Post-translational modifications of the PAX3::FOXO1 oncoprotein
Mentor Name: Charles Keller
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and often also occurs in adolescents and young adults. Clinically, survival amongst metastatic RMS patients has remained dismal yet unimproved for decades. RMS harboring the driver PAX3::FOXO1 chimeric oncoprotein is generally not survivable when metastatic. A curative-intent clinical trial for relapsed and refractory rhabdomyosarcoma has not been open for enrollment since July 20, 2013 (12 years ago), reflecting a paucity of biological insights and new therapeutic options. This unmet clinical need is the reason for this back-to-basics research proposal. For this benchmarking study of PAX3::FOXO1 post-translational modifications (PTMs), we hypothesize that specific PTMs uniquely account for the protein stability, cell-cycle dependence of expression, and transcriptional activity of PAX3::FOXO1 in rhabdomyosarcoma tumor cells. Thus, we will pursue (Aim 1) the delineation of the post-translational modifications of PAX3::FOXO1 especially related to cell-cycle dependent degradation by determining the overall degree of basal post-translational modifications, enabling site-specific identification of basal PAX3::FOXO1 PTMs, determining the types of post-translational modifications associated with proteasomal degradation, and identifying the native E3 ubiquitin ligase(s) controlling PAX3::FOXO1 stability. We will also (Aim 2) define the functional effects of post-translational modifications of PAX3::FOXO1 by determining transcriptional activity of PAX3::FOXO1 with and without phosphorylation at observed sites and determining the ubiquitin site and cognate E3 ligases for PAX3::FOXO1. Knowing these PTMs and their consequences will help design treatment interventions that target PAX3::FOXO1 stability or activity for the new era of PROTAC protein degraders and molecular glue protein degraders.
