PROJECT SUMMARY The retinoblastoma (Rb) protein is a prototypical tumor suppressor due to its role in restricting proliferation. In its active form, Rb functions as the key regulator of the G1/S cell cycle transition by physically binding to E2F transcription factors, an interaction that inhibits activation of genes necessary for DNA replication. To relieve transcriptional repression of pro-proliferative genes and promote cell cycle progression, Rb is inactivated through its phosphorylation by the cyclin-dependent kinases 4 and 6 (Cdk4/6). This inactivation is the direct target of the Cdk4/6 inhibitors that are currently used in combination with anti-hormone therapy for treatment of hormone- receptor positive, HER2-negative metastatic breast cancer. Despite the clinical success of Cdk4/6 inhibitors, primary and acquired resistance in patients remain a major challenge, and these drugs are ineffective against other tumor types. Identifying other mechanisms that regulate Rb and mediate sensitivity to Cdk4/6 inhibitors should provide avenues for addressing some of these clinical challenges. I have preliminary data that suggests Rb is subject to rapid turnover throughout the cell cycle, and I have found that Rb protein levels are decreased following Cdk4/6 inhibition. Finally, I have found that Rb protein levels can be stabilized and rescued by disrupting components of the ubiquitin-proteasome system, indicating that the Rb protein is regulated by this proteolytic pathway. Collectively, these findings challenge our current understanding of Rb as a stable protein and are clinically relevant because a change in Rb protein abundance following Cdk4/6 inhibition can potentially compromise the ability of Rb to restrict proliferation and maintain proper cell cycle arrest. Thus, the overall goal of this proposal is to understand how ubiquitin signaling affects Rb’s tumor suppressive function. Based on my preliminary findings and current understanding about the role of Rb in cell cycle control, my central hypothesis is that the regulation of Rb protein by the ubiquitin-proteasome system contributes to proper cell cycle progression and mediates response to Cdk4/6 inhibition. In Aim 1, I will use mass-spectrometry and a CRISPR/Cas9 approach to identify the ubiquitin ligase (E3) that degrades Rb and live-cell imaging to determine the timing of Rb degradation in the cell cycle. In Aim 2, I will investigate how the degradation of Rb affects cell cycle progression using various biochemical, genetic and fluorescent imaging approaches. By identifying a non- degradable Rb mutant and engineering cells lines that express an inducible form of this mutant, I will be able to determine the extent to which disrupting Rb degradation affects sensitivity to Cdk4/6 inhibition. Completion of these aims will provide me with diverse training experiences in cell, systems and cancer biology and shed light on how Rb is regulated beyond phosphorylation-dependent inactivation.