Ipatasertib, an oral AKT inhibitor, in combination with paclitaxel exhibits anti-proliferative and anti-tumorigenic effects in preclinical studies for endometrioid endometrial cancer: Endometrial cancer molecularly targeted therapy consortium (227)

Abstract

<b>Objectives:</b> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. The PI3K/ AKT pathway is altered in the vast majority of endometrioid endometrial cancers (ECs). A recent clinical trial demonstrated that IPAT combined with paclitaxel showed a significant prolonged overall survival in breast cancer patients. Thus, we aimed to evaluate the antitumorigenic effects of IPAT in combination with paclitaxel in EC cell lines and an EC mouse model. <b>Methods:</b> The human endometrioid EC cell lines Ishikawa <i>(PTEN</i> mutant), and HEC-1A (<i>PTEN</i> wild type, <i>PIK3CA</i> mutant, <i>KRAS</i> mutant) were exposed to varying concentrations and durations of IPAT (Genentech) and paclitaxel. Cell proliferation was assessed by MTT assay. To assess the synergy of IPAT and paclitaxel, the combination index (CI) was calculated by the method of Chou and Talalay. Apoptosis was assessed by cleaved caspase-3 assay. Western immunoblotting determined the effects of IPAT and paclitaxel on apoptosis (MCL, BCL-xl), cellular stress (ATF, PERK, Bip, PDI, IER), and DNA damage (RAD50). LKB1^fl/^flp53^fl/fl mice were used to evaluate the effect of IPAT, paclitaxel, and the combination of these two drugs on EC growth. The mice were treated with placebo, IPAT (50mg/kg daily orally for four weeks), paclitaxel (10 mg/kg IP weekly), or IPAT/paclitaxel starting eight weeks after tumor induction via AdCre injection. <b>Results:</b> IPAT and paclitaxel inhibited cellular proliferation in a dose-dependent fashion in both cell lines after 72 hours of treatment. The median IC50 for IPAT was 0.13µM for Ishikawa and 4.9µM for HEC-1A, respectively. The median IC50 for paclitaxel was 5.6nM for Ishikawa and 1.9nM for HEC-1A, respectively. Simultaneous exposure of cells to various doses of paclitaxel in combination with IPAT resulted in a significant synergistic antiproliferative effect (CI<1). IPAT and paclitaxel induced the activity of cleaved caspase-3 by 3.5-fold in Ishikawa and 1.12-fold in HEC-1A, while simultaneously increasing expression of the apoptotic proteins MCL-1 and BCL-xl. IPAT and paclitaxel also increased Bip, PERK, ATF4, PDI, and IER protein expression. Treatment of cells with IPAT and paclitaxel decreased phosphorylated-S6 (p-S6) expression and induced the expression of p-AKT and RAD50. Compared to control mice, treatment of LKB1^fl/flp53^fl/fl mice with IPAT reduced tumor weight by 62.1%, and treatment with paclitaxel decreased tumor weight by 73.9% (p<0.05). The combination of IPAT and paclitaxel reduced tumor growth by 78.3% and showed improved efficacy over IPAT alone (p<0.05), but not over Taxol alone. <b>Conclusions:</b> The combination of IPAT and paclitaxel demonstrates synergy in human endometrioid EC cell lines and improved efficacy over IPAT alone in the LKB^1fl/fl/p53^fl/fl mouse model of EC. These results suggest that IPAT with paclitaxel is worthy of further exploration in clinical trials for EC.