PO-482 Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer

Abstract

IntroductionHuman BAD (hBAD) is a pro-apoptotic Bcl-2 family member, whose apoptotic functions can be inactivated through phosphorylation of specific residues including Ser99. Clinically, BAD phosphorylation has been reported to indicate poor survival and cisplatin resistance in ovarian cancer patients. Herein, we investigated the therapeutic potential of inhibiting BAD Ser99 phosphorylation in ovarian cancer.Material and methodsNPB, a novel small molecule which specifically inhibits BAD Ser99 phosphorylation, has been developed in our laboratory. Cell function assays performed include Alamar Blue cell viability assay, caspase3/7 assay, PI-Annexin V apoptosis assay, cell cycle analysis and 3D growth in Matrigel. The CI values are calculated by CompuSyn using Chou-Talalay method. The cancer stem cell-like cell population was examined by ALDEFLOUR and sphere formation assays.Results and discussionsThe level of BAD Ser99 phosphorylation is negatively correlated with cisplatin sensitivity in a panel of ovarian cancer cell lines. BAD Ser99 phosphorylation is increased upon acute cisplatin treatment of both sensitive and resistant ovarian cancer cells. The inhibition of BAD Ser99 phosphorylation by NPB alone increased apoptosis, and decreased cell viability, 3D growth, and anchorage-independent growth of both parental and cisplatin resistant ovarian cancer cells. In particular, NPB increased the sensitivity of the parental cells, and partially re-sensitised cisplatin resistant ovarian cancer cells towards cisplatin, with the combination of NPB and cisplatin showing a synergistic effect. An upstream BAD kinase, AKT has also been reported to mediate cisplatin resistance in ovarian cancer cells. Correspondingly, the combination of NPB and AKT inhibitor AZD5363 exhibited strong synergistic effects in both parental and cisplatin resistant cell lines. We also demonstrated that both the phosphorylation of BAD and its upstream kinase AKT was increased in this CSC-like population. NPB treatment alone was observed to decrease the CSC-like population, while the combination of AZD5363 and NPB produced a synergistic decrease in the CSC-like population.ConclusionOur preclinical data suggests that NPB, as a novel inhibitor of BAD Ser99 phosphorylation, can potentially be used in combination with cisplatin as a therapy for naïve ovarian cancer patients to increase cisplatin sensitivity. Furthermore, the combination of NPB and AKT inhibitor AZD5363 is a potential therapeutic approach in the treatment of cisplatin resistant ovarian cancer.