Abstract Double/triple-hit lymphomas (DHL/THL, harboring concurrent MYC, BCL2 and/or BCL6 rearrangements) and double-expressor lymphomas (DEL, with MYC/BCL2 co-overexpression without underlying rearrangements) account for 7-12% and 20-30% of diffuse large B-cell lymphomas (DLBCLs), respectively, and have poor outcome after standard/intensive immunochemotherapy (Petrich, 2014; Smith, 2018). More effective and less toxic treatments for these aggressive B-cell lymphomas represent a significant unmet medical need. Venetoclax, a BCL2 inhibitor, significantly inhibits the proliferation of DLBCL cells in culture but has less promising clinical efficacy as a single agent. CG-806, a FLT3/BTK inhibitor, exhibits potent antitumor activity against cell lines and primary cells over a range of hematologic malignancies by suppressing the driver and compensatory pathways (inhibits phosphorylation of BTK, FLT3, PDGFRα, SYK, SRC, ERK, MAPK, STAT5 and AKT, and decreases MYC level). CG-806 is currently in a Phase I a/b trial in patients with CLL/SLL or non-Hodgkin's lymphomas (NCT03893682). We sought to investigate the antitumor effect of CG-806 alone or in combination with venetoclax on DHL/THL/DEL cells using MTS based proliferation assay, flow cytometry assays of apoptosis and cell cycle, and immunoblotting. In DHL/THL cells VAL, SU-DHL6 and DOHH2 that overexpress MYC and BCL2, CG-806 inhibited their proliferation with IC50s of 0.17, 0.15 and 0.005 µM, respectively. CG-806 was 5-20X more potent than venetoclax and exposure to CG-806 significantly enhanced sensitivity to venetoclax. SU-DHL2, a DHL cell line that does not overexpress MYC or BCL2, was inhibited by CG-806 but not venetoclax (IC50 0.9 vs 6.3 µM, respectively). In the DEL cell line U2933, CG-806 and venetoclax had similar potency as single agents (IC50 1.6 µM); when combined, the IC50 was markedly improved to 0.05 µM. Meanwhile, the combination of CG-806 with venetoclax enhanced induction of apoptosis in DHL/THL/DEL cells in a dose and time-dependent manner as detected by Annexin V positive, cleavage of PARP and Caspase 3. Recent studies ascribed the resistance of DLBCLs to venetoclax to upregulation of MCL1 and MAPK signaling. our study showed that CG-806 inhibited expression of the anti-apoptotic protein MCL1, overcame the effect of venetoclax-induced MCL1 and increased the level of proapoptotic BIM. While venetoclax produced no effect on MAPK signaling, CG-806 alone and in combination with venetoclax reduced p-MEK1/2, p-JNK and MYC in a cell line dependent manner. In conclusion, CG-806 inhibits driver and rescue pathways to directly and potently kill the aggressive B-cell lymphomas including DHL/THL/DEL and enhances the proapoptotic effect of venetoclax, thereby highlighting CG-806 as a promising candidate for the treatment of patients harboring unfavorable BCL2/MYC/BCL6 translocations and/or overexpression and supporting clinical development of CG-806 in patients with B-cell malignancies intolerant, resistant, or refractory to venetoclax. Citation Format: Nasrin Rastgoo, Matthew Thayer, Khalid Benbatoul, Stephen Howell, William Rice, Hongying Zhang. CG-806, a first-in-class FLT3/BTK inhibitor, and venetoclax synergize to inhibit cell proliferation and to induce apoptosis in aggressive B-cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4225.