Abstract 3805: Loss of NSD2 induces acquired resistance to EZH2 inhibitors in B-cell lymphoma by disrupting enhancer function

Abstract Introduction: Epigenetic enzymes that add or remove histone post-translational modifications are essential for cellular identity and frequently mutated in cancer. For example, in B-cell lymphomas, change-of-function mutations in EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), result in increased H3K27me3 levels and transcriptional silencing of key genes required for B-cell differentiation. However, the efficacy of EZH2 inhibitors in clinical trials has been limited by acquired resistance. Understanding the mechanisms of resistance to EZH2 inhibitors is crucial for improving treatment outcomes. Methods: Genome-wide CRISPR screens were performed in an EZH2-mutant B-cell lymphoma cell line treated with and without the FDA approved EZH2 inhibitor Tazemetostat. Flow cytometry-based GFP reporter assays using CRISPR sgRNAs, and viability assays using NSD2 PROTAC degraders, were employed to validate resistance mechanisms. Co-immunoprecipitation and mass spectrometry (IP-MS) of endogenous NSD2 was performed to identify potential protein interactors. Chromatin binding and histone post-translational modifications were mapped using quantitative CUT&RUN. RNA-seq was performed to identify transcriptional changes upon NSD2 loss. Results: A genome-wide CRISPR screen of EZH2-mutant lymphomas revealed that loss of either NSD1 or NSD2 confers resistance to EZH2 inhibition. This was validated in independent cell competition assays on an extended cohort of B-cell lymphoma cell lines. Furthermore, PROTAC mediated degradation of NSD2 also renders EZH2-mutant lymphomas resistant to EZH2 inhibition. While endogenous IP-MS of NSD2 indicates it functions in isolation, its loss in EZH2-mutant B-cell lymphoma cells leads to genome-wide decreases in its H3K36me2 modification, as expected. These global decreases in H3K36me2 led to consequent increases in the genome-wide binding of PRC2. The consequent increased deposition of PRC2 H3K27 methylations led to focal reductions in H3K27ac at enhancer elements. Furthermore, NSD2 binding increased at active enhancer regions in EZH2 inhibitor treated cells. Taken together, our results indicate that loss of NSD2 in B-cell lymphoma confers resistance to EZH2 inhibitors by altering the balance of repressive and active histone post translational modifications at key regulatory elements in the genome. Conclusion: We report a novel mechanism by which loss of NSD2 confers resistance to EZH2 inhibitors in EZH2-mutant B-cell lymphoma. Loss of NSD2 leads to impaired activation of critical enhancers required for response to EZH2 inhibitors. Our results indicate that EZH2 inhibitors should not be used to treat cancers with loss of function mutations in NSD1 or NSD2 and indicate NSD2 has a role in regulating enhancer functions. Citation Format: Daniel Angelov, Darragh Nimmo, Gráinne Holland, Molly Davies, Eugene Dillon, Eric Conway, Elisabeth Vandenberghe, Adrian P. Bracken. Loss of NSD2 induces acquired resistance to EZH2 inhibitors in B-cell lymphoma by disrupting enhancer function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3805.