The genomic and functional status of TP53 in ovarian cancer :biomarker for chemotherapy outcome and determinant of response to MDM2 inhibitors

Abstract

Background: Mutation and loss of TP53 function is one of the most frequent genetic abnormalities in ovarian cancer. TP53 genomic and functional status have been shown to provide potentially prognostic and predictive value in ovarian cancer; however, the results are controversial and evaluation in the context of a controlled clinical trial with single agent treatment have been lacking. Reactivation of p53 using MDM2-p53 antagonists is a promising therapeutic target for most patients with type I epithelial ovarian cancer and those left from type II harbouring wild-type TP53. BRCA1/2 mutations are present in 70-85% of germline mutations in patients with inherited ovarian cancer, and deficiencies in homologous recombination repair (HRR) account for up to 50% of epithelial ovarian cancer, indicating the possible sensitivity of ovarian cancer patients to PARP inhibitors. MDM2-p53 antagonists and PARP inhibitors are now undergoing clinical trials as targeted therapy for different types of cancer. The effect of RG7388 on its own and in combination with cisplatin, and combined treatment between MDM2-p53 antagonists and PARP inhibitors have not been investigated in ovarian cancer. Hypotheses: 1) Different genomic and functional status of p53 and some of its downstream targets such as p21WAF1, MDM2 and WIP1 can be used as prognostic and predictive biomarkers for the outcome of chemotherapy and overall survival in ovarian cancer. 2) Reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and RG7388, will result in p53-mediated growth arrest and apoptosis in wild-type TP53 ovarian cancer cells, and combination of them with current therapeutic agents or rucaparib increases growth inhibition and/or apoptosis in ovarian cancer cell lines compared to either agent alone. Methods: TP53 was sequenced in 260 ovarian cancer samples from the ICON3 trial using Sanger sequencing and Next Generation Sequencing (NGS) methods. The prognostic value of the expression levels of p53, p21WAF1, MDM2 and WIP1 was investigated using immunohistochemistry (IHC). The effect of MDM2-p53 antagonists, Nutlin-3/RG7112/RG7388, and PARP inhibitor, rucaparib, as single agents and in combination with cisplatin or together were investigated on a panel of ovarian cancer cell lines. Sensitivity was measured by growth inhibition, clonogenic cell survival assay, apoptosis assays including caspase 3/7 activity and flow cytometry. The effect on the p53 molecular pathway and p53-regulated candidate gene expression were investigated by western blotting and Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) respectively. Results: Patients from the ICON3 clinical trial treated with carboplatin whose tumours harbour wild-type TP53 had a significantly better overall survival based on both univariate and multivariate analysis compared to those with mutant TP53 regardless of sequencing method. Adding paclitaxel to the platinum-based treatment showed a trend in favour of greater benefit for those with mutant TP53, although this failed to reach statistical significance (p>0.05). Overexpression of p53 has potential prognostic value for overall survival of ovarian cancer patients. Ovarian cancer cell lines with wild-type TP53 were sensitive to MDM2-p53 antagonists, Nutlin-3/RG7112/RG7388, while those with mutant TP53 were resistant to MDM2 inhibitors. Among the individual cell lines, A2780 and MDAH-2774 were sensitive and other cell lines (IGROV-1, OAW42, CP70, MLH1-corrected CP70+ and SKOV-3) were resistant to rucaparib regardless of BRCA1/BRCA2 status or deficiencies in HRR reported for these cell lines. Combination of Nutlin-3/RG7388 with cisplatin or rucaparib has synergistic and/or dose reduction potential dependent on cell genotype and the type of MDM2-p53 antagonist. Combined treatments using Nutlin-3/RG7388 and cisplatin led to greater levels of p53 stabilisation and upregulation of p21WAF1 and MDM2, and higher expression of p21WAF1 was associated with a greater synergistic effect for growth inhibition. In combination treatment with rucaparib and Nutlin-3/RG7388, rucaparib showed no increase in the effect of MDM2 inhibitors on the p53 pathway, indicating that the mechanism of observed synergy does not involve enhancement of p53 pathway activation by MDM2 inhibitors. Nutlin-3/RG7388 in combination with cisplatin or rucaparib resulted in changes in cell cycle distribution, SubG1 events and caspase 3/7 activity in a cell type, time and compound-dependent manner. The fold changes in expression of candidate genes in response to MDM2 inhibitors were less in A2780 cells than IGROV-1 and OAW42. The balance of activity between growth inhibitory/pro-survival and pro-apoptotic genes dominates a small increase in the expression of several DNA repair genes as an explanation for the synergy observed for treatment with cisplatin and MDM2 inhibitors. Conclusions: The genomic and functional status of TP53 have potentially important prognostic and predictive values in ovarian cancer. Targeting the interaction between MDM2 and p53 using MDM2-p53 antagonists is a promising therapeutic strategy for ovarian cancer patients with wild-type TP53 tumours, and combination treatment with them and cisplatin or rucaparib