Abstract 5483: Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC)
Authors: del Rosario Chica-Parrado Maria , Kin Gun Min , Lin Chang-Ching , Lee Kyung-min , Napolitano Fabiana , Ye Dan , Bikorimana Emmanuel , Mendirata Saurabh , Hanker Ariella , Arteaga Carlos L. 
Published: June 2023

Abstract
The LAR subtype of TNBC is enriched for targetable biomarkers, including androgen receptor (AR) expression, PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combinations of inhibitors of CDK4/6 (palbociclib), AR (enzalutamide), and PI3K-AKT (alpelisib/capivasertib) against preclinical models of LAR TNBC. MDA-MB-453 and MFM-223 (both AR positive/Rb-intact/PIK3CA-mutant) LAR TNBC cells were treated with each inhibitor alone or in different combinations. Drug sensitivity was measured as growth in 2D, colony formation, and CellTiterGlo viability. Synergistic drug effects were expressed as the combination index (CI) calculated by CompuSyn methods. Activation of cell cycle and PI3K-AKT signaling molecules in response to different inhibitors or siRNAs was measured by immunoblot analysis. An androgen response element (ARE) luciferase reporter assay was used to evaluate AR transcriptional activity. MDA-MB-453 and MFM-223 cells were sensitive to single-agent palbociclib, alpelisib, or capivasertib (IC50 ~500 nM) but not enzalutamide (IC50 15-25 µM). Palbociclib combined with either the PI3Kαi alpelisib or the AKTi capivasertib synergistically inhibited growth of both cell lines (CI values, 0.07-0.86). AR transcriptional reporter activity was not altered by any of these treatments. Treatment with palbociclib monotherapy (24 h) suppressed p-Rb and increased phosphorylation of AKTS473 and its substrates FOXO3A, GSK3β, and PRAS40, suggesting PI3K/AKT signaling mediates an adaptive response to CDK4/6 blockade. In MDA-MB-453 cells, palbociclib-induced phosphorylation of AKT substrates was suppressed by treatment with capivasertib but not with alpelisib alone or with the PI3Kβ/δ inhibitor AZD8186, suggesting that the compensatory activation of AKT was due to a mechanism independent of the PI3K isozymes. We next used RICTOR siRNAs to block mTORC2-mediated phosphorylation AKTS473 after palbociclib treatment. Gene silencing of RICTOR reduced levels of the mTORC2 substrate p-SGK1S422 but did not block phosphorylation of AKTS473 and its substrates in palbociclib-treated cells, suggesting that this adaptative response was mTORC2-independent. Mean CI values showed that the combination of palbociclib/capivasertib was clearly more synergistic than palbociclib/alpelisib in both cells lines (mean CI, 0.29 vs. 0.78). Currently, we are comparing both combinations in mice bearing established MDA-MB-453 xenografts. Further investigation of the mechanisms of adaptive activation of AKT upon blockade of CDK4/6 in TNBC cells are in progress. In sum, addition of an AKT inhibitor, but not PI3K inhibitors, to palbociclib suppressed the rebound activation of AKT following treatment with the CDK4/6i, supporting a testable therapeutic strategy in LAR TNBC with intact Rb.
Citation Format: Maria del Rosario Chica-Parrado, Gun Min Kin, Chang-Ching Lin, Kyung-min Lee, Fabiana Napolitano, Dan Ye, Emmanuel Bikorimana, Saurabh Mendirata, Ariella Hanker, Carlos L. Arteaga. Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5483.