MYCN amplification is a major biomarker of poor prognosis, occurring in 25–30% of neuroblastomas. MYCN has contradictory roles in promoting cell growth and sensitizing cells to apoptosis. We have recently shown that p53 is a direct transcriptional target of MYCN in neuroblastoma and that p53-mediated apoptosis may be an important mechanism of MYCN-induced apoptosis. Although p53 mutations are rare in neuroblastoma at diagnosis, the p53/MDM2/p14^ARF pathway is often inactivated through MDM2 amplification or p14^ARF inactivation. We hypothesized that reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and MI-63, will result in p53-mediated growth arrest and apoptosis especially in MYCN-amplified cells. Using the SHEP Tet21N MYCN-regulatable system, MYCN(−) cells were more resistant to both Nutlin-3 and MI-63 mediated growth inhibition and apoptosis compared with MYCN(+) cells and siRNA-mediated knockdown of MYCN in four MYCN-amplified cell lines resulted in decreased p53 expression and activation, as well as decreased levels of apoptosis following treatment with MDM2-p53 antagonists. In a panel of 18 neuroblastoma cell lines treated with Nutlin-3 and MI-63, the subset amplified for MYCN had a significantly lower mean GI₅₀ value (50% growth inhibition) and increased caspase 3/7 activity compared with the non-MYCN-amplified group of cell lines, but p53 mutant cell lines were resistant to the antagonists regardless of MYCN status. We conclude that amplification or overexpression of MYCN sensitizes neuroblastoma cell lines with wild-type p53 to MDM2-p53 antagonists and that these compounds may therefore be particularly effective in treating high-risk MYCN-amplified disease.

Pre-clinical efficacy and synergistic potential of the MDM2-p53 antagonists, Nutlin-3 and RG7388, as single agents and in combined treatment with cisplatin in ovarian cancer

Cutaneous melanoma is the most serious skin malignancy and new therapeutic strategies are needed for advanced melanoma. TP53 mutations are rare in cutaneous melanoma and hence activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma. Here, we investigated the WIP1 inhibitor, GSK2830371, and MDM2–p53 binding antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells. A panel of three p53^WT (A375, WM35 and C8161) and three p53^MUT (WM164, WM35-R and CHL-1) melanoma cell lines were used. The effects of MDM2 and WIP1 inhibition were evaluated by growth inhibition and clonogenic assays, immunoblotting, qRT–PCR gene expression profiling and flow cytometry. GSK2830371, at doses ( ⩽ 10 μM) that alone had no growth-inhibitory or cytotoxic effects on the cells, nevertheless significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53^WT but not p53^MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner. The siRNA-mediated knockdown of p53 provided further evidence to support the p53 dependence. GSK2830371 increased p53 stabilisation through Ser15 phosphorylation and consequent Lys382 acetylation, and decreased ubiquitination and proteasome-dependent degradation when it was combined with MDM2 inhibitors. These changes were at least partly ATM mediated, shown by reversal with the ATM inhibitor (KU55933). GSK2830371 enhanced the induction of p53 transcriptional target genes, cell cycle arrest and apoptosis. GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors by increasing phosphorylation, acetylation and stabilisation of p53 in cutaneous melanoma cells in a functional p53-dependent manner.

Gemcitabine-based chemotherapy is the current standard treatment for biliary tract cancers (BTCs) and resistance to gemcitabine remains the clinical challenge. TP53 mutation has been shown to be associated with poor clinicopathologic characteristics and survival in patients with BTCs, indicating that p53 plays an important role in the treatment of these cancers. Herein, we comprehensively reviewed previous BTC preclinical research and early clinical trials in terms of p53, as well as novel p53-targeted treatment, alone or in combination with either chemotherapy or other targeted therapies in BTCs. Preclinical studies have demonstrated that p53 mutations in BTCs are associated with enhanced gemcitabine resistance, therefore targeting p53 may be a novel therapeutic strategy for treatment of BTCs. Directly targeting mutant p53 by p53 activators, or indirectly by targeting cell cycle checkpoint proteins (Chk1, ataxia telangiectasia related (ATR), and Wee1) leading to synthetic lethality, may be potential future strategies for gemcitabine-resistant p53 mutated BTCs. In contrast, for wild-type p53 BTCs, activation of p53 by inhibition of its negative regulators (MDM2 and wild-type p53-induced phosphatase 1 (WIP1)) may be alternative options. Combination therapies consisting of standard cytotoxic drugs and novel small molecules targeting p53 and related signaling pathways may be the future key standard approach to beat cancer.

Sensitivity to MDM2 inhibitors is widely different among responsive TP53 wild-type cell lines and tumors. Understanding the determinants of MDM2 inhibitor sensitivity is pertinent for their optimal clinical application. Wild-type p53-inducible phosphatase-1 (WIP1) encoded by PPM1D, is activated, gained/amplified in a range of TP53 wild-type malignancies, and is involved in p53 stress response homeostasis. We investigated cellular growth/proliferation of TP53 wild-type and matched mutant/null cell line pairs, differing in PPM1D genetic status, in response to Nutlin-3/RG7388 ± a highly selective WIP1 inhibitor, GSK2830371. We also assessed the effects of GSK2830371 on MDM2 inhibitor-induced p53Ser15 phosphorylation, p53-mediated global transcriptional activity, and apoptosis. The investigated cell line pairs were relatively insensitive to single-agent GSK2830371. However, a non–growth-inhibitory dose of GSK2830371 markedly potentiated the response to MDM2 inhibitors in TP53 wild-type cell lines, most notably in those harboring PPM1D-activating mutations or copy number gain (up to 5.8-fold decrease in GI50). Potentiation also correlated with significant increase in MDM2 inhibitor–induced cell death endpoints that were preceded by a marked increase in a WIP1 negatively regulated substrate, phosphorylated p53Ser15, known to increase p53 transcriptional activity. Microarray-based gene expression analysis showed that the combination treatment increases the subset of early RG7388-induced p53 transcriptional target genes. These findings demonstrate that potent and selective WIP1 inhibition potentiates the response to MDM2 inhibitors in TP53 wild-type cells, particularly those with PPM1D activation or gain, while highlighting the mechanistic importance of p53Ser15 and its potential use as a biomarker for response to this combination regimen. Mol Cancer Ther; 15(3); 379–91. ©2016 AACR.