Abstract 2989: An Intact G1/S checkpoint determines response to CDK4/6 inhibitor in breast cancer
Authors: Vijayaraghavan Smruthi , Keyomarsi Khandan 
Published: July 2016

Abstract
Deregulation of the cell cycle machinery is a hallmark of most cancers, making the positive regulators of cell cycle such as Cyclin dependent kinases (CDKs) as attractive and druggable targets. Palbociclib or PD-0332991, a potent CDK4/6 inhibitor is an anti-proliferative agent that induces G1 arrest, senescence and inhibits tumor growth in breast cancer. Based on the drug's success in phase II clinical trials, Palbociclib was recently awarded FDA approval for the treatment of metastatic ER+ breast cancer. However, one of the main limitations of this treatment is the associated adverse events of grade 3&4 neutropenia (62%) and grade 3&4 leukopenia (25.2%). Moreover, despite promising results in clinical trials, little is known about palbociclib's mechanism of action and mode(s) of resistance in ER+ breast cancer. A clear understanding of these mechanisms is critical to identify biomarkers and understand the biology of treatment response, drug resistance and combination strategies, and this study is aimed at addressing these gaps in knowledge.
In order to examine the mechanism of action and identify nodes of sensitivity to Palbociclib, we used a model system of two ER+ breast cancer cell lines (MCF7, T47D) and a mammary epithelial cell line (MCF10A). Results from drug response studies, cell cycle analysis and senescence assay revealed that treatment with palbociclib induces a dose-dependent sustained growth inhibition and G1 arrest specifically in the ER+ cell lines. Additionally, palbociclib induced senescence in a dose-dependent manner in the ER+ cancer cells and not in MCF10A.
Further, to interrogate if deregulation of the G1/S checkpoint can render cells resistant to palbociclib, we examined the effect of knocking down Rb and overexpressing the oncogenic low molecular weight isoforms of cyclin-E (LMW-E), which are known to induce constitutive phosphorylation of Rb, in our model system. Drug response, cell cycle analysis and senescence assay revealed that both downregulation of Rb and overexpression of LMW-E expression significantly diminished the sensitivity of breast cancer cells to palbociclib (∼6 to 7 fold) and its ability to induce senescence.
Moreover, while Palbociclib is known to be a specific CDK4/6 inhibitor, drug response studies showed that downregulation of either kinase in our model system did not completely recapitulate the drug's anti-tumor effects. This suggests that Palbociclib could inhibit another target, albeit, at a higher concentration, and this would be the subject of our future studies.
Collectively, our study indicates that the CDK4/6 inhibitor, Palbociclib, acts in a dose-dependent manner to induce senescence, specifically in breast cancer cells. Further, our results suggest that the presence of an intact G1/S checkpoint is necessary for response to palbociclib, providing rationale to utilize Rb positivity and absence of LMW-E as biomarkers of response to the drug in ER+ breast cancer patients.
Citation Format: Smruthi Vijayaraghavan, Khandan Keyomarsi. An Intact G1/S checkpoint determines response to CDK4/6 inhibitor in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2989.