Abstract 2819: Characterization of the mechanisms of early and later stages of resistance to the selective CDK4/6 inhibitor palbociclib
Authors: Bouchekioua-Bouzaghou Katia , Lenihan Catherine , Shia Alice , Wilkes Edmund , Casado-Izquierdo Pedro , Cutillas Pedro , Schmid Peter 
Published: July 2016

Abstract
Background: Dysregulation of the cyclin D-CDK4/6-Rb axis occurs in a substantial proportion of ER-positive (ER+) breast cancers promoting proliferation and resistance. Adding the CDK4/6 inhibitor palbociclib to endocrine treatment has led to a substantial improvement of the outcome of patients with ER+ metastatic breast cancer. However, with the expected clinical implementation of CDK4/6 inhibitors, acquired resistance is emerging as a major clinical challenge. Using long-term culture with palbociclib, we have established MCF7 and T47D cell lines with 8-10 fold increased IC50 values as a model of acquired resistance. Characterization of resistant cell lines using phosphoproteomic analysis and RNAseq revealed increased activity in the PI3K/AKT/mTOR pathway which can be targeted therapeutically by co-treatment with CDK4/6 inhibitors and PI3K/AKT/mTOR inhibitors (Lenihan C, et al, San Antonio Breast Cancer Symposium 2015). The present work addresses whether palbociclib still has target effects and/or residual activity in resistant cells, to characterize cell cycle pathways in resistant cells, to establish the role of ER-signaling in resistant cells, and to study if palbociclib resistance might be associated with a more aggressive phenotype.
Results: Western blot analysis of newly generated resistant clones demonstrated significant reduction of phospho RB (ser780), in keeping with maintained target inhibition with palbociclib. Although total RB decreased in some clones, the protein remained detectable in all resistant clones. Cumulative population doubling confirmed palbociclib still has an effect on cell proliferation in resistant clones. Subsequent long-term culture in the presence of palbociclib altered the effect of the drug; these cells gradually lose the inhibition of pRB, as well as the residual anti-proliferative effect of palbociclib, suggesting potential loss of palbociclib target effects. Mutational analysis of RB is ongoing. Cell cycle analysis of resistant clones demonstrated an increase in cyclin E expression suggesting up-regulation of alternative cell cycle pathways. Morphological changes of resistant clones and multiple phosphorylation changes in the Rho/Rac pathway showed by our phosphoproteomic analysis suggested that resistant clones might exhibit a more aggressive phenotype. Tumor spheroid-based invasion assays and transwell migration assays confirmed increased migration and invasion of resistant clones compared to sensitive cells.
Conclusions: Whilst Palbociclib has sustained target effects and anti-proliferative activity during early stages of resistance, these effects gradually diminish during later stages of acquired resistance. Palbociclib resistant breast cancer cells also exhibit a more aggressive phenotype. In vivo investigation is ongoing to better characterize the escape mechanisms involved in CDK4/6 inhibitors resistance.
Citation Format: Katia Bouchekioua-Bouzaghou, Catherine Lenihan, Alice Shia, Edmund Wilkes, Pedro Casado-Izquierdo, Pedro Cutillas, Peter Schmid. Characterization of the mechanisms of early and later stages of resistance to the selective CDK4/6 inhibitor palbociclib. [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 2819.