Abstract 3659: Alyref is a novel binding partner and co-factor for MYCN-driven oncogenesis in neuroblastoma

Abstract

MYCN amplification is a poor prognostic indicator in neuroblastoma associated with high-risk disease. Therapies that directly repress the MYCN oncogenic signal in neuroblastoma are limited. We and others have shown that MYCN requires multiple cofactors to increase its protein stability in neuroblastoma cells, so that the very high MYCN levels required to drive tumourigenesis can be achieved. Here, we have identified ALYREF, a nuclear molecular chaperone protein, as a novel regulator of MYCN function in neuroblastoma. High expression of ALYREF predicted poor neuroblastoma patient survival and substantially correlated with MYCN levels in a large dataset (n=649) of human neuroblastoma tumour samples. ALYREF mRNA expression was also significantly increased in ganglia cells from the homozygous TH-MYCN neuroblastoma mouse in comparison to ganglia from wild-type littermates. Using co-immunoprecipitation and mass spectrometry, we identified ALYREF, as a direct binding partner of nuclear MYCN protein. Chromatin immunoprecipitation showed that MYCN bound the ALYREF gene promoter, and knockdown MYCN by MYCN siRNAs decreased ALYREF expression. A set of overexpression and knockdown experiments in MYCN-amplified neuroblastoma cells revealed that MYCN and ALYREF form a positive forward feedback expression loop. Overexpression of ALYREF further increased MYCN expression and protein stability in MYCN-amplified neuroblastoma cells. We found that ALYREF had a critical function in regulating the turnover of MYCN protein through transcriptional repression of the E3 protein ubiquitin ligase, NEDD4. The stabilized N-Myc oncoprotein enhanced ALYREF expression and stimulated cell growth of neuroblastoma. Additionally, we demonstrated that ALYREF plays a significant role in maintaining cell viability and proliferation of MYCN-amplified neuroblastoma cells. Taken together, our findings demonstrate a crucial role for ALYREF in regulating MYCN function and suggest that ALYREF-mediated stabilization of MYCN protein contributes to the development and progression of the disease. Thus inhibition of ALYREF activity using small molecules is a potential therapy for MYCN-amplified tumours. Citation Format: Zsuzsanna Nagy, Maxwell Kanikevich, Jessica Koach, Chelsea Mayoh, Daniel Carter, Tao Liu, Yanhua Du, Cizhong Jiang, Michelle Haber, Murray Norris, Belamy Cheung, Glenn Marshall. Alyref is a novel binding partner and co-factor for MYCN-driven oncogenesis 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 3659.