Rationale for targeting the pre-B cell receptor signaling pathway in

Rationale for targeting the pre-B cell receptor signaling

pathway in acute lymphoblastic leukemia

Markus Müschen

Department of Laboratory Medicine,

University of California San Francisco, San Francisco, CA 94143, USA For correspondence: Markus Müschen Department of Laboratory Medicine University of California San Francisco 513 Parnassus Ave, San Francisco CA 94143 E-mail: [email protected] Running title: Targeting pre-BCR signaling in ALL

Abbreviations: ALL, acute lymphoblastic leukemia; BCR, B cell receptor; BTK, Bruton’s tyrosine kinase;

D, diversity; DLBCL, diffuse large B cell lymphoma; J, joining; μHC, immunoglobulin μ heavy chain;

PBML, primary mediastinal B cell lymphoma; V, variable

Blood First Edition Paper, prepublished online April 15, 2015; DOI 10.1182/blood-2015-01-567842

Copyright © 2015 American Society of Hematology

For personal use only.on February 21, 2018. by guest www.bloodjournal.orgFrom

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2

Abstract

Inhibitors of B cell receptor (BCR) and pre-BCR signaling were successfully introduced into patient care for

various subtypes of mature B cell lymphoma, e.g. Ibrutinib (BTK) and Idelalisib (PI3Kδ). Acute

lymphoblastic leukemia (ALL) typically originates from pre-B cells that critically depend on survival signals

emanating from a functional pre-BCR. However, whether patients with ALL benefit from treatment with

(pre-)BCR inhibitors has not been explored. Recent data suggest that the pre-BCR functions as tumor

suppressor in the majority of cases of human ALL. However, a distinct subset of human ALL is selectively

sensitive to pre-BCR antagonists.

Introduction

Acute lymphoblastic leukemia (ALL) represents the most common cancer in children, accounting for 26% of

malignancies diagnosed in ages 0-14. An estimated 2,670 children and 410 adolescents have been diagnosed

with ALL in the US in 20141. Outcomes for patients with pre-B ALL have substantially improved over the

past decades, reaching overall survival rates of 90% for children2 and 45% for adults3. Owing to its frequent

occurrence in children, ALL remains one of the leading causes of person-years of life lost in the US (362,000

person-years of life lost in 2010)1. In addition, ~20% of patients experience a bone marrow relapse after

initially successful treatment and more than 60% of these patients will die from their disease.

Cellular origins define oncogenic signaling requirements of ALL cells

With the goal to decrease the frequency of ALL relapse and reduce side-effects of cytotoxic therapy, recent

efforts have introduced targeted therapies that focus on specific vulnerabilities of ALL cells. The basic

premise for these studies has been that oncogenes in ALL will promote growth factor-independence by

delivering survival and proliferation signals that are normally provided by a favorable environment or as the

outcome of positive selection . ALL typically originates from pro- and pre-B cells during early B cell

development, i.e. cell types that critically depend on survival signals that emanate from an active cytokine

receptor (e.g. IL7R) and/or an active pre-BCR.

Recent studies revealed that a defined subset of ALL (termed Ph-like) is indeed driven by and particularly

dependent on oncogenic cytokine receptor signaling (e.g. through lesions of IL7R, CRLF2, JAK1, JAK2,

JAK3, EPOR, PDGFR)4-9. While genetically heterogeneous, this group of ALL has in common oncogenic

activation of STAT56,7 and a characteristic, STAT5-dependent, gene expression pattern8. ALL driven by

oncogenic cytokine receptor signaling accounts for ~10% of ALL cases in children and ~25% in young

adults and is associated with poor clinical outcome10-11. Importantly, ALL cells in this subgroup were found

to be sensitive to lesion-specific tyrosine kinase inhibitors (TKI) including dasatinib, ruxolitinib and

crizotinib10-11. While the concept of lesion-specific TKI-treatment of ALL driven by oncogenic cytokine




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receptor signaling is strongly supported by experimental and clinical evidence, targeted therapy of pre-BCR

signaling has not been explored in human ALL.

Pre-BCR checkpoint control during normal B cell development

During early B cell development, developing pre-B cells are selected for productive rearrangement of VHDJH

gene segments, which enables expression of a functional pre-BCR on the cell surface. Early pre-B cells are

programmed to die unless they are rescued by ‘tonic’ survival signals from a productively assembled pre-

BCR12. In mice, bone marrow progenitor cells produce approximately 10 million pre-B cells daily13, the vast

majority of which is eliminated at the pre-BCR checkpoint14. Hence, in mouse mutants unable to express a

pre-BCR, B cell development is blocked at the pro-B cell stage of differentiation15-16. The differentiation

block at the pre-BCR checkpoint, however, can be rescued by transgenic introduction of productively

rearranged VHDJH gene segments, encoding a functional immunoglobulin μ heavy chain (μHC)17-18. Two

models have been proposed for the initiation of pre-BCR signaling: Earlier work identified Galectin-1

expressed on the surface of bone marrow stroma cells as a potential ligand of the pre-BCR19. However, a

more recent study demonstrated autonomous, ligand-independent activation of the pre-BCR20. This is based

on the interaction between the surrogate light chain of the pre-BCR with a conserved asparagine (N)-linked

glycosylation site at position 46 (N46) in the first conserved domain of the μHC.

Tonic survival signals from the pre-BCR are transduced by the Igα and Igβ signaling chains21-22. Hence, B

cell development in mice lacking functional Igα21 and Igβ22 expression is arrested at the pre-BCR

checkpoint. Conditional ablation of tonic BCR signaling results in rapid B cell depletion23-24, highlighting the

critical importance of tonic pre-BCR and BCR signaling at the pre-BCR checkpoint and later stages of B cell

development. Interestingly, loss of tonic pre-BCR signaling can be rescued by activation of PI3K-AKT

signaling25, identifying PI3K-AKT as a central survival pathway downstream of the (pre-) BCR. Tonic pre-

BCR signaling involves constitutive activity of the proximal pre-BCR-associated SRC family kinases Lyn,

Fyn and Blk26 as well as the SYK and ZAP70 tyrosine kinases27, which then activate PI3K28-29. In addition

to its well established role in mature B cell survival23, recent work highlighted the role of PI3K signaling and

in particular the PI3K p110δ (PIK3CD) isoform for pre-BCR signaling and pre-B cell survival during early

B cell development30.

In addition to activation of a downstream signaling cascade, tonic pre-BCR signaling induces important

transcriptional changes that mediate positive selection and survival at the pre-BCR checkpoint. De novo

expression of the pre-BCR induces strong expression of BCL6, which promotes survival by BCL6-mediated

repression of CDKN2A and TP5331. In the absence of BCL6, positive selection at the pre-BCR checkpoint is

compromised and even pre-B cells that express a functional pre-BCR die from CDKN2A- and p53-mediated




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apoptosis32. Interestingly, BCL6 has the same function in human ALL cells and mediates survival and drug-

resistance33. At the pre-BCR checkpoint and in human ALL cells, BCL6 is opposed by BACH2, a

transcription factor that mediates negative pre-B cell selection and reverses BCL6-mediated survival signals

and drug-resistance34-35.

Targeting the BCR signaling pathway in mature B cell lymphoma

Unlike BCR signaling in mature B cell lymphoma, targeting of pre-BCR signaling has not been explored as a

treatment option for patients with ALL. The discovery that most subtypes of B cell lymphoma critically

depend on BCR signaling36-37 has led to the development of new targeting strategies that focus on BCR

signaling at the level of SRC kinases (Lyn, Fyn and Blk), SYK/ZAP70, BTK and PI3Kδ38-42.

The majority of mature B cell lymphoma subtypes depend on tonic or chronic active BCR signaling (~85%),

however, classical Hodgkin’s disease43, angioimmunoblastic lymphoma44, AIDS-related B cell lymphoma45

and primary mediastinal B cell lymphoma (PBML)46-47 together account for ~15% of human B cell

lymphoma that lack BCR function. In many cases, tumor clones show evidence of negative selection of a

functional BCR43-44. The classification of B cell lymphoma based on (i) chronic active, (ii) tonic and (iii)

lack of BCR signaling informed the development of new treatment strategies and has led to the successful

introduction of BTK (chronic active)48-50 and PI3Kδ (tonic BCR signaling)51 inhibitors into patient care.

Small molecule inhibition of BTK (e.g. Ibrutinib) and PI3Kδ (Idelalisib) in activated B cell-like (ABC)

diffuse large B cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) and mantle cell lymphoma

(MCL) has achieved major clinical successes in the treatment of these diseases48-51. It should be noted,

however, that clinical responses to inhibition of BTK (e.g. Ibrutinib) and PI3Kδ (Idelalisib) in patients are

not exclusively dependent on the role of BTK and PI3Kδ in BCR signaling. Since BTK and PI3K signaling

is also active in chemokine and integrin receptor signaling, inhibition of these pathways may contribute to

the effects observed in patients.

Tumor suppressor role of pre-BCR signaling in cytokine receptor/Stat5-dependent subsets of ALL

Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival

signals emanating from a functional (pre-)BCR. In apparent contradiction to this notion, a series of previous

studies suggested a role of pre-BCR signaling as tumor suppressor in ALL cells. For instance, in BCR-

ABL1-driven (Ph+) ALL, the BCR-ABL1 kinase activity mimics and supplants activity of the pre-BCR52-55.

While pre-BCR signaling in ALL cells is frequently inactivated through multiple defects, including deletion

of the surrogate light chain segment VPREB156, reconstitution of pre-BCR expression54 or its signaling chain

Igα55 causes cell death. In agreement with a tumor suppressor role of the pre-BCR, a number of studies




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identified defective expression of the pre-BCR signaling molecules BLNK (SLP-65)57 and BTK58 in human

ALL and demonstrated in genetic mouse models that Blnk and Btk cooperate in preventing malignant

transformation of pre-B cells59-60 (Table 1). Importantly, pre-BCR signaling via BLNK negatively regulates

STAT5 activity, which represents a central mediator of oncogenic cytokine receptor signaling in ALL cells61.

Thereby, BLNK binds to and inactivates JAK3 upstream of STAT561. Besides the pre-BCR and BLNK, also

transcription factors (e.g. PAX5 and EBF1) that drive expression of BLNK60 and other components of the

pre-BCR signaling pathway results in suppression of cytokine receptor/Stat5 signaling in mouse models of

ALL7 (Table 1). Besides PAX562, IKZF1 is a strong transcriptional activator of pre-BCR signaling63. While

genomic lesions of BLNK (~2% of ALL cases) are relatively rare, deletion of transcription factors that

promote pre-BCR expression and activity are frequent in ALL. Deletions of PAX5 occur in up to 25% of

ALL cases64 and IKZF1 deletions, resulting in expression of a dominant-negative protein, are found in >80%

of cases of Ph+ ALL65. Studying 830 ALL cases from four clinical trials, in the majority of cases, ALL cells

lacked pre-BCR signaling (pre-BCR¯ )54,66, which represents a functional equivalent to classical Hodgkin’s

lymphoma and PBML among mature B cell malignancies. Pre-BCR¯ tumors account for ~85% of ALL

cases: virtually all cases of BCR-ABL1, Ph-like (CRLF2, JAK, EPOR, PDGFR) and ETV6-RUNX1 and the

vast majority of MLL-rearranged and hyperdiploid ALL cases lack expression of a functional pre-BCR.

Among the COG P9906 and AALL0232 cohorts, in 101 of 486 ALL cases (21%) abnormal cytokine

receptor signaling was observed (Table 1). Oncogenic hyperactivation of cytokine receptor signaling was the

consequence of CRLF2 overexpression or rearrangement (n=59; 12%), FLT3 mutation (n=12; 2.5%), IL7R

mutation (n=9; 2%) or rearrangement of other cytokine receptors including PDGFRB (n=4; 1%) and EPOR

(n=1; 0.2%). In other cases, oncogenic cytokine receptor signaling was caused by JAK1, JAK2 or JAK3

mutation or rearrangement (n=35; 7%), ABL1 gene rearrangement (n=5; 1%) or SH2B3 mutation or deletion

(n=9; 2%). In 28 cases multiple lesions were detected. ALL clones that are driven by oncogenic cytokine

receptor signaling typically express constitutively active STAT5 (Table 1). Consistent with pre-BCR-

mediated attenuation of cytokine receptor/Stat5 signaling7,60,67, tumor clones are selected for defective

expression of the pre-BCR in cytokine receptor/Stat5-dependent subsets of ALL.

Identification of a pre-BCR-dependent subset of human ALL

In ~85% of human ALL cases, the dominant leukemic clones lack expression of a functional pre-BCR.

However, we and others recently identified a distinct subset of human ALL that is selected for expression

and activity of a functional pre-BCR54,66,68. In about 13.5% of human ALL cases (112 of 830 cases

studied)54,66, ALL cells exhibit tonic pre-BCR signaling (pre-BCR+), and were highly sensitive to inhibition

of SYK, SRC and BTK tyrosine kinases66,68 as well as PI3Kδ inhibition66. In analogy to mature B cell

lymphoma, patient-derived pre-BCR+ ALL cells responded to treatment with Ibrutinib and Idelalisib in vitro.

This group includes the ALL subset with TCF3-PBX1 rearrangement, which is selectively sensitive to




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Ibrutinib69. Treatment with the dual ABL1/BTK-SRC kinase inhibitor dasatinib induced leukemia regression

and extended overall survival of NOD/SCID mice that were transplanted with patient-derived pre-BCR+

ALL cells64. In up to 50% of pre-BCR+ ALL cells, the leukemia cells carry an activating lesion of the

homeodomain transcription factor PBX1, mostly through TCF3-PBX1 rearrangement66,68 and in some cases

through duplication of a fragment at 1q23 encompassing PBX1. PBX1 is critical for normal B

lymphopoiesis70 and ChIP-seq and gene expression analyses revealed that PBX1 directly binds to and

activates multiple components of the pre-BCR signaling pathway, including the surrogate light chain

segments VPREB1 and IGLL1 (λ5), CD79A, CD79B, BLNK and BTK66. Given its critical role during early B

cell development and ability to activate pre-BCR expression, it is conceivable that gain of function lesions of

PBX1 contribute to the development of pre-BCR+ ALL. Deletion of 6q21, encompassing PRDM1 (BLIMP1)

represents a second recurrent genetic lesion in pre-BCR+ ALL cells (Table 1). Of note, lesions of PRDM1 are

frequently observed in ABC-DLBCL71, which are -like pre-BCR+ ALL- sensitive to treatment with Ibrutinib

and Idelalisib. In human ALL, deletions of 6q21 are rare (~1%), likewise, TCF3-PBX1 rearrangements only

account for approximately 3-5% of all cases of human ALL (Table 1). For this reason, it is likely that other

recurrent genetic lesions that define pre-BCR+ ALL (~10-15%) still remain to be identified.

BCL6 as biomarker for pre-BCR-dependent cases of human ALL

B cell lymphoma cells express high levels of BCL6 in the context of tonic BCR signaling72. Likewise, tonic

pre-BCR signaling in ALL cells induces strong expression of BCL631. Consistent with induction of BCL6 by

tonic pre-BCR signaling, all pre-BCR+ ALL cases studied showed constitutive and pre-BCR-dependent

expression of BCL6, which was sensitive to small molecule inhibition of pre-BCR-signaling66 (Figure 1).

Unlike pre-BCR+ ALL, cytokine receptor-dependent subsets of ALL express active STAT5 (Table 1), a

negative regulator of BCL6. Illustrating the divergent biological behavior of pre-BCR+ ALL (e.g. TCF3-

PBX1) and cytokine receptor/Stat5-dependent ALL (e.g. BCR-ABL1), the dual ABL1/SRC-BTK inhibitor

dasatinib abolished BCL6 expression in pre-BCR+ ALL but induced de novo expression of BCL6 in Ph+

ALL cells (Figure 1). Since functional assays to measure tonic pre-BCR signaling in ALL patient samples

may not be practical for diagnostic purposes, immunostaining for BCL6 and immunoglobulin µ heavy chain

(µHC) expression may be a feasible alternative to rapidly identify patients that might benefit from treatment

with inhibitors of tonic pre-BCR signaling (e.g. SYK, SRC, BTK, PIK3δ). Besides BCL6/µHC double-

strainings, additional markers, including phospho-STAT5 are currently being tested with the goal to reliably

identify patients with pre-BCR+ ALL. Our current cytogenetic and phenotypic definition of pre-BCR+ ALL is

still incomplete and the identification of additional genetic and phenotypic characteristics will not only

further elucidate the biology of pre-BCR+ ALL but also help to identify patients that may benefit from

treatment strategies targeting pre-BCR signaling.




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Acknowledgements. I would like to thank Drs. Hassan Jumaa (Ulm, Germany), Ari Melnick (New York,

NY), Art Weiss (San Francisco, CA), Rudi W. Hendriks (Rotterdam, Netherlands), Louis M. Staudt

(Bethesda, MD) for critical discussions and encouragement. This work is supported by grants from NIH/NCI

through R01CA137060, R01CA139032, R01CA157644, R01CA169458, R01CA172558, the William

Lawrence and Blanche Hughes Foundation, the California Institute for Regenerative Medicine (CIRM; TR2-

01816), Leukaemia and Lymphoma Research, Cancer Research UK. M.M. is a Scholar of the Leukemia and

Lymphoma Society and a Senior Investigator of the Wellcome Trust.

M.M. developed the concept and wrote the manuscript.

M.M. has no conflicts of interest.

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Table 1: Characteristics of pre-BCR+ and pre-BCR- ALL subsets

Gene Function Type of defect Functional outcome Phenotype in disease model

Frequency in human ALL [%] Reference

Total Pre-BCR+ Pre-BCR-

CRLF2 Cytokine receptor Overexpression Rearrangement

Cytokine receptor signaling ↑ Propensity to ALL 12a 0 15 9-11

FLT3 Cytokine receptor Point mutation Cytokine receptor signaling ↑ Propensity to leukemia 2.5 0 3 9-11 IL7R Cytokine receptor Point mutation Cytokine receptor signaling ↑ Propensity to ALL 2 0 2 9-11 PDGFRB Cytokine receptor Rearrangement Cytokine receptor signaling ↑ Propensity to leukemia 1 0 1 9-11

EPOR Cytokine receptor Rearrangement Cytokine receptor signaling ↑ <1 0 <1 9-11 JAK1, JAK2, JAK3

Tyrosine kinase Point mutation Rearrangement

Cytokine receptor signaling ↑ Propensity to ALL 7a 0 9 9-11

ABL1 Tyrosine kinase BCR-ABL1

NUP214, ETV6, RCSD1

Cytokine receptor signaling ↑ ALL

Propensity to leukemia

3a-27b

1

0

0

4-35

2

9-11

9-11 SH2B3 Linker Deletion Cytokine receptor signaling ↑ pro-B cell block 2 0 2 9-11 IGHM Ig heavy chain Non-productive V(D)J No functional pre-BCR B cell autoimmunity 83 0 >80 52, 64 VPREB1 surrogate light chain Focal deletion No functional pre-BCR Pro-B cell block 15 0 20 54 CD79A (pre-) BCR signaling chain Transcriptional silencing No functional pre-BCR Pro-B cell block 80 0 >80 52, 53

CD79B (pre-) BCR signaling chain Transcriptional silencing No functional pre-BCR Pro-B cell block 80 0 >80 52, 53 SYK (pre-) BCR tyrosine kinase Aberrant splicing, mutation Pre-BCR signaling ↓ Pro-B cell block <10 0 <10 52 BLNK (pre-) BCR linker Aberrant splicing

Focal deletion Pre-B cell differentiation ↓ Cytokine receptor signaling ↑

Pre-B cell block Propensity to ALL

40 2

0 ~50 2

51, 55 62

BTK (pre-) BCR tyrosine kinase Aberrant splicing Pre-B cell differentiation ↓ Pre-B cell block Propensity to ALL

30 0 ~25 56-58

PAX5 Transcription factor Deletion, Point mutation Cytokine receptor signaling ↑ Propensity to ALL ~25 10 ~25 62 BACH2 Transcription factor Deletion 6q15 Pre-BCR signaling ↓ Propensity to ALL 20 0 ~25 32 IKZF1 Transcription factor Deletion, Point mutation Pre-BCR signaling ↓ Propensity to ALL 20-84c 0 ~25 62, 63

IKZF3 Transcription factor Deletion Pre-BCR signaling ↓ Propensity to ALL 2 0 2 62, 63 EBF1 Transcription factor Deletion, Point mutation Pre-BCR signaling ↓ Propensity to ALL 6 0 8 62, 63 PBX1 Transcription factor TCF3-PBX1 rearrangement

1q23 duplication Pre-BCR signaling ↑ Propensity to ALL 3a-5b

1 40 5

0 0

64, 66 64

PRDM1 Transcription factor Deletion 6q21 Pre-BCR signaling ↑ Plasma cell defect 1 5 0 64

Signaling Function Abnormality Functional outcome Phenotype in disease model

Frequency in human ALL [%] Reference

Total Pre-BCR+ Pre-BCR-

STAT5 Transcription factor Hyperphosphorylation Cytokine receptor signaling ↑ pro-B cell survival ↓ ~40 0 >50 9-11, 64 BCL6 Transcription factor Overexpression Pre-BCR signaling ↑ pre-B cell survival ↓ 13.5 >95 0 29-31, 64

Notes: aPediatric ALL, badult ALL, cPh+ ALL (BCR-ABL1)

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Figure legend

Figure 1: BCL6 as biomarker to identify pre-BCR- and pre-BCR+ cases of human ALL

A schematic summarizing normal cytokine-dependent survival signaling in pro-B cells (pre-BCR-) and pre-

BCR-dependent survival signaling in pre-B cells (a). SRC and BTK are targets of Dasatinib, BTK is a target

of ibrutinib and PI3Kδ is the target if idelalisib (a), which are currently being used in clinical trials for

patients with mature B cell lymphoma. Functional assays to measure tonic pre-BCR signaling in ALL patient

samples may not be practical for diagnostic purposes. Given that tonic pre-BCR signaling results in strong

expression of BCL631, immunostainings for BCL6 and immunoglobulin µ heavy chain (µHC) expression

represents a feasible alternative to rapidly identify patients that might benefit from treatment with inhibitors

of pre-BCR signaling (e.g. Ibrutinib, Idelalisib). Double stainings for BCL6 (brown; D8; Santa Cruz

Biotech) and µHC (red G20-127; BD Biosciences) were performed on paraffin-embedded sections from

bone marrow clots. Two patient derived ALL cases were studied and counterstained with H&E (100 x

magnification)66. One pre-BCR- ALL case (BCR-ABL1, left) and one pre-BCR+ ALL case (TCF3-PBX1,

right) is shown. The lower panel shows a Western blot analysis of BCL6 expression (D8; Santa Cruz

Biotech) in pre-BCR- (left) and pre-BCR+ (right) and ALL cells, with and without treatment with the dual

ABL1/SRC-BTK inhibitor dasatinib (25 nmol/l for 24h), using β-actin (ab8227, Abcam) as loading control.







doi:10.1182/blood-2015-01-567842Prepublished online April 15, 2015;

Markus Müschen lymphoblastic leukemiaRationale for targeting the pre-B cell receptor signaling pathway in acute

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