Abstract 2620: m6A demethylase ALKBH5 regulates medulloblastoma growth and progression

Introduction: Medulloblastoma (MB) is pediatric brain cancer originate from the cerebellum, and accounts for large number of all childhood cancer related deaths. MB grows vigorously, and spreads to central nervous system (CNS) through cerebrospinal fluid, leading to leptomeningeal metastases in 66% brain cancer patients. MB are commonly treated with a combination of surgery, radiation, and chemotherapy drugs like vincristine. Unfortunately, at the young age, radiation exposure and chemotherapy treatment lead to high toxicity which ultimately compromise the quality of life for those kids. Therefore, it is critical to understand the molecular mechanism that regulates MB tumor growth, metastasis and relapse so that safe and novel therapeutics can be developed to treat pediatric MB. In this study, we investigated the role of RNA demethylase ALKBH5, in regulating MB growth and progression. Methods: Medulloblastoma cell lines including HD-MB03, D556, D425 and DAOY were purchased from the ATCC. MB cells were transfected either with ALKBH5 overexpression (OE) plasmid or siRNA (KD). These OE/KD MB cells were analyzed for cell viability, migration, invasion, colony formation, cell cycle, apoptosis assays, RNA sequencing, RT-qPCR, western blotting, RNA immunoprecipitation, and in vivo tumor xenograft study. Results: Our results showed that depletion of ALKBH5 significantly reduced short term cell viability, migration, invasion and colony forming ability of MB cells. ALKBH5 knockdown MB cells showed increased apoptosis. Further, our results show that ALKBH5 silencing suppressed the self-renewal/proliferation of MB cancer stem cells (MBCSC), including medullosphere formation. In orthotopic xenograft study, ALKBH5 knockdown cells intracranially injected into mouse showed reduced tumor growth compared to the scrambled siRNA transfected MB cells. To understand the molecular mechanism of ALKBH5, we performed transcriptome analysis of ALKBH5 depleted MB cells. Silencing of ALKBH5 showed alterations in several metabolism related pathways. Further, ALKBH5-depletion led to significantly decreased levels of cancer stem cell marker proteins including Nanog, OCT4 and SOX9. Conclusion: Together, our data suggests that m6A RNA demethylase ALKBH5 may play a critical role in MB growth and tumor progression by regulating MB tumor initiating stem cells and ALKBH5 serves as a potent therapeutic target to treat MB. Citation Format: Panneerdoss Subbarayalu, Daisy Medina, Pitta V. Prabhakar, Shahad Abdulsahib, Saif R. Nirzhor, Desiree Denman, Santosh Timilsina, Deepika Singh, Phat Do, Krishna Evani, Dhiya Billa, Esha Reddy, Meera Nair, Peter Houghton, Yogesh Gupta, Yidong Chen, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi, Gangadhara R. Sareddy, Andrew Brenner, Manjeet K. Rao. m6A demethylase ALKBH5 regulates medulloblastoma growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2620.