Rational Combination Of Mtor And Aurora Kinase A Inhibition In Preclinical Models Of Triple-Negative Breast Cancer

Background: Alisertib is a highly selective inhibitor of Aurora kinase A and has antiproliferative and proapoptotic activity in a subset of triple-negative breast cancer (TNBC) cell lines and patient-derived tumor xenograft (PDX) models. Cellular senescence and increased expression of genes in the PI3K/AKT/mTOR pathway has been observed in TNBC models following treatment with alisertib that demonstrate de novo or acquired resistance. The purpose of this study was to investigate the combination of alisertib and the TORC1/2 inhibitor TAK-228 in preclinical TNBC models. Methods: MDA-MB-468, HCC1187, HCC1937, CAL51, and BT20 cells were plated in 96 well plates and exposed to increasing concentrations of alisertib (25nM-125nM), TAK-228 (25nM-125nM), or the combination and proliferation was assessed using the Cell Titer-Glo (CTG) assay. Apoptosis was assessed using long-term live cell microscopy and caspase 3/7 staining. Western blotting was used to assess changes in pS6, p4EBP1, and survivin expression. TNBC p53 wildtype CAL51 cells were transfected with the fluorescent ubiquitin cell cycle indicator (FUCCI) reporters and exposed to increasing concentrations of alisertib, TAK-228, or the combination to evaluate the effect on cell cycle progression, growth, and apoptosis. TNBC PDX models CU_TNBC_004 and CU_TNBC_007 were treated with vehicle, alisertib (30mg/kg), TAK-228 (0.5mg/kg), or the combination and assessed for tumor growth inhibition and translational markers by immunofluorescence (IF) and senescence-associated- s-galactosidase (SA-s-gal) staining. Results: A combination effect was observed for alisertib and TAK-228 in vitro with a decrease in cellular proliferation with the combination as measured by CTG. We observed an increase in cell death with the combination, as opposed to cell cycle arrest with single-agent treatment. Alisertib treatment was associated with an increase in survivin not observed with combination treatment. TAK-228 treatment was associated with a decrease in pS6 and p4EBP1 as a single agent or in combination. The combination of TAK-228 and alisertib resulted in greater tumor growth inhibition in vivo as compared to either single agent alone, accompanied by an increase in apoptosis as measured by BAX and DR5 expression and a decrease in senescence as evaluated by SA-s-gal and phenotypic changes. Single agents in the CAL51 FUCCI system resulted in a dose-dependent effect on cell cycle progression and apoptosis by live cell microscopy. The combination, however, led to a complete block of cell growth and simultaneous apoptosis, leading to no expansion of cells after treatment and a gradual loss of the cell population. Conclusions: The combination of alisertib and TAK-228 in vitro and in vivo in TNBC models resulted in greater antiproliferative and proapoptotic activity. This combination is currently being investigated in a phase I dose escalation trial in patients with advanced solid tumors with a planned expansion cohort in metastatic TNBC to further evaluate the mechanism of the combination (NCT02719691). Citation Format: Jennifer R. Diamond, James D. Orth, Anastasia Ionkina, Kyrie Dailey, Todd M. Pitts, Anna Capasso, Joshua M. Marcus, Russell T. Burke, Sarah L. Davis, Jiyhe Kim, Aik-Choon Tan, Sue G. Eckhardt, John J. Tentler. Rational combination of mTOR and Aurora kinase A inhibition in preclinical models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B175.