Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease group with variable clinico-pathologic features. Based on gene expression profiles, TNBCs are grouped into 6 major subtypes. The Luminal androgen receptor (LAR) subtype is enriched for potentially targetable biomarkers, including high androgen receptor (AR) expression, high rates of PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combinations of CDK4/6, AR, and PI3K-AKT inhibitors in pre-clinical models of LAR TNBC for future clinical trial design. Methods: MDA-MB-453 and MFM-223 (both Rb-intact/PTEN-intact/PIK3CA-mutant) and CAL-148 (Rb-null/PTEN-null/PIK3CA-mutant) LAR TNBC cell lines were treated with the CDK4/6 inhibitor palbociclib, the PI3Kα inhibitor alpelisib, the AKT inhibitor capivasertib, and the AR antagonist enzalutamide, each alone or in different combinations. Drug sensitivity was determined by coulter counter cell counts in 2D, colony formation, and the CellTiterGlo cell viability assay. The combination index (CI) which defines synergism (CI < 1), additive effect (CI = 1) and antagonism (CI > 1), calculated by the CompuSyn method, was used to evaluate the synergistic effects of drug combinations. Expression of cell cycle and PI3K-AKT downstream signaling molecules was measured by western blot analysis. An androgen response element (ARE) luciferase-based reporter assay was used to evaluate AR transcriptional activity. Results: Rb-intact LAR TNBC cell lines were sensitive to single-agent palbociclib, alpelisib or capivasertib (IC50, ~500 nM). Enzalutamide had minimal growth inhibitory activity (IC50, 15-25 μM). Palbociclib combined with either alpelisib or capivasertib synergistically inhibited proliferation of LAR TNBC cells (CI values, 0.07-0.86). Treatment of Rb-intact LAR TNBC cells with palbociclib monotherapy suppressed Rb phosphorylation and resulted in adaptive phosphorylation/activation of S473 AKT and AKT substrates GSKβ and PRAS40 at 24h. These responses were not observed in Rb-null CAL-148 cells. Palbociclib-induced phosphorylation of AKT substrates as well as induction P-S6 and P-4EBP1 were better suppressed by capivasertib than by alpelisib over a dose range. Addition of the PI3Kβ/δ inhibitor AZD8186 to alpelisib markedly enhanced the inhibition of P-AKT, P-PRAS40 and P-Sin, suggesting inhibition of PI3Kα is inadequate to block the adaptive response to palbociclib in these cells. Mean CI values showed that the combination of palbociclib/capivasertib was more synergistic against LAR TNBC cells compared to palbociclib/alpelisib (mean CI, 0.29 vs. 0.78). ARE reporter activity did not change upon inhibition of PI3K or AKT with alpelisib or capivasertib, respectively. Conclusions: Our results suggest that addition of an AKT inhibitor to palbociclib suppresses the rebound activation of AKT following treatment with the CDK4/6i and is effective in LAR TNBC with wild type Rb. In vivo studies are underway to investigate the antitumor activity of the combination of palbociclib and capivasertib in LAR TNBC xenografts.

Citation Format: Gun Min Kim, Kyung-min Lee, Dhivya Sudhan, Albert Lin, Arnaldo Marin, Sumanta Chatterjee, Dan Ye, Vishal Kandagatla, Saurabh Mendiratta, Ariella Hanker, Carlos Arteaga. Combined inhibition of CDK4/6 and AKT is effective in Rb-intact triple-negative breast cancer of the luminal androgen receptor (LAR) subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-07.