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Multiple myeloma (MM) is marked by several genetic abnormalities, including chromosome translocation t(11;14). Overexpression of anti-apoptotic BCL-2 in t(11;14) MM promotes disease progression, prompting clinical use of the BH3 mimetic and BCL-2 inhibitor venetoclax in combination with proteasome inhibitor therapy. Despite high initial response rates and prolonged progression-free survival, patients commonly relapse. To delineate mechanisms contributing to acquired drug resistance we modeled responses to venetoclax in two highly sensitive MM cell lines (KMS27 and KMS-12PE). Colonies generated from a surviving cell were cultured in high-dose venetoclax to generate monoclonal drug-tolerant expanded persister (DTEP) clones. To determine whether venetoclax resistance in DTEP clones is mediated by transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprogramming, we conducted whole-genome sequencing (WGS) and RNA-seq of the clones. WGS analysis did not show significant differences between parental and resistant clones, but transcriptomic analysis showed shared and unique transcriptome signatures in DTEP clones. Gene set enrichment analysis of the common significantly modulated genes in resistant clones revealed that PKA-ERK-CREB and K-Ras pathway genes were significantly upregulated, whereas apoptotic genes were downregulated in resistant clones compared to parental cells. Importantly, ectopically expressed ERK in venetoclax-sensitive cells conferred a resistant phenotype that was rescued using two specific ERK inhibitors in DTEP clones. These data confirm a key role for ERK activation in acquired venetoclax resistance. Resistant clones were further characterized by reduced mitochondrial priming assessed by dynamic BH3 profiling, with altered expression of anti-apoptotic regulators including MCL-1, BCL-xL, and BCL-W and the replaced BCL-2: BIM complex by both MCL-1 and BCL-xL. Because these data suggested a functional substitution between anti-apoptotic BCL-2 family members in cells with acquired resistance to venetoclax, we next evaluated if MCL-1 or BCL-xL are codependent in MM cells that are insensitive or resistant to venetoclax. Simultaneous inhibition of MCL-1 (via S63845) or BCL-xL (via A155463) and BCL-2 (via venetoclax) increased BIM release and enhanced cell death in resistant clones (vs single agents), with combination index values < 0.3 in all doses. Upregulation of BCL-xL or MCL-1 in MM cells also mediated primary venetoclax resistance independent of genetic hallmarks (e.g. t [11;14]-translocated cells). Thus, simultaneous inhibition of MCL-1 or BCL-xL and BCL-2 triggered synergistic cytotoxicity in MM cell lines intrinsically resistant to venetoclax. These data suggest that combined inhibition of BCL-2 and BCL-xL may overcome venetoclax resistance. However, the dependence of BCL-xL in mature platelets had triggered thrombocytopenia for patients under therapy using BCL-xL inhibitor. To further explore the potential clinical application of targeting BCL-xL, we employed novel BCl-2/BCL-xL dual inhibitor, BH3 mimetic pelcitoclax (APG-1252). Using pro-drug strategy for design, pelcitoclax has limited cell permeability during circulation, and was converted to a more potent metabolite APG-1252-M1 in tumors/tissues. APG-1252-M1 was thus used for in vitro cell based assays. We discovered that APG-1252-M1 induced cytotoxicity in MM cell lines intrinsically resistant to venetoclax (regardless of genetic background or BCL-2:BCL-xL ratio) and also significantly reduced MM cell viability in clones with acquired venetoclax resistance, overcoming ERK activation and decreasing BIM sequestration by BCL-xL. In vivo study using pelcitoclax is ongoing and will be presented at the meeting. In conclusion, we report that venetoclax resistance in MM evolves from outgrowth of persister clones displaying activation of the ERK pathway and a shift in mitochondrial dependency towards BCL-xL, which can potentially be effectively targeted via the novel BCL-2/BCL-xL inhibitor pelcitoclax (APG-1252), which is currently in clinical investigation for solid tumors (NCT03080311). Disclosures Deng: Ascentage Pharma Group: Current Employment. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Novartis: Consultancy; Janssen: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharm: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Amgen: Consultancy; Abbvie: Consultancy; Legend: Consultancy; Pfizer: Consultancy.

Natural killer/T-cell lymphoma (NK/TCL) is one of the most common subtypes (10.4%) of peripheral T-cell lymphoma, which in turn accounts for 10% to 15% of all cases of non-Hodgkin lymphoma. NK/TCL occurs more frequently in Asia and Latin America than other regions, and although associated with Epstein-Barr virus (EBV) infection, NK/TCL has an unclear pathogenesis of genetic and molecular alterations. Asparaginase-based chemotherapy regimens are frequently used, typically with unsatisfactory outcomes. Novel targeted therapies, such as histone deacetylase (HDAC) inhibitors and programmed death-1 antibodies, are reported effective either as single agents or in combination with other agents. Studies have also shown that EBV-positive NK/TCL cell lines are B-cell lymphoma-extra large (BCL-xL) dependent. Other preclinical experiments have shown that BH3-mimetic drugs targeting BCL-xL-induced cell death in NK/TCL cell lines were effective in NK/TCL xenograft models. BCL-xL inhibitors have shown narrow therapeutic windows in clinical trials because of dose-limiting on-target thrombocytopenia. Pelcitoclax (APG-1252) is a novel dual BCL-2/BCL-xL inhibitor under clinical development for solid tumors. As a result of its unique prodrug design, APG-1252 can overcome the undesired platelet toxicity. This study evaluated the potential antitumor effect of APT-1252 in preclinical models of NK/TCL. Cell-based antiproliferation studies showed activity of APG-1252 and its more potent metabolite APG-1252-M1 toward NK/TCL cell lines that overexpressed BCL-xL. Half-maximal inhibitory concentrations (IC 50) for APG-1252 in SNK-1, SNK-6, and SNK-8 (EBV-positive NK/TCL) cell lines were 2.652 ± 2.606 µM, 1.568 ± 1.109 µM, and 0.557 ± 0.383 µM, respectively. Corresponding values for APG-1252-M1 were 0.133 ± 0.056 µM, 0.064 ± 0.014 µM, and 0.020 ± 0.008 µM, respectively. Mechanistic studies demonstrated that APG-1252 and APG-1252-M1 disrupted the complex of BCL-xL/BCL-2-associated X protein (Bax) and BCL-xL/BCL-2 homologous antagonist killer protein (Bak) in SNK-6 cells, thereby liberating these proapoptotic proteins and further activating downstream apoptosis pathways by cleaving poly-ADP ribose polymerase-1 (PARP-1) and caspase-3. In an SNK-6 xenograft model, administration of APG-1252 at 65 mg/kg and 100 mg/kg either twice or once weekly resulted in significant antitumor effects, with tumor growth rate (T/C%) values ranging from 13.7% to 30.7%. Furthermore, the combination of APG-1252 with HDAC inhibitor chidamide or DDGP (dexamethasone, cisplatin, gemcitabine, and pegaspargase) chemotherapy demonstrated synergistic effects. Pharmacokinetic assessment in mice showed that APG-1252 had a long half-life in plasma (127 hours) and tumor tissues (25.2 hours), justifying intermittent dosing schedules used in vivo. Importantly, the transformation of APG-1252 to APG-1252-M1 was 16 times higher in tumor tissues compared to plasma (22% vs. 1.3%) after administration of APG-1252, thereby suggesting that APG-1252 can reduce platelet toxicity caused by APG-1252-M1 in plasma. In conclusion, APG-1252 has promising antitumor effects in NK/TCL, either as a single agent or in combination with an HDAC inhibitor or chemotherapy. These findings provide evidence to further evaluate APG-1252 as a potential treatment for NK/TCL. Disclosures Wang: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Liang: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Ming: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Rui: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Tang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. LV: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Ge: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Zhang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Wang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Shang: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding.

Background: Pelcitoclax (APG-1252), a novel dual inhibitor of Bcl-2/Bcl-xL, is active as monotherapy in patients with advanced solid tumors and well tolerated up to 240 mg twice weekly (NCT03387332). Preclinical data suggest that cells with Janus-associated kinase-2 (JAK2) mutations, including those associated with bone marrow fibrosis, are dependent on Bcl-2/Bcl-xL for survival and that addition of BH3 mimetics targeting Bcl-2/Bcl-xL induces apoptosis. Furthermore, in JAK2‒mutated cell models, apoptotic synergy is demonstrated when a JAK2 inhibitor and Bcl-2/Bcl-xL inhibitor are combined, as inhibition of Bcl-xL overcomes resistance to JAK2 inhibitors. Taken together, APG-1252 could overcome resistance to JAK2 inhibitors, and the combination could augment clinical benefit in patients with suboptimal responses to JAK2 inhibitor‒based therapy. Study Objectives: The primary objective of this open-label trial is to evaluate the safety and efficacy of APG-1252, as monotherapy and when combined with ruxolitinib, in adults with histologically or cytologically confirmed MF who require therapy and are ineligible for JAK2 inhibitors (and can receive single-agent APG-1252) or have had inadequate responses to ruxolitinib-based therapy (and can receive this treatment plus APG-1252). Secondary objectives include APG-1252 pharmacokinetics, time to response, and duration of response. Exploratory objectives include changes in cytogenetics and molecular mutations, bone marrow fibrosis, and cytokines on treatment. Study Design: The study is divided into Part 1 (APG-1252 monotherapy) and Part 2 (APG-1252 plus ruxolitinib). For Part 1, the key inclusion criterion is ineligibility for JAK2 inhibitors and for Part 2, inadequate responses to prior ruxolitinib-based therapy. A standard 3+3 dose-escalation design is being implemented to determine the maximum tolerated dose (MTD) of APG-1252 monotherapy in Part 1 and APG-1252 combined with ruxolitinib in Part 2. APG-1252 will initially be administered at 160 mg intravenously by 30-minute injection once weekly in a 28-day cycle. The dose can be escalated to a maximum of 240 mg or reduced to a minimum of 80 mg, depending on tolerability. Part 2 will begin once the MTD and recommended phase 2 dose (RP2D) of APG-1252 monotherapy have been determined. In Part 2, ruxolitinib will be administered orally twice daily per the package insert. After the MTD for APG-1252 monotherapy has been determined, no additional patients will be enrolled in Part 1; however, up to 15 to 30 additional patients can be enrolled in Part 2, to further evaluate the safety and anticancer activity of the combination at MTD or RP2D. Patients will continue treatment until disease progression or unacceptable toxicity. Clinical responses are being assessed every 12 weeks according to criteria from the International Working Group‒Myeloproliferative Neoplasms Research and Treatment and European LeukemiaNet panels, while optimal clinical benefit will be evaluated at 24 weeks. Enrollment will be from September 2020 and preliminary results estimated in October 2022. For further information, contact: yzhai@ascentage.com. Registration: ClinicalTrials.gov Identifier NCT04354727. Disclosures Pemmaraju: Pacylex Pharmaceuticals: Consultancy; Roche Diagnostics: Honoraria; LFB Biotechnologies: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; Celgene: Honoraria; AbbVie: Honoraria, Research Funding; MustangBio: Honoraria; Affymetrix: Other: Grant Support, Research Funding; Cellectis: Research Funding; Daiichi Sankyo: Research Funding; Plexxikon: Research Funding; Samus Therapeutics: Research Funding; DAVA Oncology: Honoraria; Blueprint Medicines: Honoraria; Novartis: Honoraria, Research Funding; Incyte Corporation: Honoraria; SagerStrong Foundation: Other: Grant Support. Mudenda:Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Wang:Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. JI:Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company. Lu:Ascentage Pharma Group: Current Employment, Current equity holder in publicly-traded company. Fu:Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Liang:Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. McClain:Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Sheladia:Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Verstovsek:Novartis: Consultancy, Research Funding; Sierra Oncology: Consultancy, Research Funding; Blueprint Medicines Corp: Research Funding; PharmaEssentia: Research Funding; ItalPharma: Research Funding; AstraZeneca: Research Funding; Protagonist Therapeutics: Research Funding; Promedior: Research Funding; Celgene: Consultancy, Research Funding; NS Pharma: Research Funding; Genentech: Research Funding; CTI Biopharma Corp: Research Funding; Incyte Corporation: Consultancy, Research Funding; Roche: Research Funding; Gilead: Research Funding. Yang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests. Zhai:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests.