Abstract 1783: Pharmacological inhibition of CDK4/6 induces G1 arrest, autophagy and senescence in ER+ breast cancer
Authors: Vijayaraghavan Smruthi , Keyomarsi Khandan 
Published: August 2015

Abstract
Deregulation of the cell cycle machinery is a hallmark of most cancers, leading to aberrant proliferation and tumorigenesis, making cell cycle proteins, particularly cyclin dependent kinases (CDKs), attractive and druggable targets. Palbociclib or PD0332991, a potent CDK4/6 inhibitor, is an anti-proliferative agent that induces G1 arrest and prevents tumor growth in breast cancer. This drug has shown tremendous success in phase II clinical trials in ER+ breast cancer and phase III trials are underway. Despite promising results in the clinic, little is known about palbociclib's mechanism of action or modes of resistance in ER+ breast cancer. A clear understanding of these mechanisms are critical to identify biomarkers and understand the biology of treatment response, drug resistance and combination strategies, and this project is aimed at addressing these gaps in knowledge.
We have used ER+ breast cancer cell lines (MCF7, T47D) to examine the mechanism of action of palbociclib and identify nodes that mediate drug resistance. Our results revealed that treatment with palbociclib induces a G1 arrest with concomitant downregulation of phospho-Rb. Furthermore, treated cells undergo autophagy and senescence in a dose-dependent manner. The autophagic response observed is a pro-survival mechanism and pharmacological inhibition of autophagy augments the cell's sensitivity to palbociclib.
Since this agent is known to be a specific CDK4/6 inhibitor, we downregulated these kinases in our model system and subjected them to drug treatment. Interestingly, knockdown of CDK4 or CDK6 did not completely recapitulate the drug's anti-tumor effects, suggesting that palbociclib could inhibit another target, albeit, at a higher concentration. We then interrogated if the Rb-FOXM1 pathway was sufficient to mediate palbociclib-induced growth inhibition in ER+ breast cancer. Results from drug-response studies revealed that knockdown of Rb reduces the sensitivity of ER+ cells by 4-6 fold, but the role of FOXM1 remains to be understood.
Lastly, to interrogate the modes of acquired resistance, we developed palbocilcib-resistant cell lines, wherein we observed an overexpression of oncogenic low molecular weight isoforms of cyclin-E (LMW-E). Additionally, drug-response studies revealed that LMW-E overexpression can mediate resistance to palbociclib, indicating that LMW-E could serve as a biomarker of resistance.
Collectively, our studies provide a rationale for utilizing palbociclib in the clinic in combination with an inhibitor of autophagy, such as hydroxychloroquine. Further, our results suggest that inhibition of G1/S transition is not the sole mode of action of palbociclib and that there could be another unidentified target that is inhibited, when the G1/S checkpoint is compromised. Identification of this target will be instrumental to overcome resistance and determine biomarkers of sensitivity to this agent in breast cancer patients.
Citation Format: Smruthi Vijayaraghavan, Khandan Keyomarsi. Pharmacological inhibition of CDK4/6 induces G1 arrest, autophagy and senescence in ER+ breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1783. doi:10.1158/1538-7445.AM2015-1783