Abstract 3274: Targeting NSD family histone methyltransferase activity with irreversible inhibitors

Abstract Nuclear SET domain containing (NSD) family member proteins are histone methyltransferases that mono- and di-methylate histone 3 at lysine 36 (H3K36) to alter gene expression in a context-dependent manner. NSD1 and NSD3 are two members of this family which share greater than 80 percent sequence similarity in the catalytic SET domain. NSD1 is found as a fusion with NUP98 in aggressive cases of pediatric leukemia, and the lysine methyltransferase (KMTase) activity of NUP98-NSD1 drives bone marrow progenitor proliferation. Similarly, high levels of enzymatically active NSD3 mRNA are correlated with reduced disease-free survival in breast cancer and catalytic NSD3 expression is sufficient to rescue tumor growth in a xenograft model of triple negative breast cancer. Selective and potent inhibitors of NSD1 and NSD3 KMTase activity are needed to further investigate the therapeutic potential of targeting this function in leukemia and/or solid cancers. Here, we report the development of irreversible small molecule inhibitors of the NSD1 and NSD3 SET domains. We utilized a NMR-based fragment screen of the NSD1 SET domain to identify the hit BT1, which binds reversibly to the SET domain of NSD1. BT1 was extensively optimized into a series of potent irreversible ligands. Mass spectrometry and structural studies revealed that these compounds react with a cysteine near the autoinhibitory loop, providing a rational for the strong inhibition of KMTase activity we observe in biochemical assays. These inhibitors also bind to the NSD1 and NSD3 SET domains in cells and exhibit potent anti-proliferative effects against leukemia cells containing NUP98-NSD1 and breast cancer cells with high levels of NSD3 expression. In summary, we present novel irreversible inhibitors of NSD1 and NSD3 KMTase activity which represent important chemical probes that can be used to investigate the therapeutic potential of targeting NSD family member catalytic activity in cancers. Citation Format: Christina Howard, Sergei Zari, Shuangjiang Li, Huang Huang, EunGi Kim, Se Ra Park, Trupta Purohit, Hongzhi Miao, Xiaotian Zhang, Caroline Nikolaidis, Hao Li, Juliano Ndoj, HyoJe Cho, Jolanta Grembecka, Tomasz Cierpicki. Targeting NSD family histone methyltransferase activity with irreversible inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3274.