Reactivating TP53 signaling by the novel MDM2 inhibitor DS-3032b as a therapeutic option for high-risk neuroblastoma

Abstract

// Viktor Arnhold 1, 2 , Karin Schmelz 1 , Jutta Proba 1 , Annika Winkler 1 , Jasmin Wünschel 1 , Joern Toedling 1 , Hedwig E. Deubzer 1, 2, 3 , Annette Künkele 1, 2 , Angelika Eggert 1, 2, 4, 5 , Johannes H. Schulte 1, 2, 4, 5 and Patrick Hundsdoerfer 1, 2 1 Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Pediatric Hematology/Oncology/Stem Cell Transplantation, Berlin, Germany 2 Berlin Institute of Health (BIH), Anna-Louisa-Karsch 2, Berlin, Germany 3 Neuroblastoma Research Group, Experimental and Clinical Research Center (ECRC), Berlin, Germany 4 German Cancer Consortium (DKTK), Heidelberg, Germany 5 German Cancer Research Center (DKFZ), Heidelberg, Germany Correspondence to: Viktor Arnhold, email: viktor.arnhold@charite.de Keywords: apoptosis; CDKN1A; MYCN; pediatric tumors; targeted therapy Received: August 23, 2017 Accepted: December 04, 2017 Published: December 18, 2017 ABSTRACT Fewer than 50% of patients with high-risk neuroblastoma survive five years after diagnosis with current treatment protocols. Molecular targeted therapies are expected to improve survival. Although MDM2 has been validated as a promising target in preclinical models, no MDM2 inhibitors have yet entered clinical trials for neuroblastoma patients. Toxic side effects, poor bioavailability and low efficacy of the available MDM2 inhibitors that have entered phase I/II trials drive the development of novel MDM2 inhibitors with an improved risk-benefit profile. We investigated the effect of the novel MDM2 small molecular inhibitor, DS-3032b, on viability, proliferation, senescence, migration, cell cycle arrest and apoptosis in a panel of six neuroblastoma cell lines with different TP53 and MYCN genetic backgrounds, and assessed efficacy in a murine subcutaneous model for high-risk neuroblastoma. Re-analysis of existing expression data from 476 primary neuroblastomas showed that high-level MDM2 expression correlated with poor patient survival. DS-3032b treatment enhanced TP53 target gene expression and induced G1 cell cycle arrest, senescence and apoptosis. CRISPR-mediated MDM2 knockout in neuroblastoma cells mimicked DS-3032b treatment. TP53 signaling was selectively activated by DS-3032b in neuroblastoma cells with wildtype TP53 , regardless of the presence of MYCN amplification, but was significantly reduced by TP53 mutations or expression of a dominant-negative TP53 mutant. Oral DS-3032b administration inhibited xenograft tumor growth and prolonged mouse survival. Our in vitro and in vivo data demonstrate that DS-3032b reactivates TP53 signaling even in the presence of MYCN amplification in neuroblastoma cells, to reduce proliferative capacity and cause cytotoxicity.