ALBERT

Description: Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. Pro-survival molecules of BCL-2 family play critical roles in leukemia transformation and chemoresistance. The anti-leukemia potency of selective BCL-2 inhibitor venetoclax (ABT-199/GDC-0199) has been demonstrated in AML models (Pan et al. Cancer Discovery 2014). However, venetoclax is often associated with resistance due to its poor inhibition of MCL-1. RAF/MEK/ERK (MAPK) pathway is commonly activated in AML, and can stabilize anti-apoptotic MCL-1 and inactivate the pro-apoptotic BIM. In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib). First, anti-leukemia activity of cobimetinib and venetoclax was examined in 18 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death, as compared to the single agent treatment (Fig 1A). Cobimetinib inhibited cell proliferation in the majority of AML cases (34.2 ± 23.7%) and the cell growth suppression was more profound in the combination group (60.2 ± 28.8%, p

Description: The early phase studies have shown the high response rates in chronic lymphocytic leukemia (CLL) patients treated with the BH3 mimetic venetoclax (ABT-199). It indicated that inhibition of BCL-2 is a viable strategy for the treatment of lymphoid malignancies. Objective anti-tumor responses were also observed in patients with other common B-cell non-Hodgkin lymphomas (NHLs) such as follicular lymphoma (FL) or diffuse large B-cell lymphoma (DLBCL), however the overall response rates are not as high as those in CLL patients. Targeting only one anti-apoptotic protein may lead to or uncover resistance owing to activity of other anti-apoptotic BCL2-family members in these settings. MCL-1 is associated with both intrinsic and acquired resistance to venetoclax and thus inhibition of MCL-1 is being explored through either direct inhibition or indirect targeting. Expression of MCL-1 is maintained via p-TEFb-mediated transcription, of which CDK9 plays a critical role. Here we aimed to investigate the combined effects of CDK9 inhibitor and venetoclax in primary DLBCL and FL cells. Inhibition of CDK9 via a small molecule A-1467729.0 (AbbVie) caused rapid loss in phosphorylation (Serine 2) of RNA polymerase II and MCL-1 expression in all tested primary cells of DLBCL and FL patients, confirming the intended effect of CDK9 inhibition. Primary samples from 12 NHL cases (6 DLBCL including 3 GCB/3 non-GCB and 6 FL) were tested for their ex vivo response to A-1467729.0 or venetoclax alone or in combination. Apoptosis assays showed negligible effects (

Description: Pro-survival molecules including BCL-2 play critical roles in leukemia transformation and chemoresistance. ABT-199/GDC-0199 (venetoclax) is an orally available BH3-mimetic that binds with high affinity to BCL-2, but lacks affinity for BCL-XL and MCL-1. We have recently demonstrated anti-leukemia potency of venetoclax in acute myeloid leukemia (AML) models (Pan et al. Cancer Discovery 2014). However, venetoclax poorly inhibits MCL-1, causing resistance in leukemia cells that rely on MCL-1 for survival. The RAF/MEK/ERK (MAPK) cascade is a major effector pathway in AML that is activated by upstream mutant proteins such as FLT3, KIT and RAS. Additionally, the MAPK pathway regulates BCL-2 family proteins by stabilizing anti-apoptotic MCL-1 and inactivating pro-apoptotic BIM. In this study, we evaluated the anti-tumor effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib).. We initially examined activity of these agents in a panel of myeloid leukemia cell lines with diverse genetic alterations (Fig. 1A). The IC50 values of cobimetinib ranged from 〈 0.01 µM to 〉 1 µM after 72 hours of drug treatment but did not correlate with the basal level of p-ERK1/2. In 7 out of 11 cell lines, combination of the agents elicited synergistic growth inhibition. Notably synergism of venetoclax with cobimetinib was observed in venetoclax-resistant cell lines (MOLM14, OCI-AML3, NB4 and THP1). Ongoing analysis of pharmacodynamic markers include transcriptome assessment by RNA sequencing, functional proteomics by reverse phase protein array (RPPA) and quantification of BCL-2:BIM and MCL-1:BIM complexes using the electrochemiluminescent ELISA assay (Meso Scale Discovery, MSD-ELISA). The preliminary MSD data revealed that BCL-2:BIM complex was disrupted in most cell lines and accumulated following cobimetinib treatment, which may be due to the disruption of MCL-1:BIM complex by inhibition of MEK (Fig. 1B). In a long-term culture of primary AML blasts in serum-free stem cell growth medium supplemented with cytokines and StemRegenin 1 (SR1) to main the immature state of leukemia cells, the combination of venetoclax and cobimetinib induced distinct apoptotic cell death, with AML #1 sensitive to venetoclax but resistant to cobimetinib. Alternatively, AML #2 and #3 samples were resistant to venetoclax but sensitive to cobimetinib and the combination of both drugs (Fig. 1C). We next investigated signaling patterns and BCL-2 family protein expression in AML stem/progenitor cells using a 34-antibody panel and time-of-flight mass cytometry (CyTOF). In AML#1, BCL-2 was expressed in leukemia blasts, with enrichment in a progenitor AML population phenotypically defined as CD45dim CD34+ CD38+ CD123+ CD33+ (Fig. 1D). The high expression level of BCL-2 and low expression of MCL-1 and BCL-XL may account for sensitivity to venetoclax in AML#1. Both basal and G-CSF- or SCF-stimulated p-ERK was efficiently down-regulated by cobimetinib; however, G-CSF-evoked p-STAT3/5 and SCF-induced p-AKT were only slightly reduced (Fig. 1E). Notably we observed increased phosphorylation of STAT5 pathway upon treatment with cobimetinib, suggesting that active MAPK signals inhibit phosphorylation of the JAK-STAT pathway, as previously reported (Krasilnikov et al. Oncogene, 2003 and Lee at al. Cancer Cell, 2014). To test the efficacy of both compounds in vivo, we injected NSG mice with genetically engineered OCI-AML3/Luc/GFP cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the cobimetinib single agent and venetoclax plus cobimetinib co-treated mice (Fig. 1F). The efficacy study is ongoing and median survival for cobimetinib and venetoclax co-treated mice has yet to be determined (Fig. 1G). In summary, our data demonstrates that combinatorial blockade of MAPK and BCL-2 pathways is synergistic in the majority of AML cell lines tested and can overcome intrinsic resistance to venetoclax. Ongoing studies will evaluate efficacy of this combination therapy in primary human AML xenografts and elucidate mechanisms of synergy. Disclosures Leverson: AbbVie: Employment, Equity Ownership. Dail:Genentech: Employment, Equity Ownership. Phillips:AbbVie: Employment, Other: Shareholder, Patents & Royalties. Chen:Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Jabbour:Pfizer: Consultancy, Research Funding. Sampath:Genentech: Employment, Equity Ownership. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Description: Acute myeloid leukemia (AML) is a clonal hematologic malignancy characterized by genomic heterogeneity and epigenetic changes, including aberrant DNA hypermethylation. Phase-Ib clinical data in relapsed/refractory AML patients indicate that combining venetoclax with the hypomethylating agents (HMAs) 5-azacitidine (5-Aza) or decitabine results in an overall response (OR) of 62% (DiNardo et al. 2018) compared to the historical OR of 28-29% with HMAs treatment alone (Kantarjian et al. 2013; Dombret et al. 2015). Subsequently, a randomized phase-III clinical trial was initiated to evaluate venetoclax activity in combination with 5-Aza in treatment-naïve AML patients ineligible for standard induction therapy (M15-656, NCT02993523). However, the underlying mechanism for the combinational activity observed between venetoclax and 5-Aza is unknown. In this study, we demonstrate that both chronic low-dose 5-Aza treatment, which induced global DNA demethylation, and acute treatment (24 hours, non-epigenetic effects) can drive combinational activity with venetoclax in AML through distinct mechanisms. Chronic culture with a low-dose 5-Aza for one week sensitized AML cell lines to venetoclax in vitro. In contrast, acute treatment with 5-Aza, activated the integrated stress response (ISR) pathway to induce expression of the BH3-only proteins NOXA (PMAIP1) and PUMA (BBC3) in human AML cell lines, independent of DNA methylation. This resulted in an increase in the amount of NOXA and/or PUMA in complex with anti-apoptotic proteins like BCL-2, BCL-XL and MCL-1, thereby "priming" AML cells for induction of apoptosis by venetoclax treatment. Priming for apoptosis resulted in significant synergistic cell death in a panel of AML cell lines treated with venetoclax and 5-Aza in vitro. In this panel of cell lines, the level of the PMAIP1, BBC3, and DDIT3 gene induction correlated with the synergy observed between venetoclax and 5-Aza. Importantly, subsequent PMAIP1 deletion significantly impacted the kinetics and depth of apoptosis induced by 5-Aza or venetoclax alone or in combination. In accordance with the in vitro combinational activity, the venetoclax/5-Aza combination provided added benefit over either agent alone in two xenograft models of AML. Together, these data provide a rationale for an ongoing randomized phase-III clinical trial evaluating venetoclax activity in combination with 5-Aza (M15-656, NCT02993523). Disclosures: DC, SJ, JP, RP, NT, YX, EB, JL, and DP are employees of AbbVie. LS is a former employee of AbbVie and was employed during the duration of this study. The design, study conduct, and financial support for this research were provided by AbbVie and Genentech. AbbVie participated in the interpretation of data, review, and approval of the publication. Disclosures Cojocari: AbbVie Inc: Employment. Jin:AbbVie Inc: Employment, Equity Ownership. Purkal:AbbVie Inc: Employment, Equity Ownership. Popovic:AbbVie Inc: Employment, Equity Ownership. Talaty:AbbVie Inc: Employment, Equity Ownership. Xiao:AbbVie Inc: Employment, Equity Ownership. Solomon:AbbVie Inc: Equity Ownership. Boghaert:AbbVie Inc: Employment, Equity Ownership. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties.

Description: All authors are employees of AbbVie and participated in the design, conduct, and interpretation of these studies. AbbVie and Genentech provided financial support for these studies and participated in the review and approval of this publication. The BCL-2-selective inhibitor ABT-199 has demonstrated efficacy in numerous preclinical models of hematologic malignancies without causing thrombocytopenia, a dose-limiting toxicity associated with the BCL-2/BCL-XL inhibitor navitoclax (Souers et al. 2013. Nat. Med. 19, 202-208). ABT-199 has also demonstrated clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (Seymour et al. 2014. J. Clin. Oncol. 32, 448s; Davids et al. 2014. J. Clin. Oncol. 32, 544s). Despite these encouraging early clinical data, some subjects do not respond to ABT-199 or progress while on treatment. Pre-clinical models indicate that both intrinsic and acquired resistance may be a consequence of MCL-1 expression. Consequently, we have explored potent and selective small molecule inhibitors of CDK9, a kinase known to maintain the expression of MCL-1 through its role in p-TEFb-mediated transcription. Inhibition of CDK9 resulted in the rapid loss in RNA polymerase II phosphorylation (Serine 5) and MCL-1 expression that was closely followed by the induction of apoptosis in MCL-1-dependent cell lines, a cellular response that could be rescued by overexpression of BCL-2. Substantial synergy was observed between CDK9 inhibitors and ABT-199 in a number of hematologic cell lines with intrinsic or acquired resistance to ABT-199. Direct inhibition of MCL-1 with the small molecule BH3 mimetic A-1210477 was also highly synergistic with ABT-199, further validating the utility of co-inhibiting MCL-1 and BCL-2 function simultaneously in ABT-199 resistant tumors. Importantly, the CDK9 inhibitor-ABT-199 combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in xenograft models of non-Hodgkin’s lymphoma (NHL) and acute myelogenous leukemia (AML). These data indicate that CDK9 inhibitors may be highly efficacious when used in combination with ABT-199 for the treatment of hematologic malignancies. Disclosures Chen: Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Tapang:abbvie: Employment, Equity Ownership. Tahir:abbvie: Employment, Equity Ownership. Smith:abbvie: Employment, Equity Ownership. Xue:abbvie: Employment, Equity Ownership. Zhang:abbvie: Employment, Equity Ownership. Gao:abbvie: Employment, Equity Ownership. Tong:abbvie: Employment, Equity Ownership. Clark:abbvie: Employment, Equity Ownership. Ricker:abbvie: Employment, Equity Ownership. Penning:abbvie: Employment, Equity Ownership. Albert:abbvie: Employment, Equity Ownership. Phillips:abbvie: Employment, Equity Ownership. Souers:abbvie: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership.

Description: Better therapeutic strategies are needed for patients with mantle cell lymphoma (MCL), an aggressive and largely incurable subtype of Non-Hodgkin Lymphoma. Concurrent expression of anti-apoptotic BCL2 family proteins in lymphoma cells contribute to their evasion of apoptosis. Therefore, targeting only one anti-apoptotic protein may lead to or uncover resistance associated with activity of other anti-apoptotic BCL2 family members. A variety of cyclin-dependent kinase (CDK) inhibitors are undergoing clinical trials either as a single agent or in combination with other approved drugs. CDK9, a portion of the elongation factor P-TEFb, phosphorylates Ser-2 in the C-terminal domain of RNA Polymerase II, which is required for transcript elongation. The effect of CDK9 inhibition is observed most immediately on those proteins with rapid turnover rates such as the BCL2 family protein MCL1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. Here we report the responses of 4 MCL cell lines (Mino, Jeko-1, CCMCL1 and JVM2) and 5 primary MCL samples (representing de novo and relapsed cases, including two relapsed cases after ibrutinib failure and a relapsed case harbor Myc rearrangement) to venetoclax and a novelCDK9 inhibitor A-1467729. Exposure of Mino and Jeko-1 cells to venetoclax rapidly induced apoptosis (IC50 at 5 hours were 235 and 955 nM, respectively). In contrast, CCMCL1 and JVM2 cells were not sensitive to venetoclax with IC50s 〉 3000 nM. However, CCMCL1 cells were more responsive to A-1467729 alone than the other 3 lines, while JVM2 cells were much less sensitive to A-1467729. All primary samples were sensitive to venetoclax, ex vivo, at the doses between 1 - 100 nM, although their IC50s were variable (range: 2-90 nM). A-1467729 at doses of 1-20 nM had modest single agent effects on the primary samples; however, its combination with venetoclax synergistically induced apoptosis and decreased the IC50 of venetoclax by 2-10 times in all cell lines and primary samples. The strongest synergy was observed in Jeko-1 cells with all combined indexes 〈 0.1. Studies on mechanisms through immunoblotting and immunohistochemical staining demonstrated that A-1467729 quickly down-regulated phospho-RNA Polymerase II (Ser2) and MCL1 protein levels. CCMCL1 cells lack BCL2 expression, while JVM2 displayed higher expression of MCL1 than other cells. The expression levels of BCL2 and MCL1 in primary samples were case-dependent as well. The expression pattern and level of anti-apoptotic BCL2 family proteins in cell lines and primary cases may be responsible for their variable reactions to these two agents. To further confirm that CDK9 inhibition was affecting cell viability at least partially through its function on MCL1, A-1210477, a MCL1 inhibitor, was applied to the same study. Strong synergistic apoptotic induction was also observed when A-1210477 was combined with venetoclax, especially in MCL1-"dependent" CCMCL1 cells as evidenced by flow cytometry based apoptotic assay and PARP cleavage. Further mechanism studies aiming the effects of CDK9 inhibitor/venetoclax on MCL1/BIM association is being under investigation. MCL mouse xenograft study for such a combined effect has been planned. In summary, the combination of a CDK9 inhibitor and venetoclax showed synergistic induction of apoptosis in both MCL cell lines and primary patient samples. These findings support further evaluation of the efficacy of such a combination in MCL, including ibrutinib-resistant MCL. Disclosures Souers: AbbVie: Employment. Phillips:AbbVie Inc.: Employment. Hsi:HTG Molecular Diagnostics: Consultancy; Abbvie: Honoraria, Research Funding; Seattle Genetics: Honoraria; Cellerant: Honoraria, Research Funding; Eli Lilly: Research Funding; Onyx Pharmaceuticals: Honoraria.

Description: Introduction: Venetoclax (VEN), a selective BCL-2 inhibitor, has yielded exceptional response rates in patients with acute myeloid leukemia (AML). VEN binds BCL-2 to directly inhibit sequestration of pro-apoptotic proteins such as the activator BIM. Free BIM can bind to BAX, enabling its oligomerization with BAK, mitochondrial outer membrane permeabilization (MOMP) and subsequent apoptosis (PMID 24074954, 9687260, 19641500, 20164920). VEN has limited efficacy in relapsed-refractory AML as a single agent, but when used in combination with DNA methyltransferase inhibitors (DNMTi; 5'azacitidine-AZA-or decitabine) or low-dose cytosine arabinoside (LDAC), 50-70% of untreated patients in recent clinical trials achieved complete remission (PMID 27520294, 30361262, 30892988). Despite this progress in AML therapy, the majority of AML patients treated with VEN ultimately relapse, and a large subset of patients never respond (PMID 30361262, 30892988). One postulated route of resistance to VEN is cellular upregulation of myeloid cell leukemia-1 protein (MCL-1), which, similarly to BCL-2, functions as an anti-apoptotic protein. VEN in combination with selective MCL-1 inhibitors has demonstrated added benefit over either agent alone in AML cells in vitro and in xenograft models but efficacy of this combination in the clinic has yet to be reported (PMID 30185627). Both MCL-1-dependent and MCL-1-independent mechanisms of VEN resistance are emerging (PMID 31048321, 30148320, 31262744) and new approaches aimed at addressing these are moving toward the clinic. Pevonedistat (PEV) was developed as a targeted inhibitor of NEDD-8 activating enzyme (NAE), which disrupts protein turnover mediated by Cullin-RING ligases (PMID 19360080) and has been shown to have activity in combination with AZA (PMID 29348128). PEV and AZA both upregulate NOXA, a BCL-2 family member that is known to suppress MCL-1 (PMID 26045051, Jin, Cojocari, Purkal et al.,unpublished data), so we postulated that PEV/AZA in combination synergizes with VEN in a triple combination with efficacy superior to VEN/AZA alone. Methods/Results: AML cell lines were treated with PEV, AZA and VEN alone, and compared with various VEN/PEV/AZA combinations revealing improved combinatorial activity with the triplet in the majority of cell lines and associated NOXA induction. Similar results were seen in primary AML patient samples ex vivo (Figure 1). These combinations were compared in vivo in an OCI-AML2 cell line xenograft model, also illustrating significantly improved response with the PEV/AZA/VEN combination (Figure 2). CRISPR/Cas 9 deletion of PMAIP1 in AML cell lines demonstrated that NOXA deletion abrogates this synergy. Discussion: Together, these results demonstrate that, in several pre-clinical models of AML, the PEV/AZA/VEN triple combination provides stronger anti-tumorigenic activity than either agent alone or the VEN/PEV and VEN/AZA combinations. Importantly, the three-drug combination may be effective in AML cells which do not respond to VEN/AZA alone. Further studies to delineate this mechanism of action, and a clinical trial (NCT03862157) testing the combination in newly diagnosed AML are underway. Figure 1 Disclosures Cojocari: AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL & DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Purkal:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL & DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Leverson:AbbVie Inc: Employment, Other: Stock or options. Boghaert:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL & DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Phillips:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL & DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Savona:AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Boehringer Ingelheim: Patents & Royalties; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees.

Description: Mutations in isocitrate dehydrogenase 2 (IDH2) promote AML pathogenesis through production of 2-hydroxyglutarate (2-HG). Enasidenib is an inhibitor of mutant IDH2 activity and induces the differentiation of IDH2-mutated leukemic blasts. In a phase I/II clinical trial, enasidenib monotherapy resulted in an overall response rate of 40% and median duration of response of 6 months in relapsed/refractory AML (Stein et. al. Blood 2017). We previously discovered that mutant IDH activity sensitizes AML cells to BCL-2 inhibition through accumulation of 2-HG (Chan et. al. Nature Medicine 2015). Pharmacologic inhibition of BCL-2 activity with venetoclax, a highly specific BH3 mimetic, preferentially targets IDH-mutated human AML cells. In a phase II clinical trial of venetoclax monotherapy, IDH-mutated relapsed/refractory AML patients had a response rate of 33% compared to 10% in IDH-wildtype patients (Konopleva et. al. Cancer Discovery 2016). Based on the above findings, we hypothesized that combination therapy with enasidenib and venetoclax may demonstrate superior anti-leukemic activity in comparison to single agents in the treatment of IDH2-mutant AML. However, given that the mechanism by which IDH mutations increase BCL-2 dependence is via 2-HG, reduction of 2-HG with enasidenib may antagonize venetoclax activity. As such, we further hypothesized that a sequential dosing schedule may be superior to concurrent dosing. To test our hypotheses, we conducted a preclinical study to evaluate the efficacy of monotherapy versus combination therapy in reducing the leukemic burden in three patient-derived xenograft (PDX) models of human IDH2-mutant AML. All three PDX models were derived from samples with co-occurring IDH2R140Q and NPM1c mutations. Engrafted animals were randomly assigned to one of six treatment arms (N=5 per arm): vehicle (arm 1), enasidenib alone (arm 2), venetoclax alone (arm 3), concurrent combination (arm 4), and sequential combinations (arms 5 and 6; see Figure 1 for details). Enasidenib and venetoclax were administered by oral gavage at a dose of 40 mg/kg twice a day and 100 mg/kg daily, respectively. Tumor burden was measured in bone marrow samples collected immediately prior to treatment and every 2 weeks during the 12-week treatment period. Concurrent combination treatment (arm 4) resulted in the greatest reduction in leukemia engraftment compared to all other treatment arms, including the sequential dosing arms, in two of the three models (#1 and #2, henceforth termed "responders"; Figure 2A). Although venetoclax monotherapy reduced engraftment in all three models, persistent disease above a threshold of 0.1% was detectable in 12 of 13 animals by flow cytometry (9 of 13 by ddPCR) after 12 weeks of treatment (Figure 2B). In contrast, disease was detectable in only 2 of 9 animals (0 of 9 by ddPCR) treated with concurrent therapy in responders. In the remaining model (#3, henceforth termed "non-responder"), combination therapy was not superior to venetoclax monotherapy but importantly, co-treatment with enasidenib did not antagonize venetoclax activity. Interestingly, enasidenib monotherapy increased expression of myeloid differentiation markers, CD15 and CD11b, only in responders, indicating that differentiation might be a precondition for responsiveness to concurrent therapy. We confirmed that the lack of response in non-responders was not due to selection of an IDH2 wildtype clone or failure to block 2-HG production by enasidenib. To gain insights into the mechanism by which enasidenib might enhance venetoclax sensitivity, we performed quantitative intracellular flow cytometry staining for the anti-apoptotic proteins BCL-2, BCL-xL and MCL-1 in leukemic cells collected after 12 weeks of treatment. Enasidenib monotherapy resulted in a significant decrease in BCL-2 expression in responders. The reduction in anti-apoptotic protein expression could potentiate mitochondrial priming and sensitization to venetoclax. In summary, our findings demonstrate that concurrent combination therapy with enasidenib and venetoclax is a promising therapeutic approach for IDH2-mutated AML. Responsiveness to combination therapy is associated with enasidenib-induced differentiation and reduction in anti-apoptotic protein expression. Our findings support ongoing and future clinical investigations in combination therapies with mutant IDH and BCL-2 inhibitors. Disclosures Cojocari: AbbVie Inc: Employment. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. MacBeth:Celgene Corporation: Employment, Equity Ownership. Nicolay:Agios: Employment. Narayanaswamy:Agios: Employment. Ronseaux:Agios: Employment. Liu:Agios: Employment, Equity Ownership. Chan:AbbVie: Research Funding; Genentech: Research Funding; Celgene: Research Funding.