Abstract Epigenetic abnormalities are common in human cancers and play a critical role in tumorigenesis via dysregulation of gene expression and by affecting chromatin function. Nuclear receptor binding SET domain protein 3 (NSD3/WHSC1L1) belongs to the family of NSD histone methyltransferases that catalyze mono- and di-methylation of lysine 36 on histone H3 (H3K36me1/2). NSD3 gene is located within 8p11-12 region amplified in approximately 15% of patients with breast carcinomas and this amplification is significantly associated with disease-specific survival and distant recurrence in breast cancer patients. NSD3 has been identified as a key oncogenic driver within 8p11-12 amplicon and knockdown of NSD3 results in profound loss of growth and survival of breast cancer cells. However, NSD3 is a complex and multidomain protein, and despite multiple efforts, no studies unambiguously validated whether the catalytic SET domain is essential for the oncogenic activity of NSD3. Here, we propose to assess the importance of the NSD3 SET domain in oncogenesis by developing small molecule chemical probes. This approach is highly novel as no NSD3 inhibitors targeting catalytic SET domain were reported in the literature. To date, we identified a class of small molecule compounds that bind selectively to the NSD3 SET domain, but not to the closely related NSD1 or NSD2. In preliminary studies, our inhibitors specifically block growth of breast cancer cells with amplifications of NSD3. The goal of this proposal is to further develop these compounds and validate whether optimized chemical probes blocking catalytic activity of NSD3 would impair development and progression of breast cancer in vivo. To accomplish this goal, we will employ extensive medicinal chemistry to improve potency, selectivity and pharmacokinetic properties of our NSD3 inhibitors. Efficacy of the most potent compounds will be evaluated in xenograft and PDX models of breast cancer. We are confident that our comprehensive plan will lead to successful development of potent NSD3 chemical probes for exploration of NSD3 biology in breast cancer and other tumors with 8p11-12 amplifications.