Abstract LB-136: Mechanistic exploration of combined CDK4/6 and ER inhibition in ER-positive breast cancer
Authors: Lee Nathan V. , Yuan Jing , Eisele Koleen , Cao Joan Q. , Painter Cory L. , Chionis John , Liu Chaoting , Shields David J. , Kan Julie L.C. , Arndt Kim , VanArsdale Todd 
Published: May 2015

The de-regulation of the Retinoblastoma tumor suppressor is widespread in cancers occurring through the direct mutation or loss of RB1, or enhanced signaling through the Cyclin dependent kinases CDK4 and CDK6 via amplification and/or over expression of D-type cyclins, or loss of p16 (CDKN2A) function. Tumors retaining intact RB1 functions therefore rely on the activity of CyclinD-CDK4/6 complexes to inactivate RB1 and promote progression through the G1 restriction point into S phase. Hormone receptor positive breast cancers are one tumor type where RB1 remains intact in most tumors and deregulation of CDK4/6-Cyclin D signaling is common. As such, dependence on CDK4/6 signaling in ER positive breast cancers has been demonstrated using the specific CDK4/6 inhibitor, PD-0332991 (palbociclib), and the combination of palbociclib and letrozole has been shown to provide significant clinical activity in ER+ breast cancer patients. To determine the mechanism of action for this combination, we investigated the effects of palbociclib with anti-estrogen therapeutics such as letrozole, fulvestrant and tamoxifen on ER+ breast cancer cell lines (MCF7, CAMA1, and T47D). Mechanistic analyses reveal that the combination of palbociclib with fulvestrant or letrozole enhanced inhibition of Rb phosphorylation leading to significantly greater loss of E2F1, FoxM1 and downstream target genes such as PLK1, SKP2 and CCNE2, leading to greater inhibition of cell proliferation. The enhanced growth arrest of the ER+ breast cell lines treated with palbociclib and ER antagonists is accompanied by increased hallmarks of cell senescence, cell enlargement and SA-β-Galactosidase staining, significantly greater than either single agent inhibitor treatment. Also, the arrest of cells following drug removal is maintained significantly longer for cells treated with the combination. To explore these activities in vivo, an ER+ breast patient derived xenograft (PDX) model was studied using the combination of palbociclib and letrozole (aromatase inhibitor). These studies recapitulate in vitro model systems, showing greater tumor growth inhibition with combination therapy, enhanced dephosphorylation of RB1 and accompanied inhibition of downstream signaling. PDX tumors treated with the combination of palbociclib and letrozole displayed reduced KI67 staining compared to single agent treatments and β-galactosidase staining revealed that cell senescence is also a component of the functional response of ER+ breast tumors to the combined inhibition of CDK4/6 and ER signaling in vivo. As apoptotic cell death was not clearly evident in any of these model systems, the mechanism of combined CDK4/6 inhibition and ER antagonism is likely driven by cellular senescence and accompanying long term arrest of tumor cells to prevent disease progression.

Citation Format: Nathan V. Lee, Jing Yuan, Koleen Eisele, Joan Q. Cao, Cory L. Painter, John Chionis, Chaoting Liu, David J. Shields, Julie L.C. Kan, Kim Arndt, Todd VanArsdale. Mechanistic exploration of combined CDK4/6 and ER inhibition in ER-positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-136. doi:10.1158/1538-7445.AM2014-LB-136