Abstract Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor and a major cause of childhood cancer mortality. It comprises four molecular subgroups: WNT, SHH, Group 3, and Group 4. Group 3 MB, often marked by c-MYC amplification, has the worst prognosis. Despite surgery, radiation, and chemotherapy, high-risk patients face low 5-year survival and frequent relapse. Current treatments cause severe side effects. Cancer stem cells (CSCs) drive MB aggressiveness, therapy resistance, and recurrence. This project aims to uncover mechanisms sustaining CSCs and MB progression. Methods: MB cell lines (HD-MB03, D556, D425, DAOY) with ALKBH5 overexpression, knockdown (siRNA), or knockout (CRISPR), along with controls, were analyzed using viability, migration, invasion, colony formation, cell cycle, and apoptosis assays. RNA sequencing identified ALKBH5-regulated genes, validated by RT-qPCR, western blot, and RNA immunoprecipitation. An orthotopic intracranial xenograft model assessed ALKBH5’s tumor-promoting role in vivo. Results: To explore the role of m6A RNA methylation in medulloblastoma (MB), we silenced key regulators (writers: METTL3, METTL14; erasers: ALKBH5, FTO) in MB cell lines. ALKBH5 depletion caused the greatest reduction in proliferation, suggesting its therapeutic potential. Analysis of pediatric cancer datasets and tissue microarrays confirmed ALKBH5 overexpression and amplification in MB. Knockdown of ALKBH5 increased global m6A levels and reduced MB cell viability, migration, invasion, and stemness (medullosphere formation and NANOG, OCT4, SOX9 expression). Apoptosis assays showed elevated Annexin V-positive cells, and in vivo studies demonstrated suppressed tumor growth in orthotopic xenografts. ALKBH5 loss also increased DNA damage markers (γH2AX, 53BP1). RNA-seq and IPA revealed downregulation of genes involved in glycolysis, lipogenesis, and c-MYC targets, including ChREBP. Western blot confirmed decreased protein levels in these pathways. Conclusion: ALKBH5 is a critical regulator of MB tumorigenesis and CSC maintenance via m6A RNA demethylation. Targeting ALKBH5 may offer a promising therapeutic strategy to inhibit MB progression, overcome therapy resistance, and improve outcomes for high-risk MB patients. Citation Format: Panneerdoss Subbarayalu, Daisy Medina, Prabhakar Pitta Venkata, Shahad Abdulsahib, Saif Nirzhor, Santosh Timilsina, Deepika Singh, Phat Do, Desiree Denman, Krishna Priya Evani, Dhiya Billa, Meera Nair, Esha Reddy, Yogesh Gupta, Peter Houghton, Yidong Chen, Suryavathi Viswanadhapalli, Gangadhara R. Sareddy, Andrew Brenner, Ratna K. Vadlamudi, Manjeet Rao. Targeting ALKBH5 mediated ChREBP signaling impairs cancer stem cell metabolism and tumor growth in medulloblastoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3492.
Subbarayalu et al. (Fri,) studied this question.