Regulation of the retinoblastoma (Rb) tumor suppressor by the ubiquitin-proteasome system

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.

Grant Number: 5F31CA288070-02
NIH Institute/Center: NIH
Principal Investigator: Amy Aponte