Author Manuscript Published OnlineFirst on September 25, 2018; DOI: 10.1158/2159-8290.CD-18-0277 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Title: Exploiting MCL-1 dependency with combination MEK + MCL-1 inhibitors leads to induction of apoptosis and tumor regression in KRAS mutant non-small cell lung cancer Varuna Nangia1*, Faria M. Siddiqui1*, Sean Caenepeel2, Daria Timonina1, Samantha J. Bilton1, Nicole Phan1, Maria Gomez-Caraballo1, Hannah L. Archibald1, Chendi Li1, Cameron Fraser3, Diamanda Rigas2, Kristof Vajda1, Lorin A. Ferris1, Michael Lanuti4, Cameron D. Wright4, Kevin A. Raskin5, Daniel P. Cahill6, John H. Shin6, Colleen Keyes7, Lecia V. Sequist8,9, Zofia Piotrowska8,9, Anna F. Farago8,9, Christopher G. Azzoli8,9, Justin F. Gainor8,9, Kristopher A. 3 10 2 1,9 2 Sarosiek , Sean P. Brown , Angela Coxon , Cyril H. Benes , Paul E. Hughes , Aaron N. Hata1,8,9 Affiliations: 1 Massachusetts General Hospital Cancer Center, Charlestown, MA 2 Department of Oncology Research, Amgen, Thousand Oaks, CA 3Department of Environmental Health, Harvard T. H. Chan School of Public Health, Boston, MA 4Department of Surgery, Massachusetts General Hospital, Boston, MA 5Department of Orthopaedics, Massachusetts General Hospital, Boston, MA 6Department of Neurosurgery, Massachusetts General Hospital, Boston, MA 7Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA 8Division of Hematology Oncology, Department of Medicine, Massachusetts General Hospital, Boston, MA 9Department of Medicine, Harvard Medical School, Boston, MA 10Department of Medicinal Chemistry, Amgen, Thousand Oaks, CA. *equal contribution Address correspondence to: Aaron N. Hata, Massachusetts General Hospital Cancer Center, 149 13th Street, Charlestown, MA 02129, USA. E-mail: [email protected]. Running Title: Combination MEK + MCL-1 inhibitors for KRAS mutant NSCLC Conflict of Interest: A.N.H. receives research support from Amgen, Novartis and Relay Therapeutics. S.C., D.R., S.B., A.C. and P.E.H. are employees of Amgen, as noted in the affiliations. Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 25, 2018; DOI: 10.1158/2159-8290.CD-18-0277 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract BH3 mimetic drugs, which inhibit pro-survival BCL-2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL-1 inhibitors, we demonstrate that concurrent MEK + MCL-1 inhibition induces apoptosis and tumor regression in KRAS mutant non-small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL-1 or BCL-XL was determined by the differential binding of pro- apoptotic BCL-2 proteins to MCL-1 or BCL-XL, respectively. The efficacy of dual MEK + MCL-1 blockade was augmented by prior transient exposure to BCL-XL inhibitors, which promotes the binding of pro-apoptotic BCL-2 proteins to MCL-1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL-2 family proteins for KRAS mutant NSCLC. Statement of Significance Defining the molecular basis for MCL-1 versus BCL-XL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-XL and MCL-1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS mutant NSCLCs treated with MEK inhibitors. 2 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 25, 2018; DOI: 10.1158/2159-8290.CD-18-0277 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Introduction 2 3 KRAS, a small GTPase that activates MAPK signaling, is one of the most frequently mutated 4 driver oncogenes (1). KRAS mutations, which are largely localized to codons G12, G13 and 5 Q61, decrease either intrinsic and/or GAP-mediated hydrolysis, causing constitutive activation of 6 MAPK and other downstream signaling pathways (2). Although effective molecular targeted 7 therapies that inhibit oncogenic mutant kinases in the RAS-MAPK pathway have been developed 8 (e.g. EGFR inhibitors for EGFR mutant NSCLC, BRAF inhibitors for BRAF mutant melanoma), 9 there currently are no approved targeted therapies for KRAS mutant cancers. KRAS mutations are 10 found in 20-25% of patients with non-small cell lung cancer (NSCLC) and predict for lack of 11 response to EGFR inhibitors (3). Attempts to target downstream MAPK signaling with inhibitors 12 of MEK1/2 have yielded disappointing results (4, 5), and strategies that simultaneously target 13 multiple signaling pathways have been limited by toxicity (6, 7). Most recently, a novel class of 14 KRAS inhibitors that covalently bind to the G12C mutant have been described (8, 9), although 15 these have yet to be tested in the clinic. Thus there remains an urgent need for new therapeutic 16 strategies that can target KRAS mutant cancers. 17 18 Several studies have shown that suppression of MAPK signaling, either by depletion of mutant 19 KRAS or by pharmacologic inhibition of downstream MEK1/2, is insufficient to induce 20 apoptosis in a significant number of KRAS mutant cell lines (10-12). Therapeutic strategies that 21 co-target kinase signaling pathways and apoptotic regulators may increase apoptosis and convert 22 cytostatic responses into tumor regressions (13). Activated kinase signaling pathways such as 23 MAPK (RAS/RAF/MEK/ERK) and PI3K/AKT converge on the BCL-2 protein family, which 24 regulates the mitochondrial or intrinsic apoptotic response (14). In cells with MAPK activation, 25 ERK phosphorylation suppresses the pro-apoptotic BH3 protein BIM by targeting it for 26 degradation (15, 16). MEK inhibition causes BIM to accumulate (16), however BIM can be 27 neutralized by pro-survival BCL-2 family members such as BCL-XL or MCL-1. Combining 28 MEK inhibitors with the BH3 mimetic navitoclax (ABT-263), which prevents the binding of 29 BIM to BCL-2 and BCL-XL, led to greater apoptosis and tumor regression in KRAS 30 experimental models compared to MEK inhibitors alone (11), and a clinical trial evaluating this 31 combination is currently on-going (NCT02079740, www.clinicaltrials.gov). To date, these 3 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 25, 2018; DOI: 10.1158/2159-8290.CD-18-0277 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 32 approaches have been limited to targeting BCL-2 and BCL-XL due to the lack of selective and 33 potent inhibitors that target other members of the BCL-2 family. 34 35 MCL-1 is frequently amplified in lung cancers (17), and the development of potent and selective 36 MCL-1 inhibitors has long been of interest. Recently, a novel MCL-1 inhibitor S63845 with in 37 vivo activity was reported (18). Significant activity was observed in leukemia, myeloma and 38 lymphoma models, and several different MCL-1 inhibitors are now currently in clinical 39 development for these malignancies (NCT02992483, NCT02979366, NCT02675452, 40 www.clinicaltrials.gov). Single agent activity of S63845 was limited in solid tumor models 41 including NSCLC and breast cancers, however combining S63845 with relevant kinase inhibitors 42 led to decreased cell viability of BRAF, EGFR and HER2-addicted cell lines in vitro, providing 43 proof of principle that MCL-1 inhibition, similar to BCL-XL inhibition, may potentiate the 44 response to kinase inhibitor targeted therapies. However, due to the lack of studies that directly 45 compare analogous combination strategies that target either MCL-1 or BCL-XL, the optimal 46 pairing of kinase inhibitors with BH3 mimetics that target different BCL-2 family proteins in 47 specific subsets of cancer remains undefined. 48 49 Here, we assessed the activity of a novel class of potent and selective spiro-macrocyclic MCL-1 50 inhibitors in combination with MEK inhibition in KRAS mutant NSCLC models and compared 51 this to the parallel strategy of MEK + BCL-XL inhibition. Distinct but overlapping subsets of 52 KRAS mutant NSCLC models were more sensitive to MEK + MCL-1 versus MEK + BCL-XL 53 inhibition, which was determined by the binding interactions between specific BCL-2 family 54 proteins. By altering the cellular localization and interactions between BCL-2 family proteins 55 with transient exposure to BCL-XL inhibitors, KRAS mutant NSCLC cells could be induced into 56 an MCL-1 dependent state with increased sensitivity to MEK + MCL-1 inhibition. These results 57 provide rationale for the clinical evaluation of MEK + MCL-1 inhibitors in KRAS mutant 58 NSCLC and suggest a broader strategy for integrating MCL-1 and BCL-XL inhibitors to 59 maximize efficacy of kinase inhibitor targeted therapies. 60 61 4 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 25, 2018; DOI: 10.1158/2159-8290.CD-18-0277 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 62 Results 63 64 Combination drug screen identifies synergistic activity between AM-8621 and MAPK pathway 65 inhibitors in cell lines with MAPK pathway activation. 66 67 Recently developed potent and selective MCL-1 inhibitors have demonstrated limited single- 68 agent activity against solid tumor malignancies (18, 19) suggesting that drug combinations may 69 be required to maximize the potential clinical benefit of this class of inhibitors for these cancers.