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Introduction: Although survival dependence on Bcl2 is a well-known aspect of the pathophysiology of chronic lymphocytic leukemia (CLL), the mechanisms of Bcl-2 dysregulation are incompletely understood. Recurrent translocations involving BCL2 and immunoglobulin genes, including t(14;18)(q32;q21) and variants such as t(2;18) or t(18;22), are classically observed in follicular lymphoma or germinal center diffuse large B-cell lymphoma (GC DLBCL), but are uncommon ( Methods: Clinically annotated primary samples from BCL2-R CLL patients identified by karyotype were obtained from the French Innovative Leukemia Organization network and Dana-Farber Cancer Institute. Primary samples from CLL without BCL2 rearrangement were used as a control (ctrl CLL). Next generation sequencing (NGS) was performed using a custom-designed panel of 29 genes, including among others: BIRC3, NOTCH1, FBXW7, MLL2, RAS pathway, SF3B1 and TP53. The mean coverage obtained was 2000X (limit of detection (LOD): 1%). Digital droplet PCR (ddPCR) was used to quantify NOTCH1 c.7544_7545delCT (LOD: 0.025%). Protein expression (Bcl2, Mcl1, Bim) was assessed by Western blot. Baseline BH3 profiling was performed as per Ryan et al., Bio Chem 2016. To mimic the lymph node microenvironment, viability assays were performed in co-culture with the stromal cell line NK.tert. Viability was assessed by AnnexinV/Hoechst staining. Ex vivo drug treatments included: BCL2i (inhibitor): venetoclax; MCL-1i: AZD5991, S63845 and BCLXLi: A133. Statistical analyses were by unpaired and paired t-test with a two-tailed nominal p ≤ 0.05 considered as significant. Results: In our cohort of 110 patients, the median age was 70 years old, and 79% were male. BCL2-R were t(14;18) in 77.2%, t(18;22) in 16.3% and t(2;18) in 6.3% of patients. The translocation involving BCL2 gene was isolated in 23.6% of cases, and was associated with trisomy 12 in 45.4% of patients. The most frequently mutated genes in this cohort were in the NOTCH pathway (NOTCH1 mutation: 43.6 %, mostly subclonal (mean of variant allelic frequency: 6.1%) and FBXW7: 4.5%)) and RAS pathway (KRAS, NRAS, BRAF: 9.1%). BCL2 mutations were observed in 22.8% of the 57 examined cases. No mutation previously described in venetoclax resistant CLL, such as F104L or G101V variant, were observed. Furthermore, MLL2 mutations were observed in 14.5% cases and were significantly associated with complex karyotype (p=0.01) and trisomy 12 (p=0.04). Others mutated genes were: BIRC3 (5.4%), TP53 (3.6%), SF3B1 (1%) and MYD88 L265P(1%). No mutations in EZH2, CREBBP or EP300 were found. In 15 CLL representative samples from each group (BCL2-R and ctrl), Bcl2 protein expression was significantly higher in BCL2-R CLL (ratio Bcl2/actin 0.94 vs 0.74, p=0.009) as was expression of the pro-apoptotic protein Bim (ratio Bim/actin: 2.059 vs 1.524, p=0.007). BH3 profiling demonstrated that BCL2-R CLL and ctrl CLL samples (n=23 in each group) had comparable overall priming (cyto-C release 66.1% vs 63.3%, ns) and Bcl-2 dependence (cyto-C release 75.4% vs 76.3%, ns). Both also had low dependence on Bcl-xL (cyto-C release 8.2% vs 8.8%, ns). In contrast, Mcl-1 dependence was found to be significantly lower in BCL2-R CLL (cyto-C release 15.6% vs 37.4%, p Conclusion: The genomic landscape of BCL2-R CLL is characterized by a high frequency of trisomy 12, subclonal NOTCH and RAS pathway mutations, as well as BCL2 and MLL2 mutations. Protein expression, BH3 profiling and viability assays data are consistent with nearly exclusive dependence on Bcl-2. Our data suggest that Bcl-2 inhibition should be favored over Mcl-1 inhibition in BCL2-R CLL. Disclosures Herbaux: Roche: Consultancy, Honoraria, Research Funding. Laribi:abbvie: Honoraria, Research Funding; amgen: Research Funding; novartis: Honoraria, Research Funding; takeda: Research Funding. Ysebaert:Roche: Consultancy; Janssen: Consultancy; AbbVie: Consultancy. Morel:Janssen: Honoraria. Guieze:abbvie: Honoraria, Other: advisory board, travel funds; janssen cilag: Honoraria, Other: advisory board, travel funds; roche: Other: travle funds; gilead: Honoraria, Other: travel funds; astrazanecka: Honoraria, Other: advisory board. Brown:Sun: Research Funding; Acerta: Consultancy; Pharmacyclics: Consultancy; Genentech: Consultancy; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other; Invectys: Membership on an entity's Board of Directors or advisory committees, Other: DSMC; Gilead: Consultancy, Research Funding; BeiGene: Consultancy; Catapult: Consultancy; Dynamo Therapeutics: Consultancy; Eli Lilly and Company: Consultancy; Juno/Celgene: Consultancy; Kite: Consultancy; MEI Pharma: Consultancy; Nextcea: Consultancy; Novartis: Consultancy; Octapharma: Consultancy; Pfizer: Consultancy; Rigel Pharmaceuticals: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Loxo: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Janssen: Honoraria; AbbVie: Consultancy.

Introduction Resistance to apoptosis is a hallmark of cancer, and modulation of BCL-2 family proteins is an important mediator of such resistance in hematologic malignancies. Despite the clinical efficacy of the BCL-2 inhibitor venetoclax (VEN), prolonged treatment may lead to resistance, such as the BCL2 G101V mutation (Blombery et al, Blood, 2020); however, over half of VEN resistant cases are not explained by known genetic mechanisms. Phosphorylation of BCL-2 at serine-70 (S70pBCL2) or of MCL-1 at threonine-163 (T163pMCL1) have been shown to increase sequestration of the pro-apoptotic protein BAX (Deng et al, JNCI, 2000) and stabilize the level of anti-apoptotic protein MCL-1 (Wang et al, Mol Cancer, 2014), respectively. We hypothesized that the increase in post-translational modifications of BCL-2 family members, in particular S70pBCL2 and T163pMCL1, are novel mechanisms of functional VEN resistance in lymphoid malignancies. We further hypothesized that the FDA-approved phosphatase activator drug FTY720 (fingolimod) would de-phosphorylate these BCL-2 family members and thereby re-sensitize malignant lymphoid cells to VEN-induced apoptosis. Methods A VEN resistant diffuse large B-cell lymphoma cell line (OCI-Ly1-R) as well as peripheral blood mononuclear cells from 12 previously untreated CLL patients co-cultured with human stromal NK-Tert cells were treated ex vivo with VEN +/- FTY720. A VEN sensitive cell line (OCI-Ly1-S) was treated with VEN +/- a phosphatase inhibitor okadaic acid (OA). Western blot was used to evaluate changes in S70pBCL2 and T163pMCL1 protein levels. BH3 profiling via flow cytometry was performed to determine the survival dependence on anti-apoptotic BCL-2 family members via cytochrome c release in response to specific BH3-only peptides and drugs such as VEN applied directly to mitochondria (Ryan et al, Biol Chem, 2016). Cell viability assays (CellTiter-Glo, Trypan Blue and Annexin/Hoechst) were employed to investigate the effects of FTY720 on OCI-Ly1-R resistance to VEN. The BCL-2-BAX interaction was investigated using co-immunoprecipitation in VEN resistant and sensitive cells following treatment with VEN +/- FTY720. T-test, ANOVA and multiple comparison with a statistical significance set at 2-tailed p ≤ 0.05 were used. Results OCI-Ly1-R cells displayed higher S70pBCL2, T163pMCL1 and MCL-1 expression compared to OCI-Ly1-S cells. Notably, the increase in S70pBCL2 was associated with reduced response of VEN-mediated BCL-2-BAX dissociation, while the increase in T163pMCL1 was accompanied by enhanced MCL-1 protein expression. Using BH3 profiling, we found that the increase in S70pBCL2, T163pMCL1 and MCL-1 expression were functionally associated with a decrease in BCL-2 survival dependence (-79.1%, 1μM VEN, P < 0.0001) and an increase in MCL-1 dependence (+52%, 10μM MS1, P < 0.0001) (Fig. A). The addition of FTY720 reversed these observations in OCI-Ly1-R cells, where we observed a decrease in S70pBCL2, T163pMCL1 and MCL-1 protein expression, BCL-2 and BAX interaction, as well as a "re-wired" functional dependence toward BCL-2 (-21.6% 10μM MS1, +27.9% 1μM VEN, P < 0.0001) (Fig. B). Importantly, pre-treatment with FTY720 re-sensitized OCI-Ly1-R cells to VEN-induced cell death (+56.1%, P = 0.0001) (Fig. C). Conversely, treatment with a phosphatase inhibitor, OA, led to an increase in S70pBCL2, T163pMCL1 and MCL-1 expression as well as reduced late death of OCI-Ly1-S cells (-60%, P = 0.0018). We validated our cell line results in primary CLL cells, and again the combination of FTY720 and VEN similarly reduced S70pBCL2, T163pMCL1 and MCL-1 expression, increased BCL-2 dependence, and enhanced VEN-induced cell death (+23.6%, P < 0.0001). Conclusion Increased S70pBCL2 and T163pMCL1 are associated with VEN resistance, in part by inhibiting VEN-induced BCL-2-BAX dissociation and switching the functional survival dependence from BCL-2 to MCL-1. FTY720 re-sensitizes VEN resistant cells by reducing S70pBCL2, T163pMCL1 and MCL-1 expression, dissociating BAX from BCL-2 and "re-wiring" the survival dependence to BCL-2. These preclinical findings support the exploration of this strategy clinically in patients with VEN resistant lymphoid malignancies. Disclosures Davids: Sunesis: Consultancy; AbbVie: Consultancy; Surface Oncology: Research Funding; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Zentalis: Consultancy; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy; Bristol Myers Squibb: Research Funding; Janssen: Consultancy; MEI Pharma: Consultancy, Research Funding; Syros Pharmaceuticals: Consultancy; Merck: Consultancy; Research to Practice: Honoraria.