Screening for inhibitors of NSD3 as a treatment for lung cancer

Abstract The nuclear receptor binding SET domain protein 3 (NSD3) belongs to the family of NSD histone methyltransferases, which are responsible for mono- and di-methylation of lysine 36 on histone 3 (H3K36me1/2). Increasing number of studies link NSD3 to various cancers, including lung, breast, pancreatic and bladder cancers. Resent study implicates NSD3 as an oncogene in aggressive lung squamous cell carcinoma (LUSC), which represents a sub-type of non-small cell ling cancer with very poor clinical outcome. NSD3 in amplified and/or overexpressed in LUSC, and its role as an oncogene in this sub-type of lung cancer has been well validated using genetic studies, both in vitro and in vivo. Furthermore, the catalytic activity of NSD3 plays a critical role in LUSC oncogenesis, supporting that small molecule inhibitors of the catalytic SET domain of NSD3 could represent new therapeutics for lung cancer patients. However, NSD3 inhibitors have not been reported to date, but are highly desired as new potential therapeutics for patients with LUSC and other cancers with NSD3 amplifications. In this project we propose to develop NSD3 inhibitors by performing high throughput screening (HTS) at CCG, University of Michigan. The HTS will focus on identification of small molecule inhibitors of the catalytic SET domain of NSD3. To date, we developed and validated a fluorescence polarization (FP) assay that will be used as a primary screening assay for HTS to identify NSD3 inhibitors. Small molecules identified by HTS will be validated for binding to NSD3 and for inhibition of its catalytic activity using a series of biochemical and biophysical experiments, including NMR, ITC, and histone methyltransferase assays. Activity of the most potent compounds will be characterized in cell-based studies to assess inhibition of NSD3 in mammalian cells and understand their mechanism of action. Selected NSD3 inhibitors will be tested in a panel of lung cancer cell lines with NSD3 amplifications and/or overexpression for their activity, selectivity, and mechanism of action. In summary, we expect to identify highly valuable chemical probes selectively targeting catalytic SET domain of NSD3, which will be suitable for mechanistic studies in cancer cells. This project will pave the way towards development of more potent NSD3 inhibitors that will be appropriate for in vivo studies in lung cancer models and for future therapeutic implications.