Abstract B016: RNA demethylase ALKBH5 drives medulloblastoma growth and progression

Abstract

Abstract Introduction: Medulloblastoma (MB) is the most common malignant childhood brain tumor. It accounts for 20% of all childhood cancer deaths. MB are very fast-growing tumors, which spreads to central nervous system through cerebrospinal fluid, leading to leptomeningeal metastases that occurs up to 66% in brain cancer patients. Despite the progress in treating MB, the 5-year survival rate for high-risk MB remains poor with high recurrence. Moreover, the quality of life for those kids who do survive is substantially reduced due to the high toxicity associated with the high radiation exposure and multiple drug chemotherapy they must endure at such an early age. Therefore, it is critical to identify novel factors and understand previously undefined mechanisms that drive MB growth and progression, so that safe and viable therapeutics can be developed for treating MB. In this study, we tested the hypothesis that epigenetic changes, specifically mRNA demethylation mediated by the RNA demethylase ALKBH5, play an important role in MB growth and tumor progression, and hence approaches aimed at inhibiting ALKBH5 activity could prove clinical utility for MB patients. Methods: Medulloblastoma cell lines (HD-MB03, D556, D425 and DAOY) were purchased from the ATCC. MB cancer stem cells (MB-CSC) were stained Aldefluor sorted by Flow cytometry and cultured in stem cell medium. 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 revealed that MB cells are highly dependent on ALKBH5 for their survival. Using multiple MB cell lines with or without ALKBH5 depletion, we discovered that of ALKBH5 inhibited both short and long-term growth of MB cells. In addition, knockdown of ALKBH5 inhibited the migration of MB cells. ALKBH5 silenced MB cells showed increased apoptosis. Importantly, we show that ALKBH5 silencing suppressed the self-renewal/proliferation of MB stem cells (including medullosphere formation). ALKBH5 knockdown HDMB03-GFP-luciferase cells injected into mouse showed reduced tumor growth compared to the control. RNA sequencing analysis of ALKBH5 depleted MB cells 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. These are significant findings as MB stem cells are considered to be the major source of MB initiation, maintenance, relapse and render MB cells resistant to radiation. Conclusion: Collectively, our study results suggest that RNA demethylase ALKBH5 may play an important role in MB growth and progression by supporting MB cancer (tumor initiating) stem cells (MB-CSC) and ALKBH5 serves as a novel therapeutic target for MB. Citation Format: Panneerdoss Subbarayalu, Daisy Medina, Pitta Prabhakar, Shahad Abdulsahib, Saif Nirzhor, Desiree Denman, Santosh Timilsina, Deepika Singh, Phat Do, Krishna Evani, Dhiya Billa, Esha Reddy, Peter Houghton, Yogesh Gupta, Yidong Chen, Suryavathi Viswanadhapalli, Ratna Vadlamudi, Andrew Brenner, Manjeet Rao. RNA demethylase ALKBH5 drives medulloblastoma growth and progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B016.