BCL-2 Dependence Research Articles

A Multicenter Phase I Study Combining Venetoclax with Mini-Hyper-CVD in Older Adults with Untreated and Relapsed/Refractory Acute Lymphoblastic Leukemia

A Multicenter Phase I Study Combining Venetoclax with Mini-Hyper-CVD in Older Adults with Untreated and Relapsed/Refractory Acute Lymphoblastic Leukemia

PKR-like Endoplasmic Reticulum Kinase Mediates Apoptosis Induced By Pharmacological AMP-Activated Protein Kinase Activation in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a myeloid progenitor-derived neoplasm of poor prognosis, particularly among the elderly, in whom age and comorbidities preclude the use of intensive therapies. Novel therapeutic approaches for AML are therefore critically needed. Adenosine monophosphate (AMP) activated protein kinase (AMPK) is a pleiotropic serine/threonine kinase promoting catabolism that represses anabolism and enhances autophagy in response to stress1. AMPK heterotrimers comprise catalytic α- and regulatory β- and γ-subunits, the latter harboring binding sites for AMP. Targets of AMPK include a host of metabolic pathway enzymes mediating carbohydrate, lipid and protein synthesis and metabolism. Accumulating evidence implicates AMPK in cancer biology, primarily as a tumor suppressor, although minimal AMPK activity may also be required for cancer cell growth under stress conditions2,3. Pharmacological activation of AMPK thus represents an attractive new strategy for targeting AML. We previously used the selective small molecule AMPK activator GSK621 to show that AMPK activation induces cytotoxicity in AML but not in normal hematopoietic cells, contingent on concomitant activation of the mammalian target of rapamycin complex 1 (mTORC1)4. However, the precise mechanisms of AMPK-induced AML cytotoxicity have remained unclear. We integrated gene expression profiling and bioinformatics proteomic analysis to identify the serine/threonine kinase PERK - one of the key effectors of the endoplasmic reticulum stress response - as a potential novel target of AMPK. We showed that PERK was directly phosphorylated by AMPK on at least two conserved residues (serine 439 and threonine 680) and that AMPK activators elicited a PERK/eIF2A signaling cascade independent of the endoplasmic reticulum stress response in AML cells. CRISPR/Cas9 depletion and complementation assays illuminated a critical role for PERK in apoptotic cell death induced by pharmacological AMPK activation. Indeed, GSK621 induced mitochondrial membrane depolarization and apoptosis in AML cells, an effect that was mitigated when cells were depleted of PERK or expressed PERK with a loss of function AMPK phosphorylation site mutation. We identified the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) as a downstream target of the AMPK/PERK pathway, as its expression was enhanced in PERK knockdown AML cells. Moreover, selective pharmacologic activation of ALDH2 by the small molecule ALDA-1 recapitulated the protective effects of PERK depletion in the face of pharmacological AMPK activation. Corroborating the impact of the AMPK/PERK axis on mitochondrial apoptotic function, BH3 profiling showed marked Bcl-2 dependency in AML cells treated with GSK621. This dependency was abrogated in PERK-depleted cells, suggesting a role for PERK in mitochondrial priming to cell death. In vitro drug combination studies further demonstrated synergy between the clinical grade Bcl-2 inhibitor venetoclax (ABT-199) and each of four AMPK activators (GSK621, MK-8722, PF-06409577 and compound 991) in multiple AML cell lines. Finally, the addition of GSK621 to venetoclax enhanced anti-leukemic activity in primary AML patient samples ex vivo and in humanized mouse models in vivo. These findings together clarify the mechanisms of cytotoxicity induced by AMPK activation and suggest that combining pharmacologic AMPK activators with venetoclax may hold therapeutic promise in AML.

Targeting Critical Kinases and Anti-Apoptotic Molecules Overcomes Steroid Resistance in Infant MLL-Rearranged Leukemia

Acute lymphoblastic leukemia (ALL) with rearrangement of the mixed-lineage leukemia (MLL) gene frequently affects infants and is associated with a poor prognosis. Standard treatment protocol for infant MLL-rearranged ALL (MLL-ALL) includes glucocorticoids (GCs). However, resistance to GCs remains a major problem. Therefore, it is important to find new treatment strategies that overcome GC resistance in MLL-ALL. To identify novel therapeutic targets to overcome GC resistance in MLL-ALL, we compared transcriptional profiles of normal cord blood CD34+CD38- hematopoietic stem and progenitor cells and GC-resistant infant MLL-ALL leukemia-initiating cells (LICs). We found enrichment of the Src family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathways in the LICs. We hypothesized that activation of these kinases may contribute to GC resistance in MLL-ALL cells. Using our previously developed infant MLL-ALL patient-derived xenograft (PDX) models (Aoki et al., Blood, 2015), we recapitulated human GC-resistance in vivo, characterized by increased hCD45+ chimerism in the peripheral blood after treatment with dexamethasone (% peripheral blood human MLL-ALL at pre-treatment: 34.1±5.2% vs. post treatment 51.3±6.6%, n=26). In order to investigate if inhibition of SFKs and FLT3 can overcome GC-resistance, we treated the MLL-ALL PDX models with our previously developed FLT3-SFK multiple kinase inhibitor RK-20449 (Saito et al., Science Translational Medicine, 2013). Combination treatment with dexamethasone and RK-20449 successfully eliminated human MLL-ALL cells from the peripheral blood (dexamethasone alone: 47.2±7.2% vs. combination: 5.6±2.8% hCD45+ cells, p=6.91E-7), bone marrow (dexamethasone alone: 72.2±4.6% vs. combination: 19.5±4.8% hCD45+ cells, p=1.79E-10), and spleen (dexamethasone alone: 52.6±4.8% vs. combination: 11.7±3.5% hCD45+ cells, p=3.75E-9). In addition, combination treatment eliminated infiltrated MLL-ALL from kidney and liver of the MLL-ALL PDX models. Bcl-2 homology domain 3 (BH3) profiling demonstrated that MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic Bcl-2 protein for their survival. Additional inhibition of Bcl-2 by ABT-199 led to complete elimination of MLL-ALL cells in vitro and in vivo in all MLL-ALL patient cases (Figure1). Taken together, in this study we demonstrated that inhibition of SFKs and FLT3 by RK-20449 overcomes GC-resistance in MLL-ALL. Further, we identified Bcl-2 dependence as a mechanism of treatment resistance in MLL-ALL. Therefore, we believe that the combined inhibition of kinase and anti-apoptotic pathways may lead to effective treatment options for highly resistant infant MLL-ALL. Aoki, et al., Identification of CD34+ and CD34- leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia. Blood, 2015: p. 967-980. Saito, Y., et al., A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo. Science Translational Medicine, 2013: p. 1-15. Disclosures No relevant conflicts of interest to declare.

Abstract 2715: Deciphering a metabolic basis for single-agent venetoclax efficacy in t(11;14) multiple myeloma

Abstract Multiple myeloma (MM) is the second most common hematologic malignancy. In 2017, MM accounted for approximately 30,770 new diagnoses and 12770 deaths in the US. Although, advancements in treatment options have increased survival rates and life expectancy, MM remains incurable due to development of resistance. Venetoclax is a highly selective, potent BCL-2 antagonist, currently in phase I/II trials for MM and FDA approved for the treatment of CLL patients exhibiting 17p deletion. Venetoclax is effective in eliciting cell death as a single agent in a subset of MM with the (11;14) translocation (which we henceforth designate “sensitive”) in contrast to the majority of MM that is resistant (which we henceforth designate “resistant”). We previously reported that glutamine deprivation increases BIM binding to BCL-2 thereby sensitizing MM to venetoclax, while alpha-ketoglutarate supplementation reversed this sensitivity. We were therefore interested to explore if there was a metabolic basis for t(11;14)-myeloma sensitivity to single agent venetoclax to aid in 1) identifying venetoclax sensitive MM and; 2) inform us of metabolic targets that could be inhibited to sensitize resistant MM to venetoclax. We first queried the CoMMpass MM patient RNAseq trial data and cell lines for electron transport chain (ETC) and TCA cycle gene expression differences in t(11;14) vs non-t(11;14) patients and cell lines. All sensitive cells exhibited varied suppression of TCA/ETC genes, and reduced TCA cycle metabolites and oxygen consumption rates (OCR) compared to resistant cells. Examination of TCA cycle activities connected to the ETC and OCR showed significant decrease in Complex II Succinate ubiquinone reductase (SQR) activity in sensitive cell lines and purified CD38+ve MM patient cells and elevated SQR activity in resistant cells. Furthermore, inhibition of SQR with thenoyltrifluoroacetone (TTFA) sensitized resistant cells to venetoclax. Consistent with SQR inhibition leading to ABT-199 sensitivity, overexpression of an SQR mutant (SDHCR72C) in SDHC-knockout resistant MM, increased venetoclax sensitivity, identifying a unique role for SQR in regulating BCL-2 dependence. In interrogating the mechanistic underpinnings of SQR inhibition-induced sensitivity to venetoclax, we identified increased expression of ATF4 and BIM upon SQR inhibition. ATF4KD or BIMKO restored viability in TTFA and venetoclax co-treated MM further confirming the role of SQR inhibition-induced ATF4 and BIM in venetoclax sensitivity. In testing translationally relevant compounds, we determined that the histone deacetylase inhibitor, panobinostat reduced SQR activity in a dose dependent manner and sensitized MM cells to venetoclax. Our study thus identifies SQR as a novel target and predictive marker to aid in identifying ABT-199-responsive MM patients in a functional biomarker informed manner. Citation Format: Richa Bajpai, Abhinav Achreja, Changyong Wei, Arusha Siddiqa, Shannon M. Matulis, Vikas Gupta, Samuel K. McBrayer, Anjali Mittal, Manali Rupji, Hsiao-Rong Chen, Jeanne Kowalski, Sagar Lonial, Ajay K. Nooka, Lawrence H. Boise, Deepak Nagrath, Mala Shanmugam. Deciphering a metabolic basis for single-agent venetoclax efficacy in t(11;14) multiple myeloma [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 2715.

ECOG-ACRIN EAA172: Phase 1/2 study of daratumumab, bortezomib, dexamethasone (DVd) with or without venetoclax in relapsed/refractory multiple myeloma (RRMM) with assessment for t(11;14) status.

TPS8052 Background: The most common translocation in multiple myeloma (MM) is t(11;14)(q13;q32), present in approximately 20% of cases. MM cells with t(11;14) usually have a favorable high BCL-2 level and inferior outcomes compared to standard risk MM. Venetoclax (VEN) is a potent, selective, orally available small-molecule BCL-2 inhibitor that induces cell death in MM cell lines and primary samples. VEN has single agent activity in relapsed/refractory MM (RRMM) with an acceptable safety profile, especially in t(11;14) MM; however, non- t(11;14) MM patients may benefit from single agent VEN and VEN incorporated in multi-agent RRMM regimens. Dexamethasone (d) promotes Bcl-2 dependence in MM resulting in sensitivity to VEN and this combination with bortezomib (Vd-VEN) has an acceptable safety profile with high response rates in heavily pre-treated MM. Combination therapy with daratumumab and bortezomib (DVd) has become a standard of care in RRMM. Our hypotheses are that the addition of VEN will improve upon this standard and be most effective in the t(11;14) positive subset. Methods: Eligibility criteria include RRMM with measurable disease, not bortezomib refractory, platelet count > 100K. t(11;14) is an integral biomarker with status (positive or negative) established at registration. After a Ph1 study to determine the recommended phase 2 VEN dose, patients are randomized to DVd +/- VEN [stratified by prior lines of therapy and R-ISS]. The primary Ph2 objectives are to compare 8-cycle minimal residual disease (MRD) negative rate and to inform the role of t(11;14) as a biomarker. The Ph2 design proposed by Freidlin et al. follows a decision algorithm as outlined in the table below. Simulations were run to establish an optimal sample size given various parameters including biomarker prevalence and power to make appropriate decisions for a Ph3 design. Target Ph2 accrual is 240 patients with a 1/3 positive:2/3 negative t(11;14) split. Clinical trial information: NCT03701321. [Table: see text]

Interactions between cancer-associated fibroblasts and tumor cells promote MCL-1 dependency in estrogen receptor-positive breast cancers

Selective inhibition of BCL-2 is expected to enhance therapeutic vulnerability in luminal estrogen receptor-positive breast cancers. We show here that the BCL-2 dependency of luminal tumor cells is nevertheless mitigated by breast cancer-associated fibroblasts (bCAFs) in a manner that defines MCL-1 as another critical therapeutic target. bCAFs favor MCL-1 expression and apoptotic resistance in luminal cancer cells in a IL-6 dependent manner while their own, robust, survival also relies on MCL-1. Studies based on ex vivo cultures of human luminal breast cancer tissues further argue that the contribution of stroma-derived signals to MCL-1 expression shapes BCL-2 dependency. Thus, MCL-1 inhibitors are beneficial for targeted apoptosis of breast tumor ecosystems, even in a subtype where MCL-1 dependency is not intrinsically driven by oncogenic pathways.

BH3-mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma treatment

AbstractBH3 mimetics are promising drugs for hematologic malignancies that trigger cell death by promoting the release of proapoptotic BCL2 family members from antiapoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival, based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL, or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3 mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addition, 35% of overall patient samples showed codependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3 mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection from cell death observed by BAK knock-down, as well as the complete and early disruption of MCL1/BAK complexes on A1210477 treatment. Interestingly, this complex was also dissociated in A1210477-resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3 mimetics for clinical evaluation in myeloma at both diagnosis and relapse.

Dynamic BH3 Profiling Predicts Patient Response and MRD Status in Chronic Lymphocytic Leukemia (CLL) Patients Undergoing Frontline Treatment with Kinase Inhibitor Plus FCR (KI+FCR)

Dynamic BH3 Profiling Predicts Patient Response and MRD Status in Chronic Lymphocytic Leukemia (CLL) Patients Undergoing Frontline Treatment with Kinase Inhibitor Plus FCR (KI+FCR)

Respective Place of Venetoclax and MCL1 BH3 Mimetics in Multiple Myeloma Treatment

Targeting anti-apoptotic proteins of the BCL2 family by BH3 mimetics is a new promising therapeutic approach in multiple myeloma (MM). The specific BH3 mimetic targeting BCL2, BCLXL or MCL1 trigger apoptosis and exploit the dependency on these different anti-apoptotic proteins to kill tumor cells. Because MM is mostly considered as dependent on MCL1, the recent clinical availability of MCL1 BH3 mimetics underlines an urgent need to better define patients that would benefit from a MCL1 targeted therapy. In the present study, we used a BH3 mimetic toolkit that includes venetoclax, A1155463 and A1210477, which target BCL2, BCLXL and MCL1 respectively to define dependencies/co-dependencies in a large cohort of 60 myeloma patients (21 at diagnosis and 39 at relapse). Alternatively, MCL1 dependency was confirmed using the S63845 MCL1 inhibitor in MM patient samples. Mononuclear bone marrow/blood cells were treated overnight with the respective BH3 mimetic and cell death was specifically measured in the tumor cell population. Primary MM cells dependencies were stratified using PCA analysis in three groups as highly dependent, intermediately dependent or not dependent. Our study demonstrated that half of patients at diagnosis were BCL2 dependent while only 10% were BCLXL dependent. The dependence on BCL2 or BCLXL was not significantly different between samples at diagnosis and relapse. Strikingly, we found that the MCL1 dependency was 33% at diagnosis while it was 69% at relapse, suggesting a significant increase in MCL1 dependency during the disease progression (p=0.01). Besides, 36% of overall patients showed co-dependencies on BCL2/MCL1. We also identified primary MM cells that did not depend on any of the three pro-survival molecules, both at diagnosis and relapse.Among this cohort of MM patients, 47 samples were further analyzed for the presence of recurrent translocations (t(11;14), t(6;14), t(4;14) and t(14;16)) allowing the analysis of dependencies in the different subgroups; these recurrent translocations lead to the overexpression of CCND1, CCND3, MMSET and MAF oncogenes, respectively. We found that BCL2 dependency was significantly higher in CCND1 subgroup (83%) compared to all other subgroups (20%, p=0.008). We also confirmed the BCL2/BCLXL mRNA ratio as a valuable biomarker to define BCL2 dependence (p=0.0001). At diagnosis, MCL1 dependency was absent in patients not harboring the common recurrent translocations while at relapse 6 out 9 patients not harboring the recurrent translocations were MCL1 dependent, indicating an increase of MCL1 dependency at relapse in this subgroup (p=0.03).Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection of cell death observed by BAK knock-down and the complete disruption of MCL1/BAK complexes upon A1210477 treatment, observed in MM cell lines and also in a patient sample. Interestingly, this complex was also dissociated in A1210477 resistant cells but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. Thus, BCLXL may act as a sink to bind freed pro-apoptotic proteins from MCL1 and limits MM cell death triggered by the specific targeting of MCL1.In conclusion, our study highlights the potential clinical use of BH3 mimetics in MM treatment guided by the practical ex-vivo testing of myeloma cell dependencies using the BH3 toolkit. This strategy could be used to identify the respective and tailored use of venetoclax, MCL1 BH3 mimetics or their combination in myeloma treatment. DisclosuresMoreau:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Combination of Enasidenib and Venetoclax Shows Superior Anti-Leukemic Activity Against IDH2 Mutated AML in Patient-Derived Xenograft Models

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. DisclosuresCojocari: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.

The Amount of Apoptosis Predicts Outcome in Ibrutinib-Treated Chronic Lymphocytic Leukemia (CLL)

BackgroundAltough the inhibitor of Bruton's tyrosyne kinase (BTK) ibrutinib has transformed the management of patients with CLL obtaining decreased tumor burden and improved progression free survival (PFS), it does not induce substantial apoptosis in vitro (Ponader, 2012). Dysregulation of the mitchondrial pathway of apoptosis is one of the hallmarks of CLL cell pathophysiology and thus targeting the anti-apoptotic protein bcl-2 with venetoclax is a promising new therapeutic strategy (Souers, 2013; Roberts, 2016). Moreover ibrutinib increases bcl-2 dependence (Deng, 2017) enhancing sensitivity to venetoclax in CLL. Therefore the basal level of apoptosis measured through bax/bcl-2 ratio before therapy may be crucial to test sensitivity to ibrutinib.AimsThe primary aims of our research were: i) to verify the correlations of bax/bcl-2 ratio with other well-known biological and clinical prognosticators in patients treated with ibrutinib; ii) to evaluate the impact of bax/bcl-2 ratio on overall response rate (ORR), PFS and overall survival (OS); iii) to test bax/bcl-2 ratio as an independent prognostic factor.Patients and MethodsTherefore, we evaluated the efficacy of ibrutinib, in a real-life contest, on 100 patients, median age 60 years (38-85), median number of previous regimens 2 (0-4; 14% previously untreated). Patients received 420 mg oral ibrutinib once daily until progression or occurrence of unacceptable side effects. Median follow up on ibrutinib was 16 months. ORR was 92% [complete response (CR): 27%, partial response (PR): 32%, PR with lymphocytosis (PR-L): 33%]. The estimate 1-year PFS and OS were 74% and 77%, respectively. Twenty-nine patients (29%) discontinued ibrutinib therapy due to progression (n=11) and serious adverse events (n=18). Five out of 6 patients with Richter's syndrome (RS) had baseline del(17p)/TP53 mutations (M). Interestingly, PFS was significantly better for patients in first/second lines (n=38) vs later lines of therapy (n=55; p=0.022). Bax/bcl-2 ratio was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on peripheral CLL cells before treatment with ibrutinib. The threshold of positivity was set at the median value higher than 1.5 (range 0.38-5.10).ResultsSixty-four patients had bax/bcl-2 ratio <1.5 (64%). Bax/bcl-2 ratio <1.5 was strongly correlated with ZAP-70 >20% (40/64; p=0.005), but not with CD49d or CD38 >30%. There was only a slight significant correlation between bax/bcl-2 ratio <1.5 and NOTCH1 mutations (M) (25/32; p=0.030). On the other hand, no significant correlations were found between bax/bcl-2 ratio and del(17)p/TP53 M or IGHV status or cytogenetic subsets. With regard to clinical outcome, lack of response/progression and PR/PR-L were significantly correlated both with bax/bcl-2<1.5 (p=0.001) and NOTCH1 M (p=0.0005), but not with del(17)p/TP53 M. Noteworthy, 20/37 del(17)p/TP53 M patients were treated in first/second lines (p=0.001). Significant shorter PFS was observed in patients with bax/bcl-2 ratio <1.5 (65% vs 92% at 24 months, p=0.015, Figure A). Moreover, patients with lower bax/bl-2 ratio showed significant shorter OS (68% vs 96% at 24 months, p=0.006, Figure B). In multivariate analysis of PFS, only bax/bcl-2 ratio < 1.5 (p=0.030), analysed together with NOTCH1 M, TP53 M, number of therapeutic lines and age, was confirmed as an independent prognostic factor. With regard to multivariate analysis of OS, bax/bcl-2 ratio (p=0.016) and number of therapeutic lines (p=0.035) remained significant.ConclusionsIn our hands, bax/bcl-2 ratio was a powerful prognosticator for patients treated with ibrutinib, particularly useful to identify subsets of CLL patients with different levels of spontaneus apoptosis and consequent different clinical outcome under ibrutinib treatment. At the light of these our recent observations, bcl-2 antagonists, such as ABT-199, should be reasonably combined with BTK inhibitors, as it is already underway in clinical protocols, in order to overcome apoptosis resistance in patients treated with ibrutinib, particularly within the CLL subset characterized by lower bax/bcl-2 ratio e poor response to therapy with ibrutinib. [Display omitted] DisclosuresDel Principe:Gilead: Membership on an entity's Board of Directors or advisory committees.

PO-072 ω3-Polyunsaturated fatty acids inhibit cell growth and invasion of human hypopharyngeal carcinoma cells

IntroductionT-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy resulting from the transformation of T-cell progenitors. Early T-cell progenitor ALL (ETP-ALL) subgroup accounts for 5% to 10% of T-ALL cases and associated with a poor prognosis and a very high risk for relapse. T-ALL leukemias develop a dependence on anti-apoptotic BCL-2 family proteins that is linked to the maturation stage of the blasts. Typical T-ALL is mostly dependent on BCL-XL, whereas ETP-ALL is more dependent on BCL-2. In recent years, there has been the development of a series of specific BH3 mimetics (ABT-199, ABT-263, WEHI 539) that can inhibit the function of anti-apoptotic BCL-2 proteins and induce apoptosis. T-ALL patients have blasts disseminated in the blood, bone marrow and spleen. Each niche may provide distinct extrinsic signals that could offer a survival advantage to leukemic cells.Material and methodsWe aim to assess in both ETP-ALL (Loucy cell line) and typical T-ALL (CEM-CCRF cell line) if distinct microenvironments affect the BCL-2 family proteins expression and the sensitivity to BH3 mimetics. To study the anti-apoptotic BCL-2 dependencies we will use the cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release.Results and discussionsWe found that ETP-ALL cell line was BCL-2 dependent and sensitive to ABT-199 (BCL-2 specific BH3 mimetic). While the Typical T-ALL cell line was BCL-XL dependent and sensitive to both ABT-263 (BCL-2,BCL-XL and BCL-w) and WEHI 539 (BCL-XL) BH3 mimetics. Interestingly, when the ETP–ALL cell line Loucy was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency, as assessed by BH3 profiling, and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF. We found an increase in BCL-2 dependence and an increase in sensitivity to ABT-199.ConclusionCurrently, we are trying to identify the signalling pathways which are activated to cause this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of a xenograft cell line isolated from the blood and spleen of animals to determine if this switiching of BCL-2 dependence occurs in vivo.

PO-068 Determining how distinct microenvironments alter BCL-2 family dependence in T-cell acute lymphoblastic leukaemia

IntroductionT-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy resulting from the transformation of T-cell progenitors. Early T-cell progenitor ALL (ETP-ALL) subgroup accounts for 5% to 10% of T-ALL cases and associated with a poor prognosis and a very high risk for relapse. T-ALL leukemias develop a dependence on anti-apoptotic BCL-2 family proteins that is linked to the maturation stage of the blasts. Typical T-ALL is mostly dependent on BCL-XL, whereas ETP-ALL is more dependent on BCL-2. In recent years, there has been the development of a series of specific BH3 mimetics (ABT-199, ABT-263, WEHI 539) that can inhibit the function of anti-apoptotic BCL-2 proteins and induce apoptosis. T-ALL patients have blasts disseminated in the blood, bone marrow and spleen. Each niche may provide distinct extrinsic signals that could offer a survival advantage to leukemic cells.Material and methodsWe aim to assess in both ETP-ALL (Loucy cell line) and typical T-ALL (CEM-CCRF cell line) if distinct microenvironments affect the BCL-2 family proteins expression and the sensitivity to BH3 mimetics. To study the anti-apoptotic BCL-2 dependencies we will use the cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release.Results and discussionsWe found that ETP-ALL cell line was BCL-2 dependent and sensitive to ABT-199 (BCL-2 specific BH3 mimetic). While the Typical T-ALL cell line was BCL-XL dependent and sensitive to both ABT-263 (BCL-2,BCL-XL and BCL-w) and WEHI 539 (BCL-XL) BH3 mimetics. Interestingly, when the ETP–ALL cell line Loucy was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency, as assessed by BH3 profiling, and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF. We found an increase in BCL-2 dependence and an increase in sensitivity to ABT-199.ConclusionCurrently, we are trying to identify the signalling pathways which are activated to cause this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of a xenograft cell line isolated from the blood and spleen of animals to determine if this switiching of BCL-2 dependence occurs in vivo.

Abstract 3909: HDAC inhibition in combination with MEK or BCL-2 inhibition as novel therapeutic strategies in multiple myeloma

Abstract Multiple myeloma (MM) is an incurable malignancy of plasma cells. It is the second most common hematologic cancer, affecting nearly 30,000 people in the United States annually. Substantial progress has been made in the past fifteen years in the treatment of MM due to the approval of several new classes of drugs. However, patients inevitably relapse and become refractory to existing therapies. Hence, there is an immediate unmet need to develop novel therapies for MM based on a better understanding of the disease biology. Mutations in RAS have been found to occur in about 40% of newly diagnosed MM patients, with the frequency increasing to around 70% in relapsed/refractory patients. Such mutations are absent in patients with the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Clearly, RAS mutations contribute to both disease progression and relapse. However, targeting the MEK/ERK pathway has been unsuccessful in MM patients to date. Given the high frequency of RAS mutations in MM, we hypothesized that targeting this pathway could still be a promising strategy when combined with existing agents that have multifaceted mechanisms to promote tumor cell death, such as the recently approved histone deacetylase (HDAC) inhibitor LBH589 (panobinostat). Our results clearly demonstrate that low doses of LBH589 in combination with the MEK inhibitor AZD6244 induce BIM-dependent synergistic cell death in several MM cell lines and patient cells. Our studies also suggest that mutations in RAS/RAF could serve as a predictive biomarker for sensitivity to AZD6244/LBH589. RAS/RAF mutations appear to confer Mcl-1 dependence in MM cells, in part by driving up the phosphorylation of Mcl-1. The AZD6244/LBH589 combination is able to decrease the phosphorylation of Mcl-1 at several sites, which dissociates BIM-Mcl-1 complexes, ultimately leading to activation of the intrinsic apoptosis pathway. Additionally, we identified that wild-type RAS/RAF cells have relatively lower levels of phospho-Mcl-1, as well as higher levels of Bcl-2 and phospho-Bcl-2 when compared to mutated RAS/RAF cells. This seems to confer functional Bcl-2 dependence. Consequently, we found that wild-type RAS/RAF cells are sensitive to the BH3-mimetic ABT199 (venetoclax) when combined with LBH589. Through ongoing experiments, we hope to further confirm the mechanism of action of both these combinations, identify the particular HDAC that is required to be inhibited for the observed synergy, and validate RAS/RAF mutational status as a biomarker for predicting sensitivity to either combination. Our findings have broad therapeutic potential given the prevalence of RAS mutations in MM. Moreover, the ABT199/LBH589 combination could emerge as a targeted therapy for wild-type RAS patients, perhaps broadening the scope and capacity of Bcl-2 inhibition in MM. Citation Format: Kevin C. Miller, Jessica Haug, Teresa Kimlinger, Sanjay Kumar, Wilson Gonsalves, S. Vincent Rajkumar, Shaji K. Kumar, Vijay Ramakrishnan. HDAC inhibition in combination with MEK or BCL-2 inhibition as novel therapeutic strategies in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3909.

Intracellular BH3 Profiling Reveals Shifts in Antiapoptotic Dependency in Human B Cell Maturation and Mitogen-Stimulated Proliferation.

Apoptosis is critical to B cell maturation, but studies of apoptotic regulation in primary human B cells is lacking. In this study, we sought to better understand the mechanisms of apoptotic regulation in normal and activated B cells. Using intracellular BH3 profiling, we defined the Bcl2 dependency of B cell subsets from human peripheral blood and tonsillar lymphoid tissue as well as mitogen-activated B cells. We found that naive and memory B cells were BCL-2-dependent, whereas germinal center B cells were MCL-1-dependent and plasma cells were BCL-XL-dependent. B cells stimulated to proliferate ex vivo by CpG or CD40L/IL-4 became more dependent on MCL-1 and BCL-XL As B cell lymphomas often rely on survival mechanisms derived from normal and activated B cells, these findings offer new insight into potential therapeutic strategies for lymphomas.

Abstract 708: Prohibitin is a prognostic marker of treatment failure and therapeutic target to block chemotherapy resistance in Wilms tumor

Abstract Wilms tumor (WT) is the most common kidney tumor of children. Over the last three decades, clinical trials employing multi-modality therapies have resulted in overall survival of greater than 90% for patients with low-risk disease. In spite of these advances, treatment of patients with advanced, anaplastic, and relapsed Wilms tumors remains challenging, with substantial rates of treatment failure and death. To improve risk stratification and identify novel therapeutic targets, we used high-accuracy mass spectrometry urine proteomics to identify urine tumors markers associated with relapsed WT and non-WT renal tumors. We measured urine proteomes at diagnosis of 54 patients with renal WT, clear cell sarcoma, rhabdoid tumor, and age-matched controls, leading to the quantitation of 6,519 urine proteins. In particular, we identified specific urine WT markers, including those that were enriched in patients with relapsed WT, such as mitochondrial regulators prohibitin and DAD1, β-catenin antagonist DACT2, and DNA repair factor SUN1. Using a specific enzyme-linked immunosorbent assay (ELISA) developed to measure urine prohibitin in an independent cohort of 139 WT and control samples, we found that urine prohibitin concentrations over 1000 ng/mL were significantly associated with the risk of disease relapse, with an odds ratio of relapse of 153 and receiver operating characteristic area under the curve of 0.77 (95% confidence interval of 0.64-0.99). Immunohistochemical tumor analysis revealed that prohibitin was highly expressed in primary Wilms tumor specimens. Importantly, using loss- and gain-of-function genetic experiments, we found that prohibitin was required for the growth and survival of Wilms tumor cells, and its overexpression conferred concomitant resistance to vincristine, doxorubicin and actinomycin D. Consistent with prohibitin’s functions in mitochondria, we are using BH3 profiling to elucidate specific intrinsic apoptotic dependencies in distinct subsets of refractory Wilms tumors, as a prelude to rational combination blockade of chemotherapy resistance, such as blockade of BCL2 dependence using venetoclax. In all, the use of urine prohibitin measurements may improve initial therapy stratification, and enable monitoring of response to therapy and early detection of relapse. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin overexpression may offer improved therapies for patients with relapsed or refractory Wilms tumors. Citation Format: Michael Vincent Ortiz, Melissa Burns, Amy Eisenberg, Saima Ahmed, Lyvia Gaewsky, Gary Bradwin, Paolo Cifani, Anton Henssen, Ian Macarthur, Michael LaQuaglia, Anthony Letai, Arlene Naranjo, Samantha Gadd, Yueh-Yun Chi, Jeffrey Dome, Elizabeth Perlman, Elizabeth Mullen, Hanno Steen, Alex Kentsis. Prohibitin is a prognostic marker of treatment failure and therapeutic target to block chemotherapy resistance in Wilms tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 708. doi:10.1158/1538-7445.AM2017-708

Crebbp loss cooperates with Bcl2 overexpression to promote lymphoma in mice

AbstractCREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 overexpression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B cells, we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, and showed Myc binding. Through the analysis of CREBBP mutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these 2 diseases, with higher frequencies of nonsense/frameshift mutations in DLBCL compared with FL. Together, our data therefore provide important links between Crebbp inactivation and Bcl2 dependence and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.

Bruton's tyrosine kinase inhibition increases BCL-2 dependence and enhances sensitivity to venetoclax in chronic lymphocytic leukemia.

Although the BTK inhibitor ibrutinib has transformed the management of patients with CLL, it does not induce substantial apoptosis in vitro, and as such the mechanisms underlying its ability to kill CLL cells are not well understood. Acalabrutinib, a more specific BTK inhibitor now in development, also appears to be highly effective in CLL, but the connection of its mechanism with CLL cell death is also unclear. Using dynamic BH3 profiling, we analyzed alterations in the function of the mitochondrial apoptotic pathway induced by ibrutinib and acalabrutinib. We studied CLL patient samples treated ex vivo with both drugs, as well as primary samples from CLL patients on clinical trials of both drugs. We found that BTK inhibition enhances mitochondrial BCL-2 dependence without significantly altering overall mitochondrial priming. Enhancement of BCL-2 dependence was accompanied by an increase in the pro-apoptotic protein BIM. In contrast, treatment with the selective BCL-2 inhibitor venetoclax enhanced overall mitochondrial priming without increasing BCL-2 dependence. Pre-treatment of CLL cells with either BTK inhibitor, whether ex vivo or in vivo in patients, enhanced killing by venetoclax. Our data suggest that BTK inhibition enhances mitochondrial BCL2 dependence, supporting the ongoing development of clinical trials combining BTK and BCL-2 inhibition.

Acalabrutinib Increases Mitochondrial Priming and Enhances Venetoclax Sensitivity in CLL Cells

IntroductionThe Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has transformed the management of patients with CLL, yet it does not induce substantial apoptosis in vitro. The mechanisms underlying the ability of BTK inhibitors to kill CLL cells are incompletely understood. Our group previously developed BH3 profiling, a functional assay that determines the proximity of a cell to the threshold of apoptosis, a property called "mitochondrial priming". A new variant of this technique, dynamic BH3 profiling (DBP), measures early drug-induced changes in mitochondrial apoptotic signaling before cell death occurs. Using DBP, we previously found that ibrutinib increases mitochondrial BCL-2 dependence in CLL cells, thereby sensitizing cells to the BCL-2 inhibitor venetoclax (formerly ABT199/GDC-0199). However, it remains unclear whether these are on-target effects of ibrutinib on BTK or off-target effects of ibrutinib on its other targets such as ITK or TEC kinase. Here, we examined acalabrutinib, a 2ndgeneration, highly specific BTK inhibitor currently in clinical trials to determine whether selective BTK inhibition increases BCL-2 dependence in CLL cells.MethodsMononuclear cells were isolated from CLL patients and treated ex vivo with acalabrutinib (provided by Acerta) alone or in combination with venetoclax (APEX Bio Technology) in the presence of NKTert stromal cells. BH3 profiling and dynamic BH3 profiling were then performed as previously described by measuring the release of cytochrome c on a BD FACS Fortessa (Montero J. et al. Cell 2015). Cell viability assays and western blot analyses were performed according to standard protocols. The student’s t-test or one-way ANOVA was used to compare drug treatments with p<=0.05 considered significant.ResultsTo validate our prior finding that ibrutinib increased mitochondrial BCL-2 dependence, we first repeated our prior ex vivo ibrutinib experiments in 3 additional patients and found similar results. Next, we performed DBP on 5 peripheral blood samples from previously untreated CLL patients using ex vivo treatment with acalabrutinib, venetoclax or the combination. Consistent with our findings with ibrutinib treatment, acalabrutinb increased BCL-2 dependence, with all patient samples showing >20% increase in priming ("delta priming") in response to BAD BH3 peptide, with no increase in overall priming (<20% in all patients) measured by BIM BH3 peptide (Figure 1A). Moreover, it did not alter BCL-xL or MCL1 dependence measured by their specific peptides HRK or MS1, respectively. In contrast, venetoclax increased the overall level of mitochondrial priming with minimal effects on BCL-2 dependence. When combining these two drugs, both overall priming and BCL-2 dependence increased. The increase in BCL-2 dependence by ibrutinib resulted in enhanced sensitivity to venetoclax (p=0.0005).To assess whether these ex vivo effects could be replicated in vivo, we next performed DBP using CLL patient samples from 5 patients on the acalabrutinib first-in-human phase I clinical trial (NCT02029443). Delta priming was calculated by comparing pre-treatment samples to samples obtained after 4 weeks of acalabrutinib monotherapy. Consistent with our ex vivo data, acalabrutinib therapy selectively increased mitochondrial BCL-2 dependence in vivo (Figure 1B) without altering BCL-XL or MCL-1 dependence, and overall priming decreased slightly. To provide additional insight into the molecular mechanism underlying the mitochondrial priming changes, we did western blot analyses for BCL-2 family proteins, and found that expression of the pro-apoptotic BIM was increased after acalabrutinib therapy.ConclusionsSelective BTK inhibition with acalabrutinib enhances mitochondrial BCL-2 dependence and increases CLL cell sensitivity to the BCL-2 inhibitor venetoclax. Induction of expression of the pro-apoptotic protein BIM provides an initial clue as to a mechanism underlying these effects. These data strongly suggest that the increase in BCL-2 dependence we previously found with ibrutinib is a BTK-specific effect. Our findings provide a strong pre-clinical rationale supporting clinical trials combining BTK and BCL-2 inhibition. More broadly, we also demonstrate that DBP can be utilized on in vivo treated patient samples to rationally construct combination regimens of targeted agents to treat cancer. [Display omitted] DisclosuresBrown:Pfizer: Consultancy; Sun BioPharma: Consultancy; Celgene: Consultancy; Roche/Genentech: Consultancy; Gilead Sciences: Consultancy; Janssen: Consultancy; Infinity: Consultancy; Abbvie: Consultancy. Letai:AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding. Davids:Gilead: Honoraria; Pharmacyclics: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; TG Therapeutics: Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Infinity: Honoraria, Research Funding.

B-Cell Markers Predict Response to Venetoclax in Multiple Myeloma

BCL2 family members such as MCL1, BCLXL, and BCL2 are critical for cancer cell survival and therefore represent promising therapeutic targets. Both B cells and CLL cells depend primarily on BCL-2 and are thus sensitive to the BCL2 specific inhibitor venetoclax, while plasma cells and multiple myeloma typically depend on Mcl-1 and would therefore be resistant to venetoclax. However, a subset of myeloma is venetoclax sensitive based on recent in vitro and clinical trial data. In preliminary results from a phase I trial of venetoclax in multiple myeloma, 40% of patients positive for t(11;14) had objective responses, while only 6% of t(11;14) negative patients responded. We have made similar observations with in vitro testing of 30 freshly isolated myeloma patient samples, identifying both non-t(11;14) samples sensitive to venetoclax as well as resistant t(11;14) positive samples. Together, these results suggest not only that a subset of multiple myeloma is co-dependent on BCL2 but also that t(11;14) is neither necessary nor sufficient for responding to venetoclax. We therefore set out to identify other factors that may predict BCL2 dependence in multiple myeloma.Previous studies of t(11;14) myeloma have noted increased expression of CD20, CD23, CD79a, and PAX5 which are typically associated with B cells prior to their differentiation into plasma cells. Based on these observations we hypothesized that venetoclax sensitivity in myeloma may be associated with the retention of B cell properties including BCL2 dependence. We probed an online expression database of myeloma cell lines for non-t(11;14) cell lines expressing CD20 and identified two cell lines, OCI-My5 and PCM6, both of which we found to have an IC50 of approximately 50nM when treated with venetoclax. We went on to characterize a panel of 13 cell lines. In addition to OCI-My5 and PCM6, 4 other cell lines were sensitive to venetoclax, all positive for t(11;14). Of the 7 venetoclax resistant cell lines, 2 were t(11;14) positive. Protein levels of MCL1, BCLXL, and BCL2 were comparable among the 13 lines and therefore anti-apoptotic expression is unlikely to be responsible for venetoclax sensitivity. Consistent with our previous co-immunoprecipitation studies, more of the pro-apoptotic BIM was bound to BCL2 in venetoclax sensitive lines compared to resistant lines. In the absence of differences in BCL2 family expression, we next sought to identify other B cell related features correlating with venetoclax sensitivity. We used RNAseq data from our 13 cell lines to compare the expression of 100 genes previously reported to be differentially expressed between normal B cells and plasma cells. Interestingly, unsupervised clustering revealed a group of venetoclax sensitive cells enriched for other B cell associated genes. GSEA revealed enrichment of genes associated with immune system activation at a p < 0.001. We also analyzed the differential expression of genes between our sensitive and resistant lines and again identified overexpression of B cell related genes such as CD20, CD79A, STAT5A, and RASGRP2 in venetoclax sensitive lines, though no single marker was present in all of the venetoclax sensitive lines. We examined the expression of CD20, CD79a, and CD79b in the CoMMpass data set (IA8) as well and found that they were not co-expressed in most patients, again suggesting that no single marker is likely to be predictive. Finally, we created a gene signature from the top differentially expressed genes to predict sensitivity or resistance to venetoclax and used this signature to evaluate a database of 68 myeloma cell lines. One of the top hits predicted to be sensitive by our gene signature is the t(11;14) negative line MOLP2, and indeed this cell line was recently reported to be highly responsive to venetoclax. In conclusion, B cell markers and our gene signature correlate with BCL2 dependence and venetoclax sensitivity independent of t(11;14). DisclosuresBahlis:BMS: Honoraria; Onyx: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria. Nooka:Spectrum, Novartis, Onyx pharmaceuticals: Consultancy. Kaufman:Pharmacyclics: Consultancy; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Lonial:Onyx: Consultancy; Onyx: Consultancy; BMS: Consultancy; Janssen: Consultancy; Merck: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Millenium: Consultancy; BMS: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Janssen: Consultancy.