Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell–like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. Significance: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549

Stable inheritance of DNA N ⁶ -methyladenine (6mA) is crucial for its biological functions in eukaryotes. Here, we identify two distinct methyltransferase (MTase) complexes, both sharing the catalytic subunit AMT1, but featuring AMT6 and AMT7 as their unique components, respectively. While the two complexes are jointly responsible for 6mA maintenance methylation, they exhibit distinct enzymology, DNA/chromatin affinity, genomic distribution, and knockout phenotypes. AMT7 complex, featuring high MTase activity and processivity, is connected to transcription-associated epigenetic marks, including H2A.Z and H3K4me3, and is required for the bulk of maintenance methylation. In contrast, AMT6 complex, with reduced activity and processivity, is recruited by PCNA to initiate maintenance methylation immediately after DNA replication. These two complexes coordinate in maintenance methylation. By integrating signals from both replication and transcription, this mechanism ensures the faithful and efficient transmission of 6mA as an epigenetic mark in eukaryotes.

N⁶-adenine DNA methylation (6mA), a rediscovered epigenetic mark in eukaryotic organisms, diversifies in abundance, distribution, and function across species, necessitating its study in more taxa. Paramecium bursaria is a typical model organism with endosymbiotic algae of the species Chlorella variabilis. This consortium therefore serves as a valuable system to investigate the functional role of 6mA in endosymbiosis, as well as the evolutionary importance of 6mA among eukaryotes. In this study, we report the first genome-wide, base pair-resolution map of 6mA in P. bursaria and identify its methyltransferase PbAMT1. Functionally, 6mA exhibits a bimodal distribution at the 5′ end of RNA polymerase II-transcribed genes and possibly participates in transcription by facilitating alternative splicing. Evolutionarily, 6mA co-evolves with gene age and likely serves as a reverse mark of endosymbiosis-related genes. Our results offer new insights for the functional diversification of 6mA in eukaryotes as an important epigenetic mark.