibitory effect on BTK
To determine whether synergy between ibrutinib and ethacridine was due to BTK
inhibition by ibrutinib, we knocked down BTK with shRNA in TEX and OCI-AML2
cells. The reduction of BTK expression was confirmed by immunoblotting (Figure 3-15A) and qPCR (not shown). Knockdown cells were then treated with
increasing concentrations of ethacridine and cell growth and viability was
assessed by Alamar Blue. Despite substantial levels of BTK knockdown by
shRNA, ethacridine treatment of BTK-knockdown cells was no more cytotoxic
than ethacridine treatment of shRNA control cells (Figure 3-154A). These
observations suggest that synergy of ibrutinib with ethacridine is independent of
its inhibitory effect on BTK.
To further examine whether synergy of ibrutinib with ethacridine is due to targets
beyond BTK, we tested the drug combination in Jurkat D1.1 cells, a T-acute
lymphoblastic leukemia cell line that does not express BTK (Figure 3-2B). The
ibrutinib-ethacridine combination synergized in Jurkat D1.1 cells, reaching EOBA
values of 0.25 (Figure 3-15B), further supporting a synergistic mechanism for
ibrutinib and ethacridine beyond AML cell lines analyzed in this study.
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Figure 3-15: Ibrutinib’s synergy with ethacridine is independent of BTK. (A) TEX and OCI-AML2 cells were transduced with 2 different shRNAs targeting BTK or a non-targeting shRNA control in lentiviral vectors. On day 4 post-transduction, cells were treated with ethacridine at concentrations previously shown to synergize with ibrutinib. Growth and viability at 72 h post-ethacridine treatment was determined by Alamar Blue and calculated relative to untreated control. BTK knockdown was confirmed by immunoblotting. (B) Jurkat cells were treated with increasing concentrations of ibrutinib and ethacridine for 72 h. Cell growth and viability was determined by the Alamar Blue assay and synergy was calculated using the EOBA formula. Data depict mean percent growth and viability ± SD from a representative experiment performed in triplicate. Data in (A) and (B) are representative of two independent experiments. In all panels, ns = not significant, based on the results of an unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%).
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Figure 3-16: Expression of TEC family kinases in AML cell lines. Expression of kinases BMX, TLK, TEC, and ITK in a panel of AML cell lines, Jurkat D1.1 and Daudi cells was determined by immunoblotting.
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3.5 Discussion
The small molecule BTK inhibitor ibrutinib has demonstrated exceptional efficacy
with minimal toxicity in several B-cell cancers. Ibrutinib is currently approved for
clinical use in CLL, mantle-cell lymphoma (MCL) and Waldenström’s
macroglobulinemia (WM), owing to its impressive patient response rates during
recent clinical trials. With widespread BTK expression across B-cell malignancies
and its role as a node in several oncogenic signaling pathways, this cytoplasmic
kinase has emerged as an attractive therapeutic target for B-lymphoid cancers.
Multiple clinical trials investigating ibrutinib alone and in combination for these
diseases are currently underway.
In addition to its expression in B-cell malignancies, BTK is also expressed in
myeloid cell lines and can be activated through mechanisms independent of the
B-cell receptor (Doyle et al., 2007; Kawakami et al., 1994; Oellerich et al., 2015).
Thus, targeting BTK with ibrutinib may have efficacy in myeloid malignancies
such as AML.
In concordance with previous work by Rushworth et al. (2014) and Oellerich et al.
(2015), we demonstrated the expression of constitutively active BTK in several
AML cell lines. However, in contrast to these other studies, in the cell lines we
tested, the cytotoxicity from ibrutinib was likely independent of its effects on BTK.
Supporting this contention, of the tested AML cell lines, KG1a cells were the
most sensitive to ibrutinib and yet lacked detectable BTK by immunoblot.
Moreover, the IC50 in TEX and OCI-AML2 leukemia cells were over 10-fold
higher than the concentration of ibrutinib required to completely repress BTK
phosphorylation and higher than the pharmacologically achievable
concentrations in humans (Appendix 1). Consistent with these observations,
ibrutinib has been reported to induce AML cell death via a BTK-independent
mechanism (Wu et al., 2015).
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Although ibrutinib was largely inactive as a single agent in the tested AML cells,
we successfully sensitized two of the more resistant AML cell lines (TEX, IC50 =
13.01 µM and OCI-AML2, IC50 = 27.44 µM) to ibrutinib by combining the drug
with the putative PARG inhibitor ethacridine. However, the observed synergistic
cytotoxicity was independent of the inhibitory effect of ibrutinib on BTK. These
findings suggest that ibrutinib may also have anti-AML activity that extends to
targets beyond BTK.
In addition to inhibiting BTK, ibrutinib cross-reacts with TEC and SRC family
kinases with similar efficacy (Honigberg et al., 2010). We noted that the SRC
inhibitor dasatinib did not recapitulate ibrutinib’s synergy with ethacridine,
however the BTK inhibitors ONO-4059 and CC-292, which have reported
inhibitory activity against TEC family kinases (Akinleye et al., 2013; Ariza et al.,
2013; Evans et al., 2013; Hendriks et al., 2014; Yoshizawa et al., 2012), did
synergize with ethacridine. We therefore favor the TEC family kinases as likely
targets of ibrutinib in its synergy with ethacridine in AML. To date, 5 TEC family
members have been identified: BTK, BMX, TEC, ITK, and RLK (Schmidt et al.,
2004a). ITK is expressed selectively in T cells and its inhibition by ibrutinib leads
to decreased STAT6, IkBa, JUNB, and NFAT activity, as well as decreased
intracellular calcium release (Dubovsky et al., 2013). BMX is expressed in
hematopoietic progenitor cells and myeloid leukemias (Kaukonen et al., 1996;
Weil et al., 1997) and has been found to mediate STAT3 activation and
subsequent transformation by Src (Tsai et al., 2000). Interestingly, BMX, TEC,
and RLK are all expressed in AML and Jurkat D1.1 cell lines (Figure 3-16). However, further investigations will be required to determine whether these TEC
family members—or different kinases altogether—are targets of ibrutinib that
explain its synergy with ethacridine. One possible strategy for ibrutinib target
determination is a synthetic-lethal human kinome shRNA array, which would
uncover kinases that when individually knocked down induce AML cell line
sensitivity to ethacridine.
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To our knowledge, this work is the first to report on the activity of PARG inhibitors
such as ethacridine and gallotannin in AML. While further work is required to fully
determine the impact of PARG inhibition in AML, it is interesting to speculate
whether some of the single agent activity of ibrutinib in primary AML might be
observed in patients with the lowest basal PARG expression.
The addition of poly(ADP-ribose) (PAR) moieties to target proteins alters their
structure, function, and localization. PAR-ylation of target proteins is mediated by
the PARP (Poly(ADP-ribose) polymerase) family of enzymes, of which PARP-1 is
the most abundant and best characterized (Durkacz et al., 1980; Luo & Kraus,
2012; Virág et al., 2013). PARP adds PAR groups to target proteins, and these
moieties are removed by PARG (Feng & Koh, 2013). Thus, genetic or chemical
inhibition of PARG leads to the accumulation of excess PAR-ylated proteins.
Through the accumulation of excess PAR-ylated proteins, PARG inhibition
reduces the proliferation of malignant cells (Erdélyi et al., 2009; Pan et al., 2012),
sensitizes cells to genotoxic stress (Cortes et al., 2004; Koh et al., 2004; Shirai et
al., 2013) and inhibits cell signaling pathways including NFκB, p38 and ERK (Pan
et al., 2012). Through these and other mechanisms, increased levels of PAR-
ylated proteins may also promote ROS generation (Krenzlin et al., 2012), which
is relevant to our observed mechanism of action of the drug combination.
Though the ibrutinib-PARG inhibitor combination produced striking synergistic
cytotoxicity in AML cell lines, it is important to note that this combination also
induced cytotoxicity in a subset of normal hematopoietic cells (Figure 3-7). This
observation highlights a potential toxicity that would need to be assessed in the
context of clinical trials of these agents.
Thus in summary, through identification of an ibrutinib combination that
sensitizes resistant AML cell lines to this kinase inhibitor, we uncovered a novel
BTK-independent role for ibrutinib in AML. Moreover, we present a potential role
for PARG inhibition as a novel target for combination therapy in AML.
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Chapter 4: Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells.
This chapter has been published as a Letter to the Editor:
Rotin LE, Gronda M, Hurren R, Wang X, Minden MD, Slassi M, Schimmer AD (2016). Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells. Leuk Lymphoma. Feb 17:1-5. Doi: 10.3109/10428194.2016.1138292 [Epub ahead of print]
Leukemia & Lymphoma © 06 Jan 2016 available: http://www.tandfonline.com/10.3109/10428194.2016.1138292
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4.1 Abstract
Bruton’s tyrosine kinase (BTK) is a potential therapeutic target in acute myeloid
leukemia (AML) and the small molecule BTK inhibitor ibrutinib has been found to
potentiate the cytotoxic activity of the anthracycline daunorubicin in AML cells
(Rushworth et al., 2014). We sought to determine whether synergy between
ibrutinib and daunorubicin was mediated by the anti-BTK activity of ibrutinib. Our
findings highlight the possibility that this synergistic mechanism is BTK-
independent and unrelated to the reported capacity of ibrutinib to enhance
cellular accumulation of other drugs. Reactive oxygen species (ROS) production
may be functionally important for synergistic cell death.
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4.2 Introduction
Ibrutinib is a well-tolerated BTK inhibitor that is used in the treatment of several
B-lymphoid malignancies, particularly chronic lymphocytic leukemia. The TEC
family kinase member BTK is expressed in most hematopoietic cell types,
including myeloid-lineage cells, and is constitutively active in primary AML and
AML cell lines (Oellerich et al., 2015; Rushworth et al., 2014). Both ibrutinib
treatment and BTK knockdown have been shown to induce cell cycle arrest
(Oellerich et al., 2015), apoptosis (Oellerich et al., 2015; Rushworth et al., 2014),
and to block migration of AML cells (Zaitseva et al., 2014). Moreover, combining
ibrutinib with first-line AML chemotherapy agents daunorubicin and cytarabine
enhanced cell killing and reduced colony formation in AML bulk cells (Rushworth
et al., 2014). Thus, ibrutinib has emerged as a possible combination therapy
candidate for this disease. The purpose of the present study was to investigate
the mechanism by which ibrutinib and daunorubicin synergize in AML cells.
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4.3 Methods
4.3.1 Radiolabelled daunorubicin accumulation assay
Radioactivity of lysed cells was quantified with a Beckman LS6000IC liquid
scintillation counter, and counts were normalized to cellular protein content.
All other experiments were carried out using materials and procedures previously
described in Chapter 3.3 (pgs 56-61).
4.4 Results & Discussion
We first validated the previously reported synergistic cytotoxicity resulting from
ibrutinib-daunorubicin and ibrutinib-cytarabine combinations in AML cell lines. We
treated the AML cell lines OCI-AML2 with wild type FLT3 (Quentmeier et al.,
2003) and TEX whose FLT3 mutation status is not known with increasing
concentrations of ibrutinib and daunorubicin or cytarabine for 72 hours and
measured cell viability using Annexin V and PI staining on flow cytometry. We
determined the extent of any resultant synergy for each of the combinations
tested with the excess-over-Bliss additivism (EOBA) formula, as previously
described (Borisy et al., 2003). The ibrutinib-daunorubicin combination yielded
synergistic EOBA scores of up to 0.33 in TEX and 0.25 in OCI-AML2 (Figure 4-1). These findings are in line with a previous report describing synergistic killing
activity between ibrutinib and doxorubicin, another anthracycline, in activated B-
cell-like subtype of diffuse large B-cell lymphoma cells (ABC-DLBCL) (Mathews
Griner et al., 2014). Meanwhile, the ibrutinib-cytarabine combination was only
borderline synergistic, producing EOBA scores no greater than 0.11 in TEX and
OCI-AML2 (Figure 4-2). Given that the ibrutinib-daunorubicin combination was
more profoundly synergistic, we decided to investigate this combination further.
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Figure 4-1: Ibrutinib and daunorubicin synergize in TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”) and daunorubicin alone and in combination for 72h. Viability was measured by Annexin V and PI staining on flow cytometry, then calculated relative to untreated cells (histograms). Excess-over-Bliss additivism (EOBA) scores for each tested combination are shown (tables); values >0.10 (grey) denote a synergistic combination, with higher EOBA values (darker grey) indicating greater synergy. Data represent mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of two independent experiments.
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Figure 4-2: Combination ibrutinib-cytarabine treatment of TEX and OCI-AML2
cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”) and cytarabine alone and in combination for 72h. Viability was measured by Annexin V and PI staining on flow cytometry and then calculated relative to untreated cells (histograms). Excess-over-Bliss additivism (EOBA) scores are shown (tables). Data represent mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate.
Figure 4-3: Ibrutinib inhibits BTK phosphorylation. TEX and OCI-AML2 cells were incubated with 1 µM ibrutinib (+) or vehicle (-) for 1h. Cells were lysed and probed for BTK-pY223, a marker of BTK activation, on immunoblot.
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To determine whether the synergy between ibrutinib and daunorubicin was
dependent upon BTK inhibition by ibrutinib, we treated BTK-knockdown TEX and
OCI-AML2 cells with daunorubicin to reproduce the sensitization observed in
combination with ibrutinib. Both cell lines express constitutively active BTK, and
treatment with ibrutinib inhibited BTK phosphorylation (Figure 4-3). We
lentivirally transduced TEX and OCI-AML2 cells with five different BTK-targeting
shRNAs, alongside one non-targeting shRNA control (GFP). On Day 3 following
puromycin selection, transduced cells were treated with increasing
concentrations of daunorubicin for 72 hours, and viability relative to respective
untreated controls was subsequently measured with Annexin V and PI staining.
Despite knockdown of BTK to undetectable levels (Figure 4-4), little to no
sensitization to daunorubicin was seen among five independent shRNA clones
targeting BTK (Figure 4-5). While sensitization to daunorubicin was observed at
single doses of daunorubicin in two shRNA clones targeting BTK, for the majority
of clones and doses of daunorubicin tested, no increased sensitization was
observed despite undetectable levels of BTK. Thus, these findings do not
convincingly support BTK inhibition as the target of ibrutinib that would explain
synergy with daunorubicin. Given that ibrutinib is known to inhibit other kinases,
including SRC family kinases and other TEC family members (Honigberg et al.,
2010), it is possible that off-target kinase inhibition by ibrutinib may explain its
synergy with daunorubicin. Possible BTK-independent explanations for profound
daunorubicin sensitization in clones shRNA-BTK508 and shRNA-BTK2490
include off-target shRNA effects (Manjunath et al., 2009).
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Figure 4-4: BTK knockdown confirmation. BTK knockdown was confirmed by immunoblotting in TEX and OCI-AML2 cells.
Figure 4-5: Daunorubicin treatment of BTK-knockdown cells. TEX and OCI-AML2 cells were transduced with 5 shRNAs targeting BTK alongside a non-targeting shRNA control (GFP) via the PLKO.1 lentiviral vector, which contains a puromycin resistance gene. On day 3 post-puromycin selection, transduced cells were treated with increasing concentrations of daunorubicin for 72h, and viability relative to respective untreated controls was determined by Annexin V/PI staining. Data depict mean percent viability ± SD from representative experiments performed in triplicate. Data are representative of two independent knockdown experiments.
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To address the possibility that a subpopulation of BTK-knockdown cells was
highly BTK-dependent and thus killed off prior to treatment with daunorubicin, we
treated BTK-knockdown TEX cells with ibrutinib and measured viability with
Annexin V and PI staining. Compared to shRNA control, neither shRNA-BTK974,
nor shRNA-BTK1066 was more resistant to ibrutinib at the concentrations
evaluated (Figure 4-6), suggesting that the overall observed lack of daunorubicin
sensitization in BTK knockdown cells was not the result of having selected for a
subpopulation of BTK-independent cells.
One of the mechanisms by which kinase inhibitors have been shown to synergize
with antineoplastic agents is through inhibition of ATP-binding cassette (ABC)
transporter-mediated drug efflux activity (Lainey et al., 2012). Ibrutinib is a
reported inhibitor of the MRP1 (ABCC1) transporter and sensitized MRP1-
overexpressing cell lines to the MRP1 substrate vinblastine by enhancing its
accumulation (Zhang et al., 2014). We sought to determine whether potentiation
of daunorubicin accumulation by ibrutinib might explain the synergistic killing by
this combination. We treated TEX cells with increasing concentrations of
radiolabelled daunorubicin in the presence and absence of ibrutinib, comparing
daunorubicin counts from lysed cells normalized to total protein content. Ibrutinib
failed to increase daunorubicin accumulation (Figure 4-7), suggesting that
inhibiting daunorubicin efflux is unlikely to be the mechanism by which ibrutinib
and daunorubicin synergize in TEX cells.
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Figure 4-6: Ibrutinib treatment of BTK-knockdown TEX cells. TEX shRNA-BTK974 and shRNA-BTK1066 cells were treated with ibrutinib alongside shRNA control and parental TEX cells for 48 hours (starting on day 3 post-puromycin selection). Viability was measured with Annexin V/PI staining and calculated relative to respective untreated controls. Data depict mean percent viability ± SD from representative experiments performed in triplicate. Data are representative of two independent knockdown experiments.
Figure 4-7: Daunorubicin accumulation in the presence or absence of ibrutinib. TEX cells were incubated with radiolabelled daunorubicin in the presence and absence of 8 µM ibrutinib for 3h. Data depict mean radioactive counts ± SD relative to protein content from an experiment performed in triplicate. Data are representative of two independent experiments.
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We have previously noted that ibrutinib synergizes with reactive oxygen species
(ROS)-inducing agents to cause cell death mediated by excessive ROS
production (Rotin et al., 2014). Because daunorubicin is also a ROS-inducer
(Gewirtz, 1999), we sought to determine whether cell death after treatment with
the ibrutinib-daunorubicin combination was ROS-dependent. TEX and OCI-AML2
cells were treated with daunorubicin and ibrutinib with and without the antioxidant
α-tocopherol. Pre-treatment with α-tocopherol dramatically rescued viability in
TEX and OCI-AML2 cells treated with ibrutinib, daunorubicin, or the combination
(Figure 4-8). We also tested whether the ibrutinib-daunorubicin combination
increased total intracellular ROS production by staining cells with carboxy-
H2DCFDA. Mildly increased carboxy-H2DCFDA staining was observed following
a 6-hour combination treatment, and this change was statistically significant
(Figure 4-9). α-tocopherol pre-treatment abrogated the slight increase in
intracellular ROS production (Figure 4-10). Examination of mitochondrial ROS
production with MitoSOX Red staining in combination-treated TEX and OCI-
AML2 cells revealed a small increase in mitochondrial ROS production following
ibrutinib treatment; however no further increases were noted when ibrutinib was
combined with daunorubicin (Figure 4-11).
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Figure 4-8: α-tocopherol rescue of combination-treated TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with 3mM α-tocopherol for 24h prior to a 72h incubation with ibrutinib and daunorubicin (“DNR”) alone and in combination, with α-tocopherol maintained at a concentration of 2.4mM. Viability relative to respective untreated controls was measured with Annexin V and PI staining. Data depict mean percent viability ± SD from an experiment performed in triplicate. Data are representative of two independent experiments.
Figure 4-9: Combination ibrutinib-daunorubicin treatment increases intracellular
ROS TEX and OCI-AML2 cells were treated with ibrutinib and daunorubicin (“DNR”) for 6h, then stained with carboxy-H2DCFDA and PI to detect intracellular ROS in live cells by flow cytometry. Fold increase in ROS was calculated relative to the geometric mean fluorescence intensities (GMFI) of carboxy-H2DCFDA staining in untreated live cells. Hydrogen peroxide treatment was included as a positive carboxy-H2DCFDA staining control. Data depict average GMFIs ± SD and are representative of a single experiment performed in triplicate. Data are representative of two independent experiments. In all panels, ns = not significant, *P<0.05, **P<0.01; ***P<0.001; ****P<0.0001, as determined by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons.
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Figure 4-10: Intracellular ROS production following combination ibrutinib-
daunorubicin treatment in the presence or absence of α-tocopherol. TEX and OCI-AML2 cells were treated with 3mM α-tocopherol prior to a 6h incubation with ibrutinib (“Ibru”), daunorubicin (DNR), or both drugs in combination. Cells were subsequently stained with carboxy-H2DCFDA and PI to measure intracellular ROS production in live cells on flow cytometry. Increases in ROS were calculated relative to the average geometric mean fluorescence intensity (GMFI) of carboxy-H2DCFDA staining in untreated TEX or OCI-AML2 cells. Hydrogen peroxide (H2O2) was included as a positive control for intracellular ROS. Data depict mean fold increase in ROS ± SD from an experiment performed in triplicate.
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Figure 4-11: Mitochondrial ROS production following combination ibrutinib-
daunorubicin treatment in TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”), daunorubicin (DNR), or both drugs in combination for 6h. Cells were stained with MitoSOX Red and Annexin V to measure mitochondrial ROS on flow cytometry. Fold increase in mitochondrial ROS production was calculated relative to the average GMFI of MitoSOX+, Annexin V- staining untreated cells. Antimycin A was included as a positive control for mitochondrial ROS staining. Results depict the mean fold increase in mitochondrial ROS ± SD from an experiment performed in triplicate. Data are representative of two independent experiments.
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A possible explanation for the discrepancy between the degree of viability rescue
by α-tocopherol and induction of ROS following combination treatment is the
length of incubation with drug (6 hours) prior to ROS measurement: previous
studies in ABC-DLBCL cells demonstrated highest ROS production by ibrutinib at
1-2 hours post-treatment, with levels significantly decreasing by four hours
(Dasmahapatra et al., 2013). Alternatively, it is possible that the modest increase
in ROS production following ibrutinib-daunorubicin treatment does not fully
account for the extent of antioxidant-mediated cell rescue: the observed
cytoprotection by α-tocopherol may have been due to an off-target effect. In
support of this possibility, a recent study reported reversal of kinase inhibitor-
mediated apoptosis and cell cycle arrest by α-tocopherol, which is independent of
its antioxidant activity (Pédeboscq et al., 2012).
In conclusion, ibrutinib potentiates the AML cell-killing activity of daunorubicin via
a mechanism that is potentially BTK-independent, and unrelated to enhancement
of intracellular daunorubicin accumulation. Incorporating ibrutinib into treatment
regimens for AML patients, regardless of BTK expression and constitutive
activation status, may be warranted.
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Chapter 5: Erlotinib synergizes with the poly(ADP-ribose) glycohydrolase inhibitor ethacridine in acute myeloid leukemia cells
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5.1 Abstract
Erlotinib is a small-molecule epidermal growth factor receptor (EGFR) inhibitor
that has demonstrated significant EGFR-independent activity against acute
myeloid leukemia (AML) cell lines and primary AML blasts in preclinical studies,
however these findings have not been reproducible in the clinical trial setting.
Combining erlotinib with other antineoplastic agents has been proposed as a
strategy for improving the clinical activity of erlotinib. With the goal of identifying
erlotinib-sensitizing drugs, we screened erlotinib against several chemical
libraries in the erlotinib-insensitive AML cell lines TEX and OCI-AML2, identifying
the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate as the top
synergistic hit common to both cell lines. The erlotinib-ethacridine combination
induced synergistic cell death, which was preceded by a profound and lethal
increase in intracellular reactive oxygen species (ROS) production. Using mass
spectrometry, we determined that erlotinib synergized with ethacridine by
potentiating ethacridine accumulation in TEX and OCI-AML2 cells. This
synergistic mechanism of action was confirmed by demonstrating that high-dose
ethacridine treatment mimics the significant increases in ROS observed following
combination erlotinib-ethacridine treatment. Thus, we have identified that erlotinib
promotes the accumulation of select drugs, thereby leading to synergism. In
addition, the potential anti-AML activity of PARG inhibitors warrants further study.
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5.2 Introduction
Erlotinib is a small-molecule epidermal growth factor receptor (EGFR) inhibitor
that reversibly blocks autophosphorylation of C-terminal EGFR tyrosine residues,
inhibiting cell proliferation and inducing apoptosis (Moyer et al., 1997). It is used
in the clinical treatment of non small-cell lung cancer (NSCLC), where EGFR is
often over-expressed or constitutively active, and thus promotes tumor cell
survival and proliferation via PI3K/AKT/mTOR, STAT, and Ras/Raf/MAPK
signaling pathways (Sharma et al., 2007; Siegelin & Borczuk, 2014).
Erlotinib has well-documented preclinical activity against acute myeloid leukemia
(AML) cells, where it induces differentiation (Boehrer et al., 2008a), cell cycle
arrest (Boehrer et al., 2008a; Boehrer et al., 2011; Lainey et al., 2011), and
apoptosis (Boehrer et al., 2008a; Boehrer et al., 2008b), yet EGFR expression in
these cells is absent (Boehrer et al., 2008a; Chan & Pilichowska, 2007;
Stegmaier et al., 2005). Several erlotinib targets have been proposed or reported
to account for its anti-leukemic effects: erlotinib was shown to inhibit SRC family
kinases (SFKs) (Boehrer et al., 2011; Weber et al., 2012), which are
constitutively active in primary AML cells and AML cell lines and mediate mTOR
complex 1 (mTORC1) signaling in these cells (Dos Santos et al., 2008). In line
with these findings, erlotinib was found to inhibit phosphorylation of mTORC1
targets and to induce autophagy in the AML cell line KG-1 (Boehrer et al., 2011).
Erlotinib has also been found to bind directly to Bruton’s tyrosine kinase (BTK)
and to decrease phosphorylation at Y551 (Weber et al., 2012); Y551
phosphorylation is required for activation of this kinase. BTK has been proposed
as a potential therapeutic target in AML because of its role as a mediator of
FLT3-ITD and TLR9 signaling in FLT3-ITD-positive and FLT3-ITD-negative AML
cell lines, respectively (Oellerich et al., 2015).
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Finally, erlotinib was shown to inhibit JAK2 Tyr1007/1008 phosphorylation and
downstream STAT5 phosphorylation at Tyr694 in KG-1 cells (Boehrer et al.,
2008a). JAK2-STAT3/5 signaling has been implicated in AML CD34+ cell survival
and colony formation (Cook et al., 2014).
Clinically, the anti-AML activity of erlotinib has been more modest. While two
case reports described AML remissions in two patients with concomitant NSCLC
and AML (Chan & Pilichowska, 2007; Pitini et al., 2008), subsequent clinical trials
examining the single-agent activity of erlotinib in AML have not yielded equally
remarkable results, with a small minority of patients exhibiting decreased
peripheral blast counts and zero patients achieving complete remission (Sayar et
al., 2015; Thepot et al., 2014). The authors of both studies suggested that
erlotinib may have better clinical utility when administered in combination with
other anti-leukemic agents (Sayar et al., 2015; Thepot et al., 2014).
In light of the limited clinical single-agent activity of erlotinib in AML, its favorable
safety profile (Gordon et al., 2005; Sayar et al., 2015; Soulieres et al., 2004;
Thepot et al., 2014), and previous reports of synergistic interactions with other
antineoplastic agents (Lainey et al., 2013a; Lainey et al., 2013b; Landriscina et
al., 2010), we sought to identify erlotinib combination candidates in AML using a
high-throughput drug screening approach.
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5.3 Materials and Methods
5.3.1 Reagents
Anti-EGFR (#2232) and anti-GAPDH (#2118) antibodies were obtained from Cell
Signaling Technology (Danvers, MA). HRP-conjugated goat anti-rabbit and goat
anti-mouse secondary antibodies were acquired from GE Healthcare
(Buckinghamshire, UK). Erlotinib was obtained from Cayman Chemical (Ann
Arbor, MI). Ethacridine lactate, sulforhodamine-B, α-tocopherol, and hydrogen
peroxide were purchased from Sigma-Aldrich (St. Louis, MO). Imatinib was
obtained from AK Scientific, Inc. (Union City, CA). Drug libraries were obtained
from MicroSource Discovery Systems, Inc. (Gaylordsville, CT), and Sequoia
Research Products (Pangbourne, UK). The kinase inhibitor library was obtained
from the Ontario Institute for Cancer Research, Toronto, Canada.
5.3.2 Cell culture
TEX cells (Warner et al., 2005) were provided by Dr. John Dick (Ontario Cancer
Institute, Toronto, Canada) and maintained in Iscove’s Modified Dulbecco’s
Medium (IMDM) supplemented with 15% fetal bovine serum (FBS)
(Seradigm/VWR, Radford, PA), 100µg/ml penicillin, 100 U/ml streptomycin, 2mM
L-glutamine (Life Technologies, Carlsbad, CA), 20ng/ml SCF (Miltenyi Biotec,
San Diego, CA), and 2ng/ml IL-3 (R&D Systems, Minneapolis, MN). OCI-AML2
and U937 cells were provided by Dr. Mark Minden (Ontario Cancer Institute,
Toronto, Canada). K562 cells were provided by Dr. Suzanne Kamel-Reid
(Ontario Cancer Institute, Toronto, Canada). OCI-AML2 and K562 cells were
grown in IMDM supplemented with 10% FBS 100 µg/ml penicillin, and 100 U/ml
streptomycin. U937 cells were grown in RPMI 1640 supplemented with 10%
FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin. MDA-468 cells were grown
in RPMI-1640 supplemented with 10% FBS. All cell lines were maintained at
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37°C and 5% CO2.
5.3.3 Primary cells
Primary bulk AML cells and peripheral blood stem cells (PBSCs) from stem cell
donors treated with G-CSF were collected from consenting patients, with the
approval of the University Health Network (Toronto, Canada) institutional review
board. Primary AML cells were isolated by Ficoll density centrifugation and
PBSCs were isolated by apheresis. Cells were maintained in IMDM
supplemented with 10% FBS, 100µg/ml penicillin, and 100U/ml streptomycin, at
37°C and 5% CO2.
5.3.4 Immunoblotting
PBS-washed cells were lysed in 1xLaemmli buffer and protein from whole cell
lysates was quantified with the DC Protein Assay (Biorad Laboratories,
Mississauga, ON, Canada). Lysates were resolved by SDS-PAGE and
transferred to a PVDF membrane. Following blocking in 5% milk-TBST for 1 hour
at room temperature, membranes were incubated with primary antibody on a
rocker overnight at 4°C. Following a 1-hour incubation with secondary antibody
(GE Healthcare, Buckinghamshire, UK), proteins were detected by
chemiluminescence.
5.3.5 Cell viability assays
Cells were treated with drugs alongside vehicle control (DMSO) in 96- or 384-
well plates over 48 or 72 hours. Cell growth and viability was measured with the
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sulforhodamine-B assay, performed as previously described (LaPointe et al.,
2005), or the Alamar Blue assay, as per manufacturer’s instructions (Life
Technologies, Carlsbad, CA). Cell viability was also measured by Annexin V and
PI staining on a BD CantoII 96w (BD Biosciences, San Jose, CA) flow cytometer,
and analyzed using FlowJo version 7.6.5 (TreeStar, Ashland, OR). For both
methods, cell viability was calculated relative to that of vehicle controls.
5.3.6 High-throughput combination drug screening & excess-over-Bliss additivism synergy calculations
High-throughput combinatorial drug screening was carried out as previously
described (Rotin et al., 2016b). The excess-over-Bliss additivism formula (Borisy
et al., 2003), which calculates the degree of cell killing unaccounted for by the
added cytotoxicities of each individual drug, was applied as previously described
(Rotin et al., 2016b).
5.3.7 Reactive oxygen species measurement
Intracellular ROS production in live drug-treated cells was measured by staining
cells with 10 µM carboxy-H2DCFDA (Molecular Probes/Life Technologies,
Eugene, OR) for 30 minutes at 37°C and 5% CO2 and subsequent staining with
propidium iodide. ROS production in stained live cells was detected with a BD
Fortessa LSR X20 (BD Biosciences, San Jose, CA) flow cytometer, and
calculated relative to the geometric mean fluorescence intensity of untreated live
cells.
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5.3.8 Mass spectrometry
Chromatographic separations were carried out on an Acquity UPLC BEH C18
(2.1 X 50 mm, 1.7 µm) column using ACQUITY UPLC II system. The mobile
phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B). A gradient starting at 95% solvent A going to 5% in 4.5
min., holding for 0.5 min., going back to 95% in 0.5 min. and equilibrating the
column for 1 min. was employed. A Waters Synapt G2S QTof mass
spectrometer equipped with an atmospheric pressure ionization source was used
for mass spectrometric analysis. MassLynx 4.1 was used for data analysis.
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5.4 Results
5.4.1 TEX and OCI-AML2 cell line sensitivity to erlotinib
To ascertain erlotinib sensitivity in the AML cell lines TEX and OCI-AML2, we
subjected these cells to treatment with increasing concentrations of erlotinib for
72 hours, and subsequently measured relative growth and viability using the
sulforhodamine-B (SRB) assay. The average erlotinib IC50s were 8.99µM and
15.61µM in TEX and OCI-AML2, respectively (Figure 5-1). These erlotinib IC50
values are significantly higher than clinically achievable concentrations
(Appendix 1) and far greater than the nanomolar-range IC50 values reported in
the NSCLC cell lines HCC827, HCC4006, HCC4011, H3255, and H292, using
the same assay (Gao et al., 2014).
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Figure 5-1: AML cell line sensitivity to erlotinib. (A) TEX and OCI-AML2 cells were treated with increasing concentrations of erlotinib for 72h. Growth and viability was measured using the SRB assay and calculated relative to vehicle-treated cells. Results depict average percent growth and viability ± SD from a representative experiment performed in triplicate. Data are representative of three independent experiments.
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5.4.2 A high-throughput combination chemical screen identifies erlotinib sensitizers in TEX and OCI-AML2 cells
With the goal of identifying synergistic combination candidates for erlotinib, we
screened this drug against three chemical libraries—the Microsource Discovery
Systems International Drug and Natural Product (“NatProd”) collections, and a
312-compound library from Sequoia Research Products—in TEX and OCI-AML2
cells. Erlotinib at its IC10 and IC25 was combined with increasing concentrations of
1,352 drugs for a total of 16,230 different assays in two cell lines. Treated cells
were incubated for 72 hours and relative growth and viability was measured with
the SRB assay. These viability data were then used to determine synergy based
on the excess-over-Bliss additivism (EOBA) formula (Borisy et al., 2003), which
calculates the difference between observed and predicted killing by a given drug
combination, with a greater positive difference indicating stronger synergy.
Compounds were plotted in order of increasing positive EOBA scores for each
drug library, in both cell lines (Figure 5-2). The most profoundly synergistic hits
from each library were individually validated using a broader range of
concentrations to more thoroughly evaluate synergy. Ethacridine lactate, a
poly(ADP-ribose) glycohydrolase (PARG) inhibitor (Bernardi et al., 1997;
Boulikas, 1990; Tavassoli et al., 1985) and abortifacient (Hou et al., 2010), was
validated as the top synergistic hit common to both TEX and OCI-AML2 cells,
generating EOBA scores of up to 0.79 in TEX and 0.69 in OCI-AML2 (Figure 5-3, top). Validation of two other screen hits, apigenin and berberine, are included
for comparison (Figure 5-3, bottom).
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Figure 5-2: Erlotinib sensitizers in TEX and OCI-AML2 cells. Erlotinib was screened against 1,352 drugs from three chemical libraries (Sequoia, International, and Natural Products A, B and C). Following a 72h incubation, EOBA synergy scores were calculated from relative growth and viability values determined by the SRB assay. Compounds were ranked in order of increasing positive EOBA score.
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Figure 5-3: Validation of synergistic hits. Synergistic hits (shown: ethacridine, apigenin, and berberine) were validated in TEX and OCI-AML2 cells using broader concentration ranges. Cells were combination-treated for 72h and percent growth and viability was measured with the SRB assay (graphs). Synergy was calculated according to EOBA criteria (tables), with EOBA values >0.1 (lightest grey) denoting a synergistic combination, while values >0.5 (darkest grey) denoting a profoundly synergistic combination. Results depict mean percent growth and viability ± SD (graphs) or mean EOBA scores (tables) from a single experiment performed in triplicate.
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5.4.3 The erlotinib-ethacridine combination synergizes in primary AML cells and other AML cell lines
To determine whether the observed synergy between erlotinib and ethacridine
extended beyond TEX and OCI-AML2 cells, we combination-treated U937 and
K562 leukemia cells over 72 hours and measured relative growth and viability
with the SRB assay. Combination erlotinib-ethacridine treatment yielded EOBA
scores in the profoundly synergistic range (>0.50) at concentrations of erlotinib
that were clinically relevant (Figure 5-4A). We also examined this combination in
5 primary AML samples and 2 samples of normal hematopoietic cells derived
from healthy volunteers donating G-CSF-mobilized peripheral blood stem cells
for allogeneic transplant (denoted as “Normal”) (see Table 5-1 for patient
demographics). For this assay, cells were incubated with increasing
concentrations of erlotinib, ethacridine, or both in combination for 48 hours.
Viability relative to untreated (vehicle) controls was measured with Annexin V
and propidium iodide (PI) staining on flow cytometry (Figure 5-4B). None of the
primary samples or normals, with the exception of AML130183, were sensitive to
single-agent erlotinib treatment. Single-agent ethacridine sensitivity was variable
amongst primary AML cells, however all were more sensitive to ethacridine killing
compared to normals. The erlotinib-ethacridine combination was synergistic in all
5 primary AML samples, most strikingly in AML130208 and AML130237, which
yielded EOBA scores in the >0.30 range (Figure 5-4C). In contrast, the
combination was not synergistic in either of the normals; the highest EOBA score
calculated was 0.11, which was only observed in one pairing (Figure 5-4C).
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Figure 5-4: Erlotinib and ethacridine synergize in additional AML cell lines and
primary AML blasts. U937, K562, primary AML blasts, and PBSCs were combination-treated with erlotinib and ethacridine for 72h (A) or 48h (B). Viability was determined by the SRB assay (A) or Annexin V and PI staining (B). Graphs depict (A) mean percent growth and viability or (B) mean percent viability ± SD from a single experiment performed in triplicate. Tables (A, C) represent mean EOBA values.
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Table 5-1: AML patient demographics.
Patient Demographics Sample ID Diagnosis Age at
Diagnosis Gender Cytogenetics Molecular
130177 AML, M4Eo 62 Male 46,XY,inv(16)(p13.1q22)[8]/46,XY[2] CBFB-MYH11 +,KIT-
130183 AML 69 Male 46,XY[20] NPM1+, FLT3-ITD+, FLT3-TKD-
130185 AML 69 Male
45,XY,der(1)t(1;?21)(p13;q11.2),del(5)(q13q33),-7,+8,-10, add(12)(p11.2),-15,add(19)(q13.3),-21,+2mar[5]/45,XY,der(1)t(1;?21)(p13;q11.2),-5,-7,+8,-10,add(12)(p11.2), -15,add(19)(q13.3),-21,+3mar[5]
Not done
130208 AML, M1 21 Male 46,XY[20] NPM1+, FLT3-ITD+, FLT3-TKD-
130237 AML 66 Male 46,XY,inv(3)(q21q26.2)[3]/45,idem,-7[8] Not done
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5.4.4 Combining erlotinib and ethacridine generates lethal levels of reactive oxygen species
To investigate the mechanism of synergistic cytotoxicity by the erlotinib-
ethacridine combination, we quantified intracellular reactive oxygen species
(ROS) production in TEX and OCI-AML2 cells following a 24-hour incubation with
erlotinib, ethacridine, and both drugs in combination. ROS was measured using
carboxy-H2DCFDA staining on flow cytometry, with dead cell exclusion by PI
staining. Fold change in ROS was calculated relative to the geometric mean
fluorescence intensity of carboxy-H2DCFDA (FITC) staining in vehicle-treated
cells. We observed a striking increase in ROS production: up to a 2-fold increase
in TEX and a 4-fold increase in OCI-AML2 cells, respectively (Figure 5-5A). ROS
induction appeared functionally important for combination-induced synergistic
cytotoxicity, as pre-treatment with the antioxidant α-tocopherol significantly
increased viability by Annexin V and PI staining following a 48-hour treatment
with the erlotinib-ethacridine combination (Figure 5-5B). Because of the reported activity of ethacridine against PARG, we sought to
determine whether gallotannin, another known PARG inhibitor (Aoki et al., 1993;
Tsai et al., 1992), synergized with erlotinib. Using Annexin V and PI staining on
flow cytometry, we observed profound synergistic activity between erlotinib and
gallotannin in TEX cells (Figure 5-5C).
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Figure 5-5: Combination erlotinib-ethacridine treatment induces lethal
intracellular ROS production. (A) TEX and OCI-AML2 cells were combination-treated with erlotinib and ethacridine for 24h. Intracellular ROS was measured with carboxy-H2DCFDA staining and dead cells were excluded by PI staining. Fold increase in ROS production was calculated relative to the geometric mean fluorescence intensity (GMFI) of vehicle-treated cells. H2O2 was included as a positive intracellular ROS control. Results depict mean fold increase in GMFI ± SD from a representative experiment performed in triplicate. Data are representative of two independent experiments. (B) TEX and OCI-AML2 cells were treated with 3mM α-tocopherol for 24h, then treated with the erlotinib-ethacridine combination for the following 48h (with α-tocopherol maintained at 2.4mM). Viability was measured with Annexin V and PI staining on flow cytometry, and calculated relative to respective (± α-tocopherol) controls. Data represent mean percent viability ± SD from an experiment performed in triplicate. These data are representative of two independent experiments. (C) TEX cells were combination-treated with erlotinib and gallotannin for 48h. Viability was determined by Annexin V and PI staining. Graph depicts mean percent
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viability ± SD from a single experiment performed in triplicate. Table represents mean EOBA values from the same experiment. Data are representative of at least three independent experiments.
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5.4.5 Ethacridine synergizes with EGFR-targeting kinase inhibitors
Given that erlotinib has been proposed to inhibit other kinases in its activity
against AML cells (Boehrer et al., 2008a; Boehrer et al., 2011; Weber et al.,
2012), we sought to determine whether off-target kinase inhibition by erlotinib
was responsible for its synergy with ethacridine. We therefore screened
ethacridine against an in-house kinase inhibitor library, comprising 480 kinase
inhibitors with a broad range of kinase targets and varying degrees of kinase
specificity. OCI-AML2 cells were incubated with ethacridine along with two
different concentrations of each kinase inhibitor for 72 hours, for a total of 2,883
different assays. Relative growth and viability was subsequently measured with
the SRB assay and synergy scores were again calculated with the EOBA formula
and plotted in order of increasing synergy score (Figure 5-6A). Erlotinib was the
second most synergistic hit, which served as further validation for our initial
combination screen. Interestingly, 4 of the 5 top synergistic hits (GW583340,
erlotinib, GW2974, and WHI-P 154) were reported EGFR inhibitors. Furthermore,
the clinically approved EGFR inhibitors gefitinib and lapatinib were also identified
as synergistic hits, with EOBA values reaching 0.18 and 0.14, respectively.
5.4.6 TEX and OCI-AML2 cell lines do not express EGFR
The observed synergy between ethacridine and multiple EGFR inhibitors
prompted us to investigate whether erlotinib might inhibit EGFR to synergize with
ethacridine in TEX and OCI-AML2 cells. We evaluated total EGFR expression in
these cell lines by immunoblot and were unable to detect expression of this
kinase in either cell line (Figure 5-6B). Likewise, EGFR expression was absent
in K562 and U937 cells, which also demonstrated erlotinib-ethacridine synergy
(Figure 5-4A). Thus, these chemical EGFR inhibitors likely synergize with
ethacridine via a common, EGFR-independent mechanism.
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5.4.7 Erlotinib potentiates ethacridine accumulation in TEX and OCI-AML2 cells.
One property common to several small molecule EGFR inhibitors is their potent
inhibition of ATP-binding cassette (ABC) transporter efflux activity, and thus their
ability to enhance the cellular accumulation of—and sensitivity to—their
substrates. We therefore investigated whether erlotinib potentiates ethacridine
accumulation in AML cells. We treated TEX and OCI-AML2 cells with 5 µM
ethacridine in the presence and absence of erlotinib for one hour. Cells were
lysed and contents were quantified by LC-MS/MS. Ethacridine concentrations
increased nearly 2-fold in the presence of as little as 1 µM erlotinib in both cell
lines (Figure 5-6C). In contrast, imatinib (which does not synergize with
ethacridine (Figure 5-7)) did not potentiate ethacridine accumulation in either cell
line (Figure 5-6C).
5.4.8 High-dose ethacridine treatment mimics ROS production observed from the erlotinib-ethacridine combination.
To determine whether erlotinib-mediated intracellular ethacridine accumulation
could be responsible for excessive ROS production, we treated TEX and OCI-
AML2 cells with high concentrations of ethacridine and measured changes in
ROS production. Cells were incubated for 24 hours with 15 µM and 20 µM
ethacridine to mimic the increase in ethacridine accumulation observed in the
presence of 3 µM erlotinib, alongside combination-treated cells. ROS production
in live cells was measured by carboxy-H2DCFDA and PI staining on flow
cytometry, and calculated relative to vehicle-treated controls. High-dose
ethacridine increased ROS production more than 2-fold in TEX cells, and greater
than 3-fold in OCI-AML2 cells (Figure 5-6D), which was comparable to the
increase in ROS production observed with combination erlotinib-ethacridine
treatment (Figure 5-5A). These findings suggest that erlotinib-mediated
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ethacridine accumulation is the mechanism that explains synergistic cell death
caused by excessive ROS production.
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Figure 5-6: Erlotinib enhances ethacridine accumulation in TEX and OCI-AML2
cells. (A) Ethacridine was screened against a 480-compound kinase inhibitor library in OCI-AML2 cells. Cells were treated for 72h. Growth and viability was measured with the SRB assay and synergy scores were calculated with the EOBA formula. Compounds were ranked in order of increasing positive synergy score. (B) EGFR expression in a panel of AML cell lines was detected by immunoblotting, with the MDA-468 cell line included as an EGFR-positive control. (C) TEX and OCI-AML2 cells were treated with ethacridine in the presence and absence of erlotinib for 1h. Cells were lysed and analyzed by LC-MS. Imatinib was included as a negative (non-synergizing) control. Data depict mean ethacridine accumulation ± SD from an experiment performed in triplicate. Data are representative of three (TEX) or two (OCI-AML2) independent experiments. (D) TEX and
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OCI-AML2 cells were treated with high-dose ethacridine or erlotinib and ethacridine in combination for 24h. Intracellular ROS was measured with carboxy-H2DCFDA staining (with PI exclusion of dead cells) and calculated relative to GMFI of vehicle-treated cells. Data depict mean fold increase in GMFI ± SD from a representative experiment performed in triplicate, and are representative of two independent experiments.
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Figure 5-7: Imatinib does not synergize with ethacridine in TEX and OCI-AML2
cells. TEX and OCI-AML2 cells were treated with increasing concentrations of imatinib and ethacridine alone and in combination. Relative growth and viability following a 72h incubation was measured with the Alamar Blue assay. Synergy was calculated with the EOBA formula. Graphs depict mean percent growth and viability ± SD from an experiment performed in triplicate. Tables depict mean EOBA scores from the same experiment. Data are representative of three independent experiments.
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5.5 Discussion
The impressive EGFR-independent antileukemic activity of erlotinib reported in
preclinical studies has not translated to significant responses in the clinical
setting, with two clinical trials reporting negligible or no single-agent erlotinib
activity against AML. The first, a Phase I/II clinical trial evaluating erlotinib use in
30 high-risk MDS and AML patients who had previously failed azacytidine
treatment, reported no responses to erlotinib treatment in those with AML (n=12)
(Thepot et al., 2014). The second study, which assessed erlotinib treatment in
nine patients with treatment-naïve AML and two patients with relapsed or
refractory AML, reported disease progression in all 11 patients, however two
patients demonstrated a significant decrease in peripheral blasts (Sayar et al.,
2015). Failure of erlotinib as a single-agent therapeutic strategy for this disease
in clinical trials has prompted interest in the investigation of this tyrosine kinase
inhibitor (TKI) as a combination candidate for AML therapy.
With the goal of identifying novel erlotinib combination candidates, we screened
this TKI against several drug libraries in two non-EGFR expressing AML cell
lines, which had erlotinib sensitivities in the micromolar range. The PARG
inhibitor ethacridine lactate was the most synergistic hit common to both TEX
and OCI-AML2 cells. We determined that synergy was due to erlotinib
potentiation of intracellular ethacridine accumulation, as evidenced by the fact
that high-dose ethacridine treatment recapitulated the lethal increase in ROS
production observed following combination erlotinib-ethacridine treatment.
Erlotinib has been previously shown to synergize with other drugs in AML cell
lines and primary AML blasts: erlotinib potentiated ATRA- and vitamin D3-
mediated differentiation of HL60 cells, an effect attributed to erlotinib’s inhibition
of SRC and p38MAPK phosphorylation (Lainey et al., 2013b). Erlotinib and
gefitinib, another EGFR inhibitor, were also found to synergistically block cell
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proliferation and induce apoptosis when combined with the hypomethylating
agent azacytidine, owing to erlotinib- and gefitinib-mediated potentiation of
intracellular azacytidine accumulation (Lainey et al., 2013a). Finally, erlotinib and
gefitinib were shown to sensitize KG-1 AML cells to antineoplastic agents such
as etoposide and doxorubicin by enhancing their accumulation through
simultaneous inhibition of P-glycoprotein (P-gp), multidrug resistance protein 1
(MRP1), and breast cancer resistance protein (BCRP)-mediated drug efflux
activity (Lainey et al., 2012). EGFR inhibitors have been extensively shown to
enhance the cytotoxicity of antineoplastic drugs through inhibition of ABC
transporter-mediated drug efflux (Dai et al., 2008; Kuang et al., 2010; Shi et al.,
2007).
While our study did not address the mechanism of intracellular ethacridine
uptake or the mechanism by which it is extruded from AML cells, the fact that the
concentrations of erlotinib required for synergy with ethacridine are in line with
those required for erlotinib synergy with other antineoplastic agents (1 to 10 µM
range (Lainey et al., 2012; Lainey et al., 2013a)) would strongly suggest that
erlotinib inhibition of one or more ABC transporters accounts for lethal levels of
ethacridine accumulation in TEX and OCI-AML2 cells.
Given the capacity of erlotinib to promote the accumulation of other drugs, it is
surprising that the combination chemical screen against erlotinib did not yield a
greater number of validated synergistic hits common to both TEX and OCI-AML2
cells. This observation may suggest that in these AML cell lines, erlotinib may
inhibit a more restricted number of transporters. This observation may also
highlight the potential therapeutic relevance of PARG inhibition in AML. PARG
hydrolyzes poly(ADP-ribose) (PAR) polymers, which are synthesized by
poly(ADP-ribose) polymerases (PARPs) in response to DNA damage and other
cellular stresses. PARG therefore quenches PARP-elicited signals, which drive
processes such as DNA repair or cell death.
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With the exception of our own previous report describing the synergistic
cytotoxicity between chemical PARG inhibitors and the Bruton’s tyrosine kinase
inhibitor ibrutinib (Rotin et al., 2016b), the role of PARG inhibition in AML has not
been investigated. PARG inhibition has been found to sensitize other cancer cell
lines to DNA-damaging and oxidative stress-inducing agents by failing to reduce
cellular PAR levels following PARP activation. Excessive PAR accumulation
induces cell death by necrosis (NAD+ is a substrate for PAR synthesis, thus
cellular ATP stores become depleted), or parthanatos (excess PAR triggers
nuclear translocation of apoptosis-inducing factor). We therefore hypothesize that
erlotinib-mediated ethacridine accumulation induces lethal levels of PAR
accumulation due to profound PARG inhibition.
In summary, we have identified the PARG inhibitor ethacridine as a novel
combination candidate for erlotinib in AML. Erlotinib synergizes with ethacridine
by potentiating its intracellular accumulation. The impact of PARG inhibition as a
therapeutic strategy in AML warrants further investigation.
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Chapter 6: General Discussion & Conclusion
135
6.1 Discussion
The goal of this project was to further elucidate the role of BTK as a therapeutic
target in AML, through the identification of drugs that sensitize AML cells to killing
by ibrutinib. We determined that both ethacridine lactate and daunorubicin
synergized with ibrutinib, however neither synergistic interaction was dependent
upon ibrutinib-mediated BTK inhibition. Further work investigating the striking
synergy between ethacridine and the kinase inhibitor erlotinib, a known inhibitor
of ATP-binding cassette (ABC) transporter-mediated drug efflux, shed light on
the possibility that ibrutinib also likely synergizes with ethacridine via this
mechanism. The mechanism by which ibrutinib and daunorubicin synergize is not
yet known.
6.1.1 BTK-independent anti-leukemic activity of ibrutinib
In demonstrating that ethacridine or daunorubicin treatment of BTK-knockdown
AML cell lines does not recapitulate the synergy observed when these drugs are
combined with ibrutinib, and that ibrutinib and ethacridine synergized in cells
lacking BTK protein expression, we provided evidence to suggest that ibrutinib
has anti-AML activity that extends beyond BTK inhibition.
6.1.1.1 Ibrutinib potentiates ethacridine accumulation
Given that ibrutinib has well-known inhibitory activity against other kinases such
as those from the TEC and SRC families (Dubovsky et al., 2013; Honigberg et
al., 2010), and because the ibrutinib concentrations at which synergy was
observed were significantly greater than those required for in vitro BTK inhibition
(Honigberg et al., 2010), we postulated that ibrutinib was inhibiting additional
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kinases, and that inhibition of the TEC family kinases in particular was
responsible for the observed synergy between this drug and ethacridine
(Chapter 3.5). However, determining that the synergy between erlotinib and
ethacridine (which was as striking as that of ibrutinib and ethacridine) was due to
erlotinib potentiation of intracellular ethacridine accumulation (Chapter 5.4) shed
light on the possibility that ibrutinib and ethacridine may synergize via this same
mechanism.
Many tyrosine kinase inhibitors (TKIs)—in particular those inhibiting ABL, EGFR,
and VEGFR kinases—have well-described activity as both substrates and, at
higher concentrations, inhibitors of ATP-binding cassette (ABC) transporters
(Deng et al., 2014). At micromolar concentrations, erlotinib has been shown to
sensitize AML cells to chemotherapy agents by blocking P-gp, MRP1, and
BCRP-mediated drug efflux and thus increasing intracellular concentrations and
cytotoxicity of these drugs, which are substrates of these transporters (Lainey et
al., 2012). In line with these observations, we demonstrated that concentrations
as low as 1 µM erlotinib were sufficient to nearly double intracellular
concentrations of ethacridine in TEX and OCI-AML2 cells, which would suggest
that ethacridine is a substrate of one or more of these ABC transporters.
Moreover, treating AML cell lines with high-dose ethacridine recapitulated the
ROS increases observed in response to combination erlotinib-ethacridine
treatment, indicating that ethacridine accumulation is responsible for the
observed synergistic cytotoxicity.
Further supporting the possibility that the mechanism accounting for ibrutinib’s
synergy with ethacridine is the same as that of erlotinib’s synergy with
ethacridine, Zhang et al. (2014) demonstrated that ibrutinib, via MRP1 inhibition,
potently sensitized MRP1-overexpressing HL60 leukemia cells to the MRP1
substrate drugs vincristine and doxorubicin, which was accompanied by an
increase in intracellular chemotherapy concentrations. We therefore anticipate
that like erlotinib, ibrutinib is potentiating ethacridine accumulation to induce ROS
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and synergistic cell death. Indeed, preliminary mass spectrometry data
generated by our group (not shown) would suggest that this hypothesis is
correct: ibrutinib enhances intracellular ethacridine accumulation and intracellular
ROS induction following high-dose ethacridine treatment parallels that observed
in response to combination treatment.
6.1.1.2 Synergy between ibrutinib and daunorubicin is mediated by a mechanism unrelated to that of ibrutinib/erlotinib and ethacridine
Ibrutinib and daunorubicin very likely synergize via a mechanism that differs from
that of the TKI-ethacridine combination. Several lines of evidence point to this
conclusion. First, the patterns of synergy were notably different: synergy between
ibrutinib and daunorubicin was far less striking, with EOBA values reaching the
0.20-0.30 range, whereas ibrutinib-ethacridine combinations reached well
beyond 0.50 in the same cell lines, at the same ibrutinib concentrations.
Furthermore, although the ibrutinib-daunorubicin combination induced statistically
significant increases in ROS, this increase was modest relative the increase
observed following ibrutinib-ethacridine treatment. Finally, and perhaps most
importantly, ibrutinib was not found to potentiate TEX cell accumulation of
daunorubicin, a mechanism that was central to the TKI-ethacridine synergistic
mechanism. This finding is difficult to reconcile, because anthracyclines, which
include daunorubicin, are reported substrates for P-gp and MRP1 (Deng et al.,
2014). However, it is important to note that the technique used to measure drug
accumulation in the presence and absence of ibrutinib was different for both
drugs (radiolabeling of daunorubicin, versus mass spectrometric measurement of
ethacridine). Thus, these findings cannot be directly compared, however it is
likely that potentiation of daunorubicin accumulation is not a significant
contributor to ibrutinib-daunorubicin synergy.
Kinase inhibitor synergy with cytotoxic agents is a commonly reported
observation in in vitro preclinical studies (Eriksson et al., 2012; Landriscina et al.,
138
2010; Li et al., 2014; Pichot et al., 2009). Ibrutinib has been found to synergize
with the anthracycline doxorubicin in activated B-cell-like subtype of diffuse large
B-cell lymphoma (ABC-DLBCL) cells (Mathews Griner et al., 2014). The
hypothesized mechanism to explain this synergistic effect in ABC-DLBCL cells is
related to NFKB inhibition by ibrutinib: DNA damage induced by
chemotherapeutic agents stimulates NFKB activation, and ibrutinib blocks this
cytoprotective response (Mathews Griner et al., 2014). Likewise, NFKB lies
downstream of BTK signaling in FLT3-ITD negative AML cells, and ibrutinib
treatment downregulates this transcription factor (Oellerich et al., 2015), which in
turn leads to decreased expression of survival genes downstream of NFKB
(Rushworth et al., 2014). Thus, a similar NFKB-mediated mechanism may
underlie the observed synergistic cytotoxicity between ibrutinib and daunorubicin
in FLT3-ITD negative AML cells, such as OCI-AML2.
6.1.2 Anti-leukemic activity of ethacridine lactate
Mass spectrometry, intracellular ROS, and antioxidant rescue experiments
confirmed that intracellular ethacridine accumulation resulted in increased ROS
production, which was functionally important for the synergistic cell death
observed following combination erlotinib-ethacridine (and ultimately, ibrutinib-
ethacridine) treatment. However, our work did not address the mechanism
responsible for the lethal effects of ethacridine.
Ethacridine lactate has several reported cellular targets with potential
antineoplastic activity. Because we also observed synergy between erlotinib or
ibrutinib and gallotannin, another reported poly(ADP-ribose) glycohydrolase
(PARG) inhibitor, we reasoned that PARG inhibition was the relevant target of
ethacridine responsible for AML cell death. In the setting of PARP1 activation,
PARG inhibition causes poly(ADP-ribose) accumulation leading to cell death due
to apoptosis-inducing factor activation or necrosis. The finding that TKI-mediated
139
ethacridine accumulation was responsible for cell killing does not preclude PARG
inhibition as the relevant target; rather, increased intracellular ethacridine may
suggest more potent PARG inhibition.
However, given that our work did not address whether on-target PARG inhibition
was responsible for ethacridine cytotoxicity, other possible reported ethacridine
targets cannot be ruled out as the cause of this drug’s lethality. Ethacridine,
among other acridine compounds, impairs RNA polymerase 1 complex formation
by degrading the RP194 subunit, which inhibits ribosomal RNA transcription and
subsequently induces ribosomal stress, leading to p53 induction (Morgado-
Palacin et al., 2014). This mechanism of p53 induction is independent of DNA
damage (Morgado-Palacin et al., 2014). Preliminary work (not shown)
investigating ethacridine-mediated p53 induction as its cytotoxic mechanism in
TEX and OCI-AML2 cells does not rule out this possibility: ethacridine
concentrations similar to those used by Morgado-Palacin et al. (2014) induced
p53 expression by immunoblot. In line with our observation that ibrutinib
potentiates ethacridine accumulation, incubation with ibrutinib and ethacridine in
combination further induced p53 expression in both cell lines, while single-agent
ibrutinib treatment did not induce p53. Interestingly, p53 induction in both cell
lines was not accompanied by increased phosphorylation of histone γH2A.X, a
marker of DNA damage. Thus, ethacridine may exert its cytotoxic effect via
ribosomal stress-mediated p53 induction.
6.1.3 Clinical relevance of BTK-independent effects of ibrutinib
The findings of this work, and of that of Zhang et al. (2014) suggest that ibrutinib
may have a role in the reversal of multidrug resistance in AML. However, there
may be some limitations to exploiting ibrutinib for its activity against this particular
target in the clinical setting. First, the ibrutinib concentrations required for synergy
140
in our cell lines (4-8 µM) and those used by Zhang et al. (2014) (1-5 µM) are
greater than what is clinically achievable in patients: 420 mg/day orally resulted
in maximum steady-state concentrations of 100 ng/mL, or ~230 nM
(Sukbuntherng et al., 2013). However, the concentrations achieved in in vivo
studies carried out by our group and Zhang et al. (2014) did demonstrate a mild-
to-moderate sensitization effect by ibrutinib, which would imply that at clinically
relevant concentrations, ibrutinib has some effect on drug sensitization.
Another reason for the limited clinical utility of this particular application of
ibrutinib is that previous efforts to sensitize AML cells to chemotherapy agents
using drug efflux pump modulators have failed to improve AML outcomes in the
clinical setting. Leukemic stem cells and blasts have well-documented
expression of P-gp, MRP1 and BCRP, and other ABC transporter proteins, and
expression of these transporters (particularly P-gp) on leukemic cells is
correlated with chemotherapy resistance and poor patient prognosis (van der
Kolk et al., 2002). This observation prompted extensive clinical evaluation of P-
gp inhibitors as adjuncts to chemotherapy in AML: the addition of the second-
generation P-gp modulator valspodar to a chemotherapy regimen consisting of
mitoxantrone, etoposide, and cytarabine did not increase complete response
rates or overall survival in patients with relapsed/refractory AML or high-risk
myelodysplastic syndrome (Greenberg et al., 2004). Similarly, adding valspodar
to AML induction regimens with daunorubicin and cytarabine in treatment-naïve
patients over the age of 60 did not improve complete response, event-free
survival, disease-free survival, or overall survival rates regardless of P-gp
expression status (van der Holt et al., 2005). Finally, valspodar did not improve
complete remission, disease-free survival, or overall survival in treatment-naïve
AML patients under the age of 60 when added to cytarabine-daunorubicin-
etoposide induction regimens (Kolitz et al., 2010). The failure of valspodar to
improve treatment efficacy in AML may be related to the presence of
compensatory chemotherapy resistance mechanisms: BCRP and MRP1 also
mediate extrusion of daunorubicin, etoposide, and/or mitoxantrone (Shaffer et al.,
141
2012; van der Kolk et al., 2002). While ibrutinib may overcome some of these
compensatory chemotherapy resistance mechanisms by potentially inhibiting
multiple ABC transporters (as opposed to P-gp alone), transporter-mediated drug
efflux is not the only cause of treatment resistance in AML (Funato et al., 2004).
Thus, the use of ibrutinib as a drug efflux modulator may have limited efficacy
against treatment resistance in AML.
Finally, perhaps the most important barrier to clinical translation of ibrutinib for
this purpose is the potential for significant hematologic toxicity. As was
demonstrated in Chapter 3.4, while combination ibrutinib-ethacridine treatment
was preferentially cytotoxic to primary AML blasts, this combination was also
cytotoxic in a subset of peripheral blood stem cell samples from healthy
volunteers. Ibrutinib combinations would therefore require careful safety
evaluation prior to its investigation as a combination candidate in clinical trials.
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6.2 Conclusion
Using a high-throughput combination screening approach to identify drugs that
synergize with ibrutinib, we have demonstrated that ibrutinib has activity against
AML cells that is not limited to its inhibitory effect against BTK. While we did not
elucidate the exact mechanism by which ibrutinib synergizes with chemical
PARG inhibitors in our study, the observation that erlotinib synergizes with
ethacridine by enhancing intracellular ethacridine accumulation sheds light on the
strong possibility that ibrutinib relies on this same mechanism to synergize with
ethacridine. Furthermore, we have demonstrated a potential role for PARG
inhibition in AML. Both the mechanism by which ibrutinib mediates ethacridine
accumulation in AML cells, and the impact of PARG inhibition in AML, warrant
further study.
143
Chapter 7: Future Directions
144
7.1 Future Directions
7.1.1 Determining the mechanism of ethacridine accumulation by erlotinib and ibrutinib
7.1.1.1 ABC transporters
Given that erlotinib is known to inhibit drug efflux by P-gp, MRP1, and BCRP in
its synergy with chemotherapy agents (Lainey et al., 2012) and that both erlotinib
and ibrutinib potentiate ethacridine accumulation, it would be worthwhile to
determine whether ethacridine accumulation is mediated by the inhibition of one
or more of these transporters. To test this possibility, one could first assess
expression of P-gp, MRP1, and BCRP by immunoblot in all of the cell lines in
which ibrutinib and ethacridine were found to synergize (TEX, OCI-AML2, HL60,
K562, U937, Jurkat D1.1). One possible method for narrowing down which ABC
transporter(s) to investigate would be to compare expression of these
transporters in cell lines exhibiting synergy to those expressed in KG1a, a cell
line in which ibrutinib and ethacridine did not synergize: transporters common to
KG1a and the other cell lines would likely not be responsible for mediating
ethacridine accumulation.
After determining which transporters are exclusively expressed in synergizing cell
lines, one could then assess the capacity of ibrutinib and erlotinib to functionally
inhibit the relevant transporters by measuring substrate extrusion in the presence
or absence of erlotinib or ibrutinib treatment. This would be carried out using the
approach undertaken by Lainey et al. (2012): flow cytometry would be used to
measure intracellular accumulation of fluorescent dyes that are preferentially
extruded by a single ABC transporter in the presence or absence of TKI
treatment. Rhodamine 123 and 3,3-diethyloxacarbocyanine iodide would be used
to assess P-gp, Hoechst 33342 would be used to assess BCRP, and calcein and
5-(and-6)-carboxy-2',7' dichlorofluorescein diacetate would be used to assess
145
MRP function.
An alternate or complementary method for assessing whether ethacridine
accumulation is caused by erlotinib or ibrutinib-mediated inhibition of these ABC
transporters would be to assess whether substitution of erlotinib/ibrutinib with
specific chemical ABC transporter inhibitors alone or in combination could
recapitulate the synergy observed with ethacridine. Cyclosporine A (P-gp
inhibitor), KO-143 (BCRP inhibitor), verapamil (P-gp and BCRP inhibitor), and
MK-571 (MRP family inhibitor) could all be used alone or in combination with
ethacridine in the synergizing AML cell lines to confirm whether inhibition of one
or more of these transporters is responsible for ethacridine accumulation by
erlotinib/ibrutinib.
To further confirm whether erlotinib/ibrutinib inhibits one or more ABC
transporters to potentiate ethacridine accumulation, one could carry out lentiviral-
mediated shRNA knockdown of ABC transporter genes in AML cell lines.
Enhanced sensitization of these cells to ethacridine, relative to parental cells,
would provide further confirmation that TKI-mediated ABC transporter efflux
inhibition is the mechanism by which these drugs synergize with ethacridine.
7.1.2 Determining the relevant target of ethacridine
7.1.2.1 PARG inhibition
To determine whether PARG inhibition is the mechanism by which ethacridine is
lethal to AML cells, one could first assess the effects on cell proliferation and
viability following shRNA knockdown of PARG in TEX and OCI-AML2 cells.
Increased PAR accumulation accompanied by reduced proliferation and viability
in clones where complete knockdown is achieved would suggest that PARG
146
activity is essential for AML cell survival, and thus a possible lethal target of
ethacridine.
One could further investigate the impact of PARG inhibition in AML cell lines by
measuring resultant PAR accumulation by immunoblotting following combination
TKI-ethacridine treatment, and then measuring nuclear translocation of
apoptosis-inducing factor (AIF) by subcellular fractionation and subsequent
immunoblotting. PAR accumulation—due to excess PARP1 activation or inhibited
PARG activity—is necessary for nuclear translocation of apoptosis-inducing
factor (AIF) from the mitochondria, which triggers nuclear condensation and
subsequent cell death (Yu et al., 2006; Yu et al., 2002; Zhou et al., 2011). Thus,
the observation that increased PAR accumulation leads to nuclear AIF
translocation in response to combination TKI-ethacridine treatment would further
support PARG inhibition as a relevant mechanism of ethacridine-mediated cell
death.
Finally, PARG inhibition could be further ruled in or out as the lethal target of
ethacridine by combination TKI-ethacridine treatment of PARP1-knockdown TEX
and OCI-AML2 cells: reduced susceptibility of PARP1 knockdown cells to
ibrutinib/erlotinib-ethacridine-induced synergistic cytotoxicity would favour PARG
inhibition as the relevant target of ethacridine, as PARP1 inhibition impairs PAR
polymer synthesis, thereby reducing the toxicity associated with PARG inhibition.
Alternatively, one could overexpress PARG in synergizing cell lines and treat
these cells with the TKI-ethacridine combination: reduced synergistic cytotoxicity
relative to parental cell lines would also implicate PARG as the relevant target of
ethacridine.
7.1.2.2 p53 induction and the ribosomal stress pathway
147
Given our preliminary data suggesting that ethacridine treatment induces p53
expression in TEX and OCI-AML2 cells, and a previous report demonstrating the
capacity of acridine compounds (including ethacridine) to induce p53 activation
via the ribosomal stress pathway (Morgado-Palacin et al., 2014), it would be
worthwhile to investigate the potential involvement of this pathway in ethacridine-
mediated AML cell death.
One could first determine whether single-agent ethacridine induces nucleolar
disruption—and whether combination ibrutinib/erlotinib-ethacridine further
potentiates nucleolar disruption—in TEX and OCI-AML2 cells by anti-
nucleophosmin staining and immunofluorescence microscopy: relocalization of
the nucleolar protein nucleophosmin to the nucleoplasm is one marker of
nucleolar disruption.
If ethacridine (or the TKI-ethacridine combination) is found to induce nucleolar
disruption in TEX and OCI-AML2 cells, one could then investigate whether this
effect of ethacridine is linked to its induction of p53: ribosomal stress causes
formation of the preribosomal complex RPL11/RPL5/5SrRNA, which binds
MDM2 and thus blocks MDM2-mediated ubiquitination of p53 (Horn & Vousden,
2008). Morgado-Palacin et al. (2014) demonstrated that the acridine compound
CID-765471 caused p53 activation by inducing RPL11 binding to MDM2. This
group further demonstrated that knockdown of RPL11 abrogated p53 induction
following CID-765471 treatment. To determine whether ethacridine induces p53
by this same mechanism, one could perform an immunoprecipitation assay to
determine whether ethacridine and/or TKI-ethacridine treatment induces RPL11-
MDM2 binding. Furthermore, reduced p53 induction by immunoblot following
ethacridine treatment of RPL11-knockdown TEX and OCI-AML2 cells (relative to
p53 induction in shRNA control TEX and OCI-AML2 cells) would also support
ribosomal stress pathway-mediated induction of p53 as a mechanism of
ethacridine toxicity.
148
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Appendix 1
Clinically achievable concentrations of tyrosine kinase inhibitors
Drug Dose (oral)
Approximate steady-state
plasma concentration
Reference
Ibrutinib 840mg/d 450nM (peak) Byrd et al. (2013)
Imatinib 400mg/d 5.3µM (peak) 2.4µM (trough) Peng et al. (2005)
Dasatinib 140mg/d (70mg BID) 140nM (peak) 20nM (trough) Demetri et al. (2009)
Erlotinib 150mg/d 3.1µM (trough) Hidalgo et al. (2001)
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