POST-TRANSLATIONAL REGULATION OF THE RETINOBLASTOMA TUMOR SUPPRESSOR IN RESPONSE TO CDK4/6 INHIBITION

Precise control of cell cycle progression is essential for normal tissue homeostasis, and its dysregulation is a defining feature of cancer. Central to regulation of the G1/S transition is the retinoblastoma tumor suppressor protein (RB), which restrains cell cycle entry by suppressing E2F- dependent transcription. In many cancers, hyperactivation of cyclin-dependent kinases 4 and 6 (CDK4/6) functionally inactivates RB, promoting uncontrolled proliferation. Small- molecule CDK4/6 inhibitors have therefore emerged as effective therapies, particularly in estrogen receptor-positive (ER+) breast cancer, where they induce durable G1 arrest in an RB-dependent manner. While the canonical mechanisms of CDK4/6 inhibition involve prevention of RB phosphorylation, the broader consequences of CDK4/6 inhibition remain incompletely understood. This dissertation investigates the regulation of the RB pathway in response to CDK4/6 inhibition, with a particular focus on post-translational mechanisms that shape RB function, stability and therapeutic response. First, a conceptual framework is established outlining the structure, regulation and functional diversity of the retinoblastoma family of proteins, emphasizing their roles beyond simple cell cycle arrest. Using ER+ breast cancer models, this work then characterizes the multi-level response to CDK4/6 inhibition by integrating transcriptional and proteomic analyses following palbociclib treatments. These studies reveal that CDK4/6 inhibition induces widespread remodeling of the cellular proteome that extends beyond transcriptional regulation and canonical G1 arrest programs, highlighting adaptive responses that are not readily inferred from gene expression alone. Building on these observations, this dissertation identifies a previously underappreciated mechanism by which CDK4/6 inhibition regulates RB protein abundance. Specifically, CDK4/6 inhibition promotes ubiquitin-proteasome-mediated destabilization of RB through a mechanism involving the deubiquitinating enzyme USP28. This finding challenges the prevailing view of RB as a predominantly stable protein regulated solely by phosphorylation and uncovers a novel layer of RB pathway control that may influence sensitivity and resistance to CDK4/6 inhibitors. Collectively, this work advances understanding of RB regulation beyond phosphorylation-centric models and demonstrates that post-translational control of RB stability is an important component of the cellular response to CDK4/6 inhibition. These findings provide new insight into CDK4/6 inhibitor biology and suggest potential strategies to improve the durability and effectiveness of cell cycle-targeted cancer therapies.