Abstract 2424: Evaluating the biochemical activity and endogenous engagement of covalent small molecule inhibitors of NSD1 and NSD3 in cancer cell lines

Abstract Background: The nuclear receptor-binding SET domain (NSD) family of proteins consists of NSD1, NSD2, and NSD3, each with histone methyltransferase (HMT) activity responsible for mono and di-methylation of histone 3 lysine 36 (H3K36me2). NSD1 expression and genetic alterations are implicated in 17% of head and neck squamous cellular carcinoma (HNSCC) patients, and knockdown of NSD1 inhibits the growth of HNSCC cell lines. Additionally, amplification of chromosome 8p11-12 (amplicon encoding NSD3 among other genes), overexpression, or activating mutations of NSD3 drive the progression of non-small cell lung cancer (NSCLC) subtypes. In NSCLC cell lines with NSD3 amplification, knockdown of NSD3 results in growth inhibition. In multiple cancer cell lines, methyltransferase activity of NSD1 and NSD3 has been implicated in promoting cell growth, rationalizing the development of small molecule inhibitors. Results: Optimization of our previously reported covalent inhibitors of the NSD1 SET domain led to the development of separate series of NSD1 and NSD3 inhibitors, which demonstrate potent growth inhibition in various cancer cell lines. To evaluate the potency of these inhibitors, we developed an in vitro engagement assay utilizing intact protein mass spectrometry. These experiments validated nearly complete covalent engagement of NSD1 and NSD3 after 2 h of incubation with small molecule inhibitors. We have also determined second order rate constant Kinact/KI confirming effective covalent inhibition of NSD1 and NSD3. To assess inhibition of NSD1 and NSD3 SET domains, we employed an enzymatic histone methyltransferase activity assay and found mid-nanomolar IC50 values. To determine the covalent engagement of endogenous methyltransferases in cancer cells, we have developed and optimized an immunoprecipitation followed by bottom-up proteomics workflow. Our current experiments evaluate the inhibition of H3K36 methylation in these cell lines. Conclusions: We have developed small molecule covalent inhibitors of NSD1 and NSD3 SET domains with strong engagement in in vitro mass spectrometry analyses and potent inhibition in histone methyltransferase assays. Furthermore, IP bottom-up proteomics methodology has enabled confirmation of covalent engagement to NSD3 in cancer cell lines, supporting on-target growth inhibitory effects of NSD3 inhibitors. These covalent inhibitors can be used as chemical probes to further study the effects of NSD1 and NSD3 inhibition in cancer and represent potential therapeutic modalities. Citation Format: Joshua Ray, Bradley Clegg, Sergei Zari, Kibum Kim, Se Ra Park, Jolanta Grembecka, Tomasz Cierpicki. Evaluating the biochemical activity and endogenous engagement of covalent small molecule inhibitors of NSD1 and NSD3 in cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2424.