PO-037 Targeting mutant p53 with COTI-2: a new approach for the treatment of patients with triple-negative breast cancer?

Abstract

IntroductionThe identification of a targeted therapy for triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. Since the p53 gene is mutated in approx. 80% of TNBC tumours, it is an attractive target. COTI-2 is a clinical stage, small molecule which claims to target p53. The aim of this study was to investigate COTI-2 as a new treatment for TNBC.Material and methodsCell viability was determined by MTT assay. p53 protein levels were quantified by ELISA and immunofluorescent staining. P53 binding kinetics were measured by Surface Plasmon Resonance. Apoptosis was measured using Annexin V-FITC Apoptosis Detection Kit. Caspase 3/7 was measured by CellEventCaspase-3/7Green Flow Cytometry Assay Kit. CI values were calculated using Calcusyn software.Results and discussionsUsing a panel of 18 breast cell lines, TNBC cell lines were more responsive to COTI-2 than non-TNBC cells (p=0.04). Lower IC50 values for COTI-2 were found in p53 mutant vs p53 WT cells (p=0.001). Additionally, the higher the endogenous p53 protein, the more sensitive the cell line was to COTI-2 (p=0.035, r=−0.51, n=18). By staining with antibodies specific for folded WT p53 (PAb1620) or unfolded mutant p53 (PAb240), we showed that COTI-2 can induce refolding of mutant p53. Moreover, by SPR, we showed that COTI-2 binds to fl-mut-p53 protein, in a concentration dependent manner.In addition to inhibiting proliferation, COTI-2 induced apoptosis in a concentration dependent manner. Furthermore, inhibition of caspase activity with Z-VAD-FMK reduced apoptosis, suggesting that COTI-2 induces caspase-dependent apoptosis. Accordingly, COTI-2 induced a significant increase in caspase 3/7.In an effort to enhance response, COTI-2 was combined with a number of cytotoxic agents. Highly synergistic growth inhibition, i.e. CI <1, was found when COTI-2 was combined with doxorubicin in 6 different cell lines. In addition, COTI-2 plus docetaxel or eribulin was synergistic in 4/6 cell lines, plus carboplatin was synergistic in 3/6, while plus cisplatin was synergistic in 2/6. Finally, we compared response to COTI-2 with that of APR-246, the best studied p53 reactivating compound. Overall, the mean COTI-2 IC50 value was 64 fold lower than that for APR-246 (p=0.0006). Furthermore, no correlation was seen between response to COTI-2 or APR-246, suggesting that the compounds act differently in inhibiting cell growth.ConclusionWe conclude that targeting mutant p53 with COTI-2 is a potential new approach for treating p53-mutated TNBC.