Abstract 2925: Safety and efficacy of EZH2 and BRD4 dual targeting in EZH2Y641mut germinal centre-derived lymphoma
Authors: Chamorro-Jorganes Aranzazu , Ribeiro Marcelo L. , Profitos-Peleja Nuria , Reyes-Garau Diana , Recasens-Zorzo Clara , Valero Juan G. , Armengol Marc , Perez-Galan Patricia , Butler Ray , Postigo Antonio , Bosch Francesc , Roue Gael 
Published: September 2020

Abstract
A significant proportion of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) patients harbor a gain-of-function, heterozygous somatic mutations of the methyltransferase gene EZH2. Despite acceptable safety profile and early signs of activity in clinical trials, single agent treatment with EZH2 inhibitors is unlikely to be curative in aggressive lymphomas. In an effort to established novel rational combinations, we have evaluated the activity and mechanism of action of the EZH2 small molecule inhibitor CPI169 as single agent and in combination with BET bromodomain inhibition, using preclinical models of DLBCL and FL with distinct EZH2 mutational status. CPI169 anti-tumor activity and specificity was assessed in vitro in 10 DLBCL and FL cell lines, including cells expressing basal or ectopic EZH2mut. Molecular bases of its activity were determined by gene expression profiling (GEP), qPCR and western blot, followed by automated exploratory data analysis. Biomarkers validation was made in vitro and in vivo, using a mouse xenotransplant model of EZH2mut DLBCL, considering both exposure to CPI169 single agent and/or combination treatment with a BRD4 inhibitor, CPI203. CPI169 induced dose-dependent proliferation blockade in EZH2mut, but not EZH2wt DLBCL and FL cell lines, independently of EZH2 expression level or basal methyltransferase activity. Loss of H3K27me3 mark upon CPI169 treatment was associated with upregulation of gene sets related to G1 cell cycle blockade, mTOR and P53 pathways, and MYC signaling. Accordingly, combination with the MYC-interfering drug, BET inhibitor CPI203, achieved a synergistic anti-proliferating activity in EZH2 mutated cases and in mice bearing EZH2mut DLBCL tumors. Activity of EZHi/BRD4i combo was characterized by lower mitotic index, increased loss of H3K27me3 mark, in association with MYC downregulation. GEP analysis, followed by automated exploratory data analysis and validation by a siRNA screening, further identified the PI3K/AKT-regulated gene and mitosis regulator, YPEL2, and the regulator of innate-like B lymphocyte maturation, KLHL14, as crucial factors involved in the efficacy of MYC/EZH2 dual targeting. In conclusion, CPI169 shows significant activity and safety as single agent in EZH2 mutated-DLBCL and FL cases and displays synergistic interaction in vitro and in vivo with BRD4 inhibition, mediated by the modulation of a limited set of EZH2-regulated genes.
Citation Format: Aranzazu Chamorro-Jorganes, Marcelo L. Ribeiro, Nuria Profitos-Peleja, Diana Reyes-Garau, Clara Recasens-Zorzo, Juan G. Valero, Marc Armengol, Patricia Perez-Galan, Ray Butler, Antonio Postigo, Francesc Bosch, Gael Roue. Safety and efficacy of EZH2 and BRD4 dual targeting in EZH2Y641mut germinal centre-derived lymphoma [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 2925.