Abstract 386: Targeting MDM4 to reactivate p53 signaling in neuroblastoma

Abstract

Abstract Introduction: Despite aggressive therapeutic regimens, long-term survival of patients with high risk neuroblastoma (NB) is less than 50%. The p53 tumor suppressor gene is mutated in more than 50% of human cancers, however, unlike in most adult tumors, p53 is wild-type and functional in 99% of NB cases at diagnosis. Reactivation of p53 tumor suppressor function by pharmacological inhibition of MDM4, a negative regulator of p53, is a novel strategy in NB treatment. We hypothesized that targeting MDM4 to reactivate p53 signaling would have effective anti-tumor effects on NB. Methods: Levels of gene expression of MDM4 and its association with event-free and overall survival in NB patients were analyzed using the publically available patient datasets in the R2 Genomics Analysis and Visualization Platform. MDM4 gene and protein expression were also examined in six NB cell lines with qPCR and immunoblotting assays. The effects of MDM4 inhibitor NSC207895 on p53 wild-type (p53wt) and mutated cell lines were analyzed for cytotoxicity, growth in soft agar, and expression of apoptosis markers and p53 downstream targets. Moreover, an orthotopic xenograft NB mouse model was used to test the in vivo efficacy of NSC207895. Results: MDM4 expression is significantly correlated with both event-free and overall survival of NB patients in the Kocak (n=476) and Oberthuer (n=251) datasets in the R2 Genomics Analysis and Visualization Platform, with higher expressions correlated with decreased survival (*** p <0.001). All six NB cell lines showed gene and protein expression of MDM4. NSC207895 had strong cytotoxic effects at relatively low concentrations (IC50 from 0.45-2.46 μM) after 48 hours of treatment with p53wt IMR-32, NGP, NB-19 and SH-SY5Y cells, but not with p53 mutant SK-N-AS (IC50:10.55 μM). NSC207895 significantly inhibited the colony formation ability of several NB cells. NSC207895 exposure also led to upregulation of p53 targets p21, Puma, and Bax in p53wt cells but not in p53 mutant cells. In addition, NSC207895 is more potent in p53wt SH-SY5Y (IC50=0.46 uM) than in the p53 knock out SH-SY5Y cells (IC50=0.98 uM). Moreover, NSC207895 significantly enhanced the cytotoxicity of doxorubicin on p53wt cells but not in p53 mutant cells. Above all, NSC207895 inhibited the tumor growth in an orthotopic xenograft NB mouse model (Mann-Whitney test, ** p <0.01) with no significant side-effects. In addition, compared with the placebo group, NSC207895 treated NB tumors demonstrated elevated expression levels of cleaved PARP, together with p53 downstream targets p21, Puma, and Bax, suggesting that NSC207895 induced p53-mediated apoptosis in vivo. Conclusion: Novel MDM4 inhibitor NSC207895 shows anti-tumor efficacy in p53 wild-type NB by reactivating p53 tumor suppressor signaling and inducing p53-mediated apoptosis, both in vitro and in vivo. Inhibition of MDM4 by small molecules is a promising treatment strategy for high risk patients with wild-type p53. Citation Format: Zhenghu Chen, Sarah E. Woodfield, Roma H. Patel, Aryana M. Ibarra, Sanjeev A. Vasudevan. Targeting MDM4 to reactivate p53 signaling in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 386.