Background & Introduction Each year approximately 220,000 people are diagnosed with non-small cell lung cancer (NSCLC) in the US alone. The LKB1 (also known as STK11) tumor suppressor is mutationally inactivated in ~30% of sporadic non-small cell lung adenocarcinomas, however there are no effective therapies for lung tumors harboring these mutations. LKB1 functions as a master regulator of cell growth, metabolism and mitochondrial homeostasis through the adenosine monophosphate activated kinase (AMPK) and the Unc-51 like kinase 1 (ULK1) pathways. Work in the Shackelford lab has identified the LKB1 signaling pathway to be critical for maintaining cellular energy balance and mitochondrial turnover known as mitophagy in both normal and lung tumor cells. Importantly, LKB1 mutant cells unable to appropriately sense metabolic stress and are selectively sensitive to energy stress inducing compounds such as phenformin. Phenformin is a mitochondrial inhibitor that induces energy stress and cell death in LKB1 mutant lung tumor cells therefore we wanted to explore the possibility that other highly potent mitochondrial inhibitors may selectively kill LKB1 mutant lung tumors. In an effort to identify selective and highly effective new therapies for the treatment of LKB1 mutant lung cancer, we investigated FDA approved mitochondrial inhibitors to be repurposed for use as anti-cancer agents.

Conclusion & Discussion: Targeted therapeutics have made significant advances in subsets of NSCLC bearing activated oncogenic targets. Currently there are limited options for LKB1-mutant tumors, but here we illustrate the hypersensitivity of LKB1-defective cells to metabolic and mitochondrial stress and test the therapeutic use of a mitochondrial inhibitor MI007 on LKB1 mutant human and mouse lung tumor lines and MEFs. MI007 selective and efficiently killed LKB1 mutant lung tumor cell lines at low, clinically relevant doses. Our results suggest that MI007 and future mitochondrial inhibitors identified in our screen may have clinical benefit in treating LKB1 and KRAS mutant lung tumors in patients. and that Grp78 may have potential be developed as a clinical biomarker to measure of mitochondrial based therapies. Future Directions: We plan to assess MI007 on a larger panel of human NSCLC tumor cell lines and begin performing preclinical studies using GEMMs of lung cancer. Development of biomarkers P-AMPK and Grp78 analyzed in this study will help to guide future in vivo pre-clinical studies.

Figure 1. Compound MI007 significantly decreases membrane potential and cell viability at low doses and in an LKB1 specific manner. (A) Measurement of MMP (ΔΨ) following treatment with mitochondrial inhibitors (MI’s) or ROS inducers (RI’s) as indicated. IC50’s of MI007 determined using KrasMUT or KrasMUT;Lkb1-/- mouse lung tumors lines in (B) and human A549 isogenic NSCLC lines in (C). IC50’s were calculated after treatment with varying doses of MI007. Viability measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Viability normalized as a percentage of no treatment (NT) control. (D). KW634 and KW821 cell lines were plated in clear-bottom 96-well plates and Red:Green ratio of JC-1 Dye was measured as described in Figure 1.B. (E). Viability was measured using the Caspase-Glo 3/7 Assay (Promega) following 6hrs of treatment with 0.5% DMSO, MI007 or phenformin. Samples were ran in triplicate. Significance was calculated using student’s t-test.

Figure 2. Biomarker analysis of K-Ras mutant, Lkb1-/- MEFs and Kras mutant lung tumors following treatment with MI007 or phenformin.

Immunoblots from whole cell lysates Kras mutant (KrasMUT), Kras mutant + Lkb1 null (Lkb1-/-;KrasMUT) wild MEFs in (A) and KrasMUT lung tumors in (B). Cells were not treated (NT) or treated with MI007 for 24 hours. Lysates were blotted and probed with antibodies against caspase 3 (CC3), a marker of apoptosis, Grp78, a marker of mitochondrial stress, phospho-AMPK-threonine 172 (p-AMPK thr172) a marker of cellular energy stress or B-actin as a loading control.

LKB1 is the 3rd most frequently mutated gene in lung adenocarcinoma

Objectives: 1) To use a high throughput screen measuring tumor cell mitochondrial membrane potential (MMP) and apoptosis to identify mitochondria inhibitors with anti-cancer properties; 2) perform functional assays on lead candidate compounds and 3) assess potential biomarkers to guide pre-clinical studies.

Results:

Summary of Results: 1) JC-1 dye accurately measures loss of ΔΨ following treatment with MI’s. 2) MI007 found to have an IC50 of 500nM in human and mouse LKB1 mutant NSCLC tumor cell lines. 3) MI007 selectively induces both loss of ΔΨ and apoptosis in LKB1 mutant NSCLC tumor cell lines. 4) Low dose MI007 induces apoptosis, energy stress and mitochondrial stress in Lkb1-/-;KrasMUT ;Lkb1-/- MEFs and mouse lung tumor cell

lines. Additionally, MI007 induces mitochondrial stress in KrasMUT and wild type MEFs represented by elevated Grp78 protein levels.

Summary of the Experimental Design: 1)  Performed a literature search of studies assessing FDA approved compounds to disrupt mitochondrial function and MMP. 2)  Performed IC50’s on lead compounds using the cell titer glo assay. 3)  Performed a 96 well formatted H.T.S screen on MI’s that measured both MMP and apoptosis. MMP was measure using the

voltage sensitive dye JC-1 and cellular apoptosis was measure by the caspase 3/7 glo assay. 4)  Performed functional assays in cell culture by immunoblotting for markers of: i) apoptosis, ii) energy stress and iii)

mitochondrial stress. 5)  We will perform PK and PD analysis of lung tissue and lung tumors in mice followed by pre-clinical studies assessing MI’s as

anti-cancer agents targeting LKB1 mutant lung cancer.

Developing a H.T.S of mitochondrial inhibitors to be repurposed for the treatment of LKB1 mutant NSCLC

Mitochondria Disrupters / ROS Inducers

NT

2 mM Phe

n

10 uM

PL

20 uM

PL

50 uM

PL

500 n

M Oligo

mycin

1 uM O

ligomyci

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5 uM O

ligomyci

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25 uM

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Red:G

reen R

atio

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KrasMUT KrasMUT ;Lkb1-/-

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96 well format

Literature Search of FDA approved MI’s

Multiplex: Measure both MMP (ΔΨ) and apoptosis

IC50’s on lead compounds

Functional assays in cell culture

PK and PD analysis

Preclinical trials

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undergo apoptosis in response to metabolic stress

AMPKα

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Mouse lung tumors

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% C

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ility

MI007 (nM)

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0 2 4 60.0

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IC50 520nM430nM570nM

A549B A549WT A549KD

IC50 = 500nM Viab

ility

MI007 (nM)

A

B C

D E

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MEFs Mouse lung tumors

Alex Yang1, Robert J. McMickle1, David B. Shackelford1

1Pulmonary and Critical Care Medicine, David Geffen UCLA School of Medicine, Los Angeles, CA 90095 *Correspondence: DShackelford@mednet.ucla.edu,

Identification of personalized therapies for LKB1 mutant lung cancer using a high throughput screen of FDA approved compounds