ALBERT

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: 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.