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Description of additional methods and procedures used in the study. Also includes Supplementary References.

Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950–63. ©2016 AACR.

Compounds used in this study.

From images to synergies (S1); Reproducibility of data from two screens (S2); Single agent responses and selectivity (S3); Screen-wide comparison of Caspase 3/7 activation and growth inhibition (S4); Heatmaps of growth inhibition and Capase 3/7 activation, and examples of broadly synergistic combinations targeting RAS/MAPK and/or PI3K/AKT pathways (S5); Combinations targeting RAS/MAPK and PI3K/AKT pathways at different nodes show similar efficacies (S6); Combinations involving RTKs (S7); Heatmaps of growth inhibition and Capase 3/7 activation, and examples of combinations targeting RAS/MAPK or PI3K/AKT pathways and other cellular processes (S8); Synergies of triple combination increase with synergies of underlying drug pairs (S9); Heatmap of synergies, growth inhibition, and Caspase 3/7 activation for triple combinations after hierarchical clustering (S10); Triple combinations targeting RAS/MAPK and PI3K/AKT pathways (S11); Triple combinations targeting RAS/MAPK and/or PI3K/AKT pathways and other cellular processes (S12); Combination targeting MDM2 and MEK in p53 wild-type models (S13); Sequential treatment of p53 wild-type lines with triple combination targeting MDM2, MEK, and BCL-2/-XL (S14); High order combinations to kill 'robust' cell lines (S15).

Supplementary Data from Phase I Study of Taminadenant (PBF509/NIR178), an Adenosine 2A Receptor Antagonist, with or without Spartalizumab (PDR001), in Patients with Advanced Non–Small Cell Lung Cancer

Supplementary Figure from Phase I Study of Taminadenant (PBF509/NIR178), an Adenosine 2A Receptor Antagonist, with or without Spartalizumab (PDR001), in Patients with Advanced Non–Small Cell Lung Cancer

Legend for Supplementary Tables S1-S6 and Supplementary Figures S1-S15.

Purpose:The adenosine 2A receptor (A2AR) mediates the immunosuppressive effects of adenosine in the tumor microenvironment and is highly expressed in non–small cell lung cancer (NSCLC). Taminadenant (PBF509/NIR178) is an A2AR antagonist able to reactivate the antitumor immune response.Patients and Methods:In this phase I/Ib, dose-escalation/expansion study, patients with advanced/metastatic NSCLC and ≥1 prior therapy received taminadenant (80–640 mg, orally, twice a day) with or without spartalizumab (anti–programmed cell death-1, 400 mg, i.v., every 4 weeks). Primary endpoints were safety, tolerability, and feasibility of the combination.Results:During dose escalation, 25 patients each received taminadenant alone or with spartalizumab; 19 (76.0%) and 9 (36.0%) had received prior immunotherapy, respectively. Dose-limiting toxicities (all Grade 3) with taminadenant alone were alanine/aspartate aminotransferase increase and nausea [n = 1 (4.0%) each; 640 mg], and in the combination group were pneumonitis [n = 2 (8.0%); 160 and 240 mg] and fatigue and alanine/aspartate aminotransferase increase [n = 1 (4.0%) each; 320 mg]; pneumonitis cases responded to steroids rapidly and successfully. Complete and partial responses were observed in one patient each in the single-agent and combination groups; both were immunotherapy naïve. In the single-agent and combination groups, 7 and 14 patients experienced stable disease; 7 and 6 patients were immunotherapy pretreated, respectively.Conclusions:Taminadenant, with and without spartalizumab, was well tolerated in patients with advanced NSCLC. The maximum tolerated dose of taminadenant alone was 480 mg twice a day, and 240 mg twice a day plus spartalizumab. Efficacy was neither a primary or secondary endpoint; however, some clinical benefit was noted regardless of prior immunotherapy or programmed cell death ligand-1 status.

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

Purpose:The adenosine 2A receptor (A2AR) mediates the immunosuppressive effects of adenosine in the tumor microenvironment and is highly expressed in non–small cell lung cancer (NSCLC). Taminadenant (PBF509/NIR178) is an A2AR antagonist able to reactivate the antitumor immune response.Patients and Methods:In this phase I/Ib, dose-escalation/expansion study, patients with advanced/metastatic NSCLC and ≥1 prior therapy received taminadenant (80–640 mg, orally, twice a day) with or without spartalizumab (anti–programmed cell death-1, 400 mg, i.v., every 4 weeks). Primary endpoints were safety, tolerability, and feasibility of the combination.Results:During dose escalation, 25 patients each received taminadenant alone or with spartalizumab; 19 (76.0%) and 9 (36.0%) had received prior immunotherapy, respectively. Dose-limiting toxicities (all Grade 3) with taminadenant alone were alanine/aspartate aminotransferase increase and nausea [n = 1 (4.0%) each; 640 mg], and in the combination group were pneumonitis [n = 2 (8.0%); 160 and 240 mg] and fatigue and alanine/aspartate aminotransferase increase [n = 1 (4.0%) each; 320 mg]; pneumonitis cases responded to steroids rapidly and successfully. Complete and partial responses were observed in one patient each in the single-agent and combination groups; both were immunotherapy naïve. In the single-agent and combination groups, 7 and 14 patients experienced stable disease; 7 and 6 patients were immunotherapy pretreated, respectively.Conclusions:Taminadenant, with and without spartalizumab, was well tolerated in patients with advanced NSCLC. The maximum tolerated dose of taminadenant alone was 480 mg twice a day, and 240 mg twice a day plus spartalizumab. Efficacy was neither a primary or secondary endpoint; however, some clinical benefit was noted regardless of prior immunotherapy or programmed cell death ligand-1 status.

Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.

Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950–63. ©2016 AACR.

EGFR targeted kinase inhibitors (TKIs) are the standard of care in non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR). Patients initially respond well to EGFR inhibitors, although the majority only achieve a partial response and a subset of drug-tolerant persister cells remain at minimal residual disease (MRD). These drug-tolerant persister cells represent a cell reservoir from which de novo genetic mutations, such as EGFRT790M or MET amplification, can arise to render the tumor fully drug-resistant. Previous studies suggest that drug-tolerant cells rely on an altered chromatin state to survive EGFR-inhibition. However, it is still unclear whether the drug-tolerant cell population emerges through selection for cells that pre-existed in that state or through and adaptation in response to drug. It is also unknown if drug-tolerant persister cells rely on a single survival mechanism that could be exploited to more effectively target this population or if multiple independent mechanisms are being utilized and need to be targeted to fully suppress drug tolerance. Despite the urgent clinical need to answer these questions, we have lacked the techniques capable of the dynamic resolution necessary to investigate the emergence of drug tolerance throughout the course of treatment within individual cell lineages. Here we present a strategy to investigate the clonal evolution of drug tolerance in EGFRmut NSCLC using an expressed molecular barcoding library coupled with single cell RNAseq (scRNAseq). We found that the cell lineages that are destined to become drug-tolerant are pre-defined, although the epigenetic drug-tolerant state does not pre-exist. We observed multiple distinct heterogeneous classes of drug-tolerant cells with unique gene expression signatures as well as distinct trajectories in response to EGFRi. We observed evidence of putative mechanisms of drug tolerance, such as EMT and adaptive MAPK signaling, in parallel trajectory classes across cell lines. Finally, we compared EGFRi/TKI drug combinations versus EGFRi/chemotherapy combinations to investigate which therapeutic approach was more efficacious in targeting multiple trajectory classes of drug tolerant cells. Taken together, our work presents a new technology that enables a comprehensive interrogation of drug response over time and provides greater insight into how drug-tolerant cells evolve over the course of drug treatment, which ultimately can help inform combination treatment strategies for patients in the clinic. Citation Format: Jennifer L. Cotton, Viveksagar Krisnamurthy Radhakrishna, Javier Estrada Diez, David A. Ruddy, Kathleen Sprouffske, Gaylor Boulay, Michelle Piquet, Joel Wagner, Youngchul Song, Xiaoyan Li, Katja Schumacher, Joshua Korn, Erick J. Morris, Peter S. Hammerman, Jeffrey A. Engelman, Matthew J. Niederst. Expressed molecular barcoding coupled with single cell RNAseq enables a high resolution investigation into the evolution of drug tolerance [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-100.

International audience; Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, welltolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

Patients with non-small cell lung cancer (NSCLC) that is driven by an activating mutation in the epidermal growth factor receptor (EGFR) are routinely treated with tyrosine kinase inhibitors (TKI) to specifically target the activated EGFR signaling pathway. EGFR-mutant NSCLC tumors initially respond well to EGFR inhibitors, however a subset of drug-tolerant persister cells remain at minimal residual disease (MRD) and represent a cell reservoir from which acquired genetic mutations, such as EGFRT790M or MET amplification, can emerge to render the tumor fully drug-resistant. Prior to the emergence of genetic mutations, little is known about how drug-tolerant persister cells are able to survive EGFR targeted therapy at MRD. To better understand this cell population, we investigated drug-tolerance using single cell cloning and scRNAseq in NSCLC cell lines. Using ClonTracer barcoding, we found that the same barcodes emerged after EGFR-inhibitor treatment across multiple replicates, indicating that drug-tolerance is both pre-defined and stable over many generations. Within each individual cell line, we observed multiple distinct heterogeneous subpopulations of drug-tolerant persister cells with unique gene expression signatures and proliferation rates. Additionally, we observed evidence of putative mechanisms of drug tolerance that were shared by persister cells across cells lines and used a drug combination treatment approach to target these distinct subpopulations of drug-tolerant persister cells. Taken together, our findings provide evidence that drug-tolerant persister cell subpopulations are both predefined and heterogeneous, as well as suggesting that drug-combination treatment approaches in the clinic would be more effective at targeting multiple persister cell survival mechanisms. Citation Format: Jennifer L. Cotton, Viveksagar KrishnamurthyRadhakrishna, Julie Chen, Michelle Piquet, Joel Wagner, Gaylor Boulay, Kathleen Sprouffske, Youngchul Song, Xiaoyan Li, Katja Schumacher, Raphael Thierry, Nathaniel D. Kirkpatrick, David A. Ruddy, Joshua Korn, Erick J. Morris, Peter S. Hammerman, Jeffrey A. Engelman, Matthew J. Niederst. Molecular barcoding and single cell approaches to investigate drug tolerance in EGFRmut NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A122. doi:10.1158/1535-7163.TARG-19-A122

Myeloid cell leukemia 1 (Mcl-1) is one of the key anti-apoptotic Bcl-2 family proteins that binds and neutralizes pro-apoptotic BIM, BAX and BAK at the mitochondrial outer membrane, preventing cytochrome c release and caspase activation. Selective upregulation of Mcl-1 functionally contributes to resistance of acute myeloid leukemias (AML) with FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) to chemotherapy (Kasper S. et al. 2012, Blood Cancer J. 2:e60, doi:10.1038/bcj.2012.5). Here we show that a novel Mcl-1 inhibitor S63845 (Kotschy A. et al. 2016, Nature 538, 477-482), has synergistic proapoptotic activity in combination with FLT3-ITD kinase inhibitor midostaurin in pre-clinical models of AML. Our studies demonstrate that S63845 has potent single agent activity in AML cell lines and primary AML samples harboring FLT3-ITD with IC50 values in low nanomolar range. Co-targeting of Mcl-1 and FLT3-ITD with S63845 and midostaurin, respectively, significantly increased apoptosis in FLT3-ITD cells with caspase-3 activation and PARP cleavage occurring rapidly within 6 hours of treatment. Consistent with markedly reduced cell growth and viability, analysis of drug combinations efficacy using Bliss independence model revealed strong synergistic interactions between S63845 and midostaurin in FLT3-ITD cell lines and primary AML samples. Midostaurin caused de-phosphorylation of FLT3-ITD and its downstream targets such as STAT5, AKT and MAPK. This was accompanied by significant downregulation of MAPK-mediated phosphorylation of Mcl-1 at Thr163 required for Mcl-1 stability. Consequently, midostaurin reduced Mcl-1 protein levels, with no major changes in antiapoptotic Bcl-2 or Bcl-XL. Importantly, midostaurin increased expression of pro-apoptotic Bim, which could in turn bind and negate residual Mcl-1 pro-survival activity. Elevated Bim was sustained upon S63845 co-treatment, suggesting that Bim plays functional role in midostaurin/S63845-mediated lethality. Dynamic BH3 profiling showed that midostaurin primed FLT3-ITD cells to Mcl-1 and Bcl-2 inhibitors and facilitated general apoptosis priming in response to Bim peptide. Importantly, given that Mcl-1 is a major contributing factor to resistance of AML to Bcl-2 selective BH3-mimetic venetoclax, S63845/midostaurin treatment induced cell death in venetoclax-resistant FLT3-ITD mutants. In summary, S63845/midostaurin is highly synergistic in FLT3-ITD mutated AML cells including those resistant to venetoclax. In vivo experiments of tolerability and efficacy are ongoing and will be reported. Citation Format: Anna Skwarska, Qi Zhang, Shelley M. Herbrich, Natalia Baran, Ensar Halilovic, Peter Ruvolo, Vivian Ruvolo, Erick Morris, Andrew Wei, Donia Moujalled, Michael Andreff, Marina Konopleva. S63845, a novel BH3 mimetic Mcl-1 inhibitor synergizes with midostaurin to induce potent apoptosis in acute myeloid leukemia cells carrying FLT3-ITD mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 342.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematologic malignancy, characterized by uncontrolled proliferation and impaired differentiation of myeloid cells. With the exception of certain subtypes, the average long-term survival rate remains low, thus underlining the need to further improve the outcome of AML patients. Since AML is one of the least mutated cancer types, the majority of AML patients may not carry targetable genetic alterations. However, the anti-apoptotic proteins of the Bcl-2 family, such as Bcl-2 and Mcl-1, are often overexpressed in AML, allowing deregulated survival; hence pro-apoptosis priming with small molecule inhibitors of Bcl-2 and Mcl-1 may provide a broader therapeutic benefit across the disease. In addition, a majority of AML patients carry wild-type p53, providing therapeutic opportunity for Hdm2 inhibitors to stabilize p53 and lead to expression of pro-apoptotic molecules (e.g., PUMA & BAX). Therefore, targeting the combined apoptosis mechanisms by inhibiting different anti-apoptotic Bcl-2 family of proteins and activating p53 concomitantly may synergistically enhance apoptotic cell death of AML tumor cells. We tested the combination of Bcl-2 inhibitors (BCL201/S55746 or venetoclax) with either MIK665/S64315, a novel and selective inhibitor of Mcl-1 or HDM201, a selective small molecule inhibitor of p53:Hdm2 interaction, in a series of in vitro and in vivo studies in AML. In vitro, strong combination synergy was observed with a remarkable induction of cell death for both combinations. In vivo, the combination of Bcl-2 inhibitors with MIK665/S64315 or HDM201 lead to complete and durable antitumor responses in a variety of p53wt AML patient-derived xenograft models of heterogeneous genetic profiles. Notably, lowering the dose of HDM201 by 4 fold from its most efficacious dose, resulted in a high degree of tumor regressions while mitigating the toxicity effects on platelets. Taken together, these data demonstrate that a combination of Bcl-2 inhibitor (BCL201/S55746 or venetoclax) with MIK665/S64315 or HDM201 provide therapeutic benefit over the monotherapy, and support a rationale for testing these apoptosis enhancing combination approaches in AML patients. Citation Format: Youzhen Wang, Shumei Qui, Sneha Sanghavi, Iain Mulford, Gaëlle Lysiak, Maïa Chanrion, Prakash Mistry, Ulrike Pfaar, Marie Schoumacher, Audrey Claperon, Laurence Kraus-Berthier, Sébastien Banquet, Alix Derreal, Claire Fabre, Heiko Maacke, Frédéric Colland, Olivier Geneste, Erick Morris, Ensar Halilovic. Targeting AML through apoptosis activation using Bcl-2/Mcl-1 or Bcl-2/Hdm2 inhibitor combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 257.

Mcl-1 is highly expressed in a variety of human cancers (including those of hematopoietic and lymphoid origin) and is exploited by cancer cells to evade cell death and to develop resistance to diverse chemotherapeutic agents. We disclose, for the first time, the structure of S64315 (also named MIK665) a highly potent and selective inhibitor of Mcl-1 with improved potency over its predecessor S63845 (Kotschy et al, Nature, 2016). S64315/MIK665 is currently in phase 1 in AML (Acute Myeloid Leukemia) and MDS (Myelodysplastic Syndrome) (EudraCT 2016-003768-38, NCT 02979366) and in MM (Multiple Myeloma) and lymphoma (NCT02992483). A fragment-based, structure-guided drug discovery effort led to the identification of S64315/MIK665 that binds to human Mcl-1 with a sub-nanomolar affinity (Ki 0.048 nM) and selectively over other anti-apoptotic Bcl-2 family members. It has similar affinity for human, rat, dog and monkey Mcl-1 but about a ten-fold lower affinity for mouse Mcl-1. S64315/MIK665 causes dose-dependent activation of the intrinsic apoptosis pathway in a Bax/Bak-dependent manner, as measured by increased caspase activity and cleaved PARP. S64315/MIK665 shows strong cell killing activity in a diverse panel of human hematological tumor cell lines, including AML, lymphoma and MM. The activity profile of S64315/MIK665 is distinct from that of venetoclax, a selective Bcl2 inhibitor. In vivo, S64315 as single agent demonstrated potent and dose-dependent apoptotic and antitumor response after intravenous administration in several human hematological tumor models grafted in immuno-compromised mice and rats. Complete regression of established tumors, at well tolerated doses, was achieved using different intravenous dosing regimens in rats as well as in mice. Finally, dual BH3-mimetic targeting approach combining S64315/MIK665 with BCL2 inhibitors showed strong and durable antitumor responses in several hematological tumor models both in vitro and in vivo. Citation Format: Ana Leticia Maragno, Prakash Mistry, András Kotschy, Zoltán Szlavik, James Murray, James Davidson, Gaëtane Le Toumelin-Braizat, Maïa Chanrion, Alain Bruno, Audrey Claperon, Heiko Maacke, Erick Morris, Youzhen Wang, Alix Derreal, Márton Csekei, Attila Paczal, Zoltán Szabo, Szabolcs Sipos, Agnes Proszenyak, Balázs Balint, Allan Surgenor, Pawel Dokurno, Natalia Matassova, Ijen Chen, Gaëlle Lysiak-Auvity, Anne-Marie Girard, Fabienne Grave, Frédéric Colland, Ensar Halilovic, Olivier Geneste. S64315 (MIK665) is a potent and selective Mcl1 inhibitor with strong antitumor activity across a diverse range of hematologic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4482.

Introduction: SCLC is an aggressive high-grade neuroendocrine malignancy in which targeting anti-apoptotic regulators such as Bcl-2 and Bcl-xL has shown efficacy in pre-clinical models but has not resulted in successful clinical trials (Rudin et al., Clin Cancer Res. 2012). Although SCLC cell-lines do not reflect the clinical impact of these inhibitors, patient-derived xenograft (PDX) models may more accurately recapitulate Bcl-2 family expression profiles and BH3 mimetic efficacy. One promising hypothesis is that the fellow anti-apoptotic protein MCL-1 rescues viability in the presence of Bcl-2/Bcl-xL antagonists. Here we evaluate the efficacy of the MCL-1 inhibitor S63845 in combination with a novel specific inhibitor of Bcl-2, BCL201/S55746, in SCLC patient-derived xenografts. Methods: BH3 mimetic compounds were tested for synergy in vitro in SCLC cell lines. A set of ten cell lines was chosen based on relative expression of BCL2, MCL1, and BCL2L1 (Bcl-xL) mRNA. Single agent and and pair-wise combinations of Bcl2 family inhibitors were compared in three-day growth inhibition assays. Loewe synergy scores were plotted versus Bcl2 family mRNA expression to identify the determinants of drug sensitivity. Based on the cell line synergy assays, a combination of BCL201/S55746 and S63845 was selected to test in PDX models of SCLC. Bcl-2 family expression was profiled across a panel of 37 SCLC PDX models generated at MGH by quantitative western blot, and standardized to the most sensitive SCLC cell line, NCI-H211. Ten models were selected based on absolute expression of Bcl-2, Bcl-xL and MCL-1. Mice were treated when subcutaneous tumors reached a volume of 400-800 cc, enabling precise measurement of tumor regression and time to tumor regrowth. Findings: Bcl-2 family dependency in SCLC cell lines was profiled with selective inhibitors as single agents or combinations. Maximum synergy was found between BCL201/S55746 and S63845 in cell lines with the highest Bcl-2:Bcl-xL expression ratio. Bcl-2 family expression was profiled across a panel of 37 PDX models of SCLC, and a representative set of 10 models was selected for in vivo testing. Consistent with cell line results, the two most sensitive models to BCL201/S55746+S63845 demonstrated the highest Bcl-2:Bcl-xL ratios, with moderate to high expression of MCL-1. In these models BCL201/S55746+S63845 resulted in a 44-70% tumor regression that was stable throughout 4 weeks of treatment. Efficacy was not dependent on MCL-1 expression, and was not strongly correlated with PDX sensitivity to platinum-etoposide. Conclusions: Combined inhibition of Bcl-2 with BCL201 and MCL-1 with S63845 is effective in SCLC tumors with relatively low Bcl-xL expression. This combination overcomes MCL-1 mediated resistance to Bcl-2 inhibitors, and represents a promising strategy to target anti-apoptotic dependency in SCLC. Citation Format: Benjamin J. Drapkin, Sneha Sanghavi, David T. Myers, Jun Zhong, Sarah Phat, Youzhen Wang, Ensar Halilovic, Javad Golji, Anna Farago, Erick Morris, Nicholas J. Dyson. Combined inhibition of Bcl-2 and MCL-1 in small cell lung cancer (SCLC) is most effective in tumors with low Bcl-xL expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 381.

One of the hallmarks of cancer is evasion of apoptosis. The B-cell lymphoma-2 (Bcl-2) family of proteins represents a crucial point of control of apoptosis. The Bcl-2 family comprises both pro- and anti-apoptotic members, the latter of which (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bcl-2A1) are often overexpressed in cancer cells, supporting their aberrant survival. Thus, these anti-apoptotic proteins have become an attractive target for cancer therapy. BH3 mimetics have been shown to bind to the BH3 binding groove of anti-apoptotic Bcl-2 family members and inhibit their function, resulting in apoptotic cell death, and one such BH3 mimetic, ABT-199 (venetoclax), has recently been approved for treatment of relapsed or refractory Chronic Lymphocytic Leukemia. We have developed two novel and potent BH3 mimetics: MIK665/S64315, a highly selective inhibitor of Mcl-1 and BCL201/S55746, a selective Bcl-2 inhibitor. Both compounds, individually induce apoptosis in hematological cancer cell lines, primary patient samples and demonstrate anti-tumor efficacy in xenograft models. MIK665/S64315 is currently in phase 1 clinical development in AML and MDS (NCT 02979366) and in MM and lymphoma (NCT02992483). Here, we describe the activity of the combination of MIK665/S64315 with BCL201/S55746 or venetoclax, both in vitro and in vivo, across a range of hematological indications (AML, MM and DLBCL). In vitro, a strong synergy was observed with these combinations, resulting in a remarkable induction of cell death in majority of cell lines tested. In vivo, MIK665/S64315 and BCL201/S55746 combinations lead to complete and durable antitumor responses in many different xenograft models in mice and rats. Taken together, these data demonstrate that a combination of MIK665/S64315 and BCL201/S55746 provide strong therapeutic benefit over either monotherapy, and support a rationale for testing Mcl-1 and Bcl-2 inhibitor combinations in patients with hematological malignancies. Citation Format: Ensar Halilovic, Maïa Chanrion, Prakash Mistry, Markus Wartmann, Shumei Qiu, Sneha Sanghavi, Yan Chen, Gaëlle Lysiak, Ana Leticia Maragno, Ulrike Pfaar, Felix Huth, Marie Schoumacher, Audrey Claperon, Laurence Kraus-Berthier, Sébastien Banquet, Alix Derreal, Heiko Maacke, Frédéric Colland, Olivier Geneste, Erick Morris, Youzhen Wang. MIK665/S64315, a novel Mcl-1 inhibitor, in combination with Bcl-2 inhibitors exhibits strong synergistic antitumor activity in a range of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4477.

9089Background: ATP is catabolized to adenosine in the tumor microenvironment, leading to excess adenosine and immunosuppressive effects via immune checkpoint protein adenosine 2A receptor (A2AR). ...

Tumors are complex biological systems that often retain proliferative capacity even when challenged with drug treatment. Given this resiliency, drug combinations may provide greater therapeutic benefit, however, which molecules to combine and how many to include in combinations for effective responses is not clear yet. Using image-based proliferation and apoptosis assays in colorectal cancer cell lines we systematically investigated combinations that ranged in number from two to six drugs and targeted critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds, but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In many cases, where cell lines were resistant to two- and three-way drug combinations, increased expression of anti-apoptotic proteins was observed and induction of cytotoxic responses required up to fourth-order combinations. Our results demonstrate that high-order drug combinations might be needed to kill cancers and show how systematic drug combination screening together with a molecular understanding of drug responses can guide their identification. Citation Format: Thomas Horn, Stephane Ferretti, Nicolas Ebel, Angela Tam, Samuel Ho, Fred Harbinski, Ali Farsidjani, Matt Zubrowski, William R. Sellers, Robert Schlegel, Dale Porter, Erick Morris, Jens Wuerthner, Sebastien Jeay, Joel Greshock, Ensar Halilovic, Levi A. Garraway, Giordano Caponigro, Joseph Lehar. High order drug combinations are required to effectively kill colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1311.

Tumors are complex and robust biological systems that harbor the potential to proliferate against various drug treatments. Drug combinations provide a promising therapeutic strategy, but it is not clear which and how many drugs are required to overcome cancers. Using image-based proliferation and apoptosis assays in colorectal cancer cells we systematically investigated complex treatments composed of two to six drugs targeting critical oncogenic pathways. Drug pairs targeting growth signaling resulted in synergies across a broad spectrum of genetic backgrounds, but often yielded cytostatic responses and failed to induce apoptosis. Enhanced, and sometimes genotype-specific cytotoxicity was seen after targeting additional mechanisms including apoptosis or cell cycle. Cells that resisted all tested drug pairs and drug triples were protected by a mechanism that prevented apoptosis. Targeted inhibition of this mechanism using combinations of up to four compounds induced cytotoxic responses in cells in vitro and in vivo. Our results demonstrate that complex combinations of targeted drugs might be required to induce killing in cancers and show how the cells' genetic alterations and a molecular understanding of drug responses can guide their identification. The identification of resistance mechanisms that are pre-existing in subpopulations of tumor cells also opens the exciting avenue of sequenced treatments, with each drug or drug combination targeting and eradicating a specific cell population. Citation Format: Thomas Horn, Stéphane Ferretti, Nicolas Ebel, Angela Tam, Samuel Ho, Fred Harbinski, Ali Farsidjani, Matthew Zubrowski, Ensar Halilovic, Erick Morris, William R. Sellers, Robert Schlegel, Jens Wuerthner, Levi A. Garraway, Sébastien Jeay, Joel Greshock, Giordano Caponigro, Joseph Lehár. Complex drug combinations can induce apoptotic killing in robust colorectal cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B04.