Multi-omics analysis of pediatric minimally differentiated acute myeloid leukemia reveals RUNX1-driven stemness and chemoresistance

Minimally differentiated acute myeloid leukemia (AML-M0) is a rare and therapeutically challenging subgroup of AML characterized by immature hematopoietic stem cell-like features. To uncover the molecular basis, we conducted a comprehensive multi-omics analysis of 23 pediatric AML-M0 cases and compared them with 1483 leukemia samples. AML-M0 formed a characteristic group that exhibited global DNA hypermethylation and transcriptional suppression, particularly downregulation of genes related to oxidative phosphorylation and ribosome assembly compared to non-M0 AML. Genomic profiling revealed frequent loss-of-function alterations in RUNX1 (26%) and ETV6 (22%), along with activating mutations in signaling pathways (83%), such as RAS, FLT3, and JAK. Notably, RUNX1 alterations were significantly associated with a poor prognosis. Functional analyses using a CRISPR/Cas9-mediated RUNX1 knockout in a pediatric AML-M0 cell line showed stem cell-like transcriptional features and reduced expression of genes related to oxidative phosphorylation and ribosomal pathways. RUNX1 disruption was also associated with reduced in vitro sensitivity to multiple drugs, including cytarabine and anthracyclines. Our study provides the most comprehensive molecular characterization of pediatric AML-M0 to date and identifies RUNX1 alterations as important biological and clinical determinants. These insights highlight the potential strategies for precision therapy, including hypomethylating agents, signaling inhibitors, and metabolic targeting, to improve outcomes.