The molecular mechanisms of progestin efficacy and resistance in EC-AEH

Abstract - Project 2: Establish the molecular signatures of progestin efficacy and resistance in EC-AEH. Progestins are synthetic steroids used to prevent the progression of atypical endometrial hyperplasia and early- stage endometrial cancer (EC-AEH) in patients for whom fertility-sparing is desirable or when surgery is not an option. Of the >20 progestins, only three are widely used to treat EC-AEH. Though initially effective, 25% of patients do not respond, and 40% of patients relapse and develop resistance to progestin treatment, limiting their widespread deployment and longer-term use. Improving progestin-based therapy is hampered by a lack of mechanistic understanding of how they work and whether other progestins may be more effective than those currently used. Canonically, progestins are thought to work cell-autonomously in tumor cells by binding the progesterone receptor (PR), which regulates genes that cause tumor regression. However, many EC-AEH tumors with low or absent PR still respond to progestins. Evidence is emerging that progestins also work cell- autonomously through non-canonical pathways, such as activating other steroid hormone receptors. Further, progestins also induce EC-AEH regression non-cell autonomously through PR in stromal and myometrial cells and by modulating the immune response in the tumor microenvironment, though it is unclear how. To improve hormone therapy, there is a critical need to decipher the mechanisms of how progestins drive tumor regression cell-autonomously and through paracrine signaling in EC-AEH. To tackle this, we propose a functional genomic approach using advanced CRISPR, mass spectrometry, and gene regulatory techniques using in vivo EC models to systematically elucidate mechanisms of resistance and response for a panel of progestins in a uterine environment. We have also developed a method to culture intact slices from EC-AEH tumors to compare the effects of different progestins on the tumor directly and all cells within the tumor microenvironment using single- cell RNA-seq. Using these methods, we will derive critical information about how different progestins work through different mechanisms to induce tumor regression and identify sources of resistance and targets to improve efficacy. The information gained from this Project is critical to the overall goals of the P01: identifying the most promising progestins and the patients most likely to benefit from their use.