Abstract 4871: The roles of E3/E4 ubiquitin ligase UBE4B and its targets p53 and Ku70 in neuroblastoma cell death and differentiation

Abstract

Abstract Children with high-risk neuroblastoma have poor survival rates and need novel treatment strategies. UBE4B is an E3/E4 ubiquitin ligase whose gene is found in the chromosome 1p36 region commonly deleted in high-risk neuroblastoma tumors and whose expression is associated with neuroblastoma patient outcomes. However, the functional roles of UBE4B in neuroblastoma pathogenesis are not known. We hypothesized that UBE4B would interact with mediators of DNA damage and repair pathways, leading to the observed associations between UBE4B and patient outcomes. Using cell lines with stable UBE4B depletion and over-expression, we evaluated the interaction of UBE4B with the E3 ubiquitin ligase ITCH/AIP4 and the effects of UBE4B expression levels on p53 and Ku70 ubiquitination and proteasomal degradation by co-immunoprecipitation and Western blots. We also evaluated the role of UBE4B in neuroblastoma differentiation, cell death, and responses to chemotherapy and HDAC inhibition using continuous live cell imaging and Western blots. We determined that UBE4B directly interacts with ITCH/AIP4 in the last WW domain of ITCH and we also determined that Ku70 directly interacts with ITCH in the three first WW domain of ITCH. We also determined that UBE4B depletion leads to reduced polyubiquitination and degradation of both p53 and Ku70 in neuroblastoma cells. UBE4B depletion also led to reduced apoptosis induced by both HDAC inhibition with vorinostat and chemotherapy treatment with carboplatin and doxorubicin via stabilizaton of the ITCH target protein c-FLIPL leading inhibition of caspase 8 activation. Our results have identified novel interactions and novel targets for UBE4B ubiquitin ligase activity and a direct role of the UBE4B-ITCH complex in HDAC inhibitor and DNA-damage induced cell death, and suggest that the association of UBE4B expression with neuroblastoma patient outcomes may be mediated by effects on responses of patient tumors to chemotherapy. Citation Format: Christophe Le Clorennec, Jacqueline Lesperance, Yuchen Huo, Peter E. Zage. The roles of E3/E4 ubiquitin ligase UBE4B and its targets p53 and Ku70 in neuroblastoma cell death and differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4871.