Genome-Wide Identification of a Chromatin-Splicing Regulatory Axis Driven by DOT1L in MLL-Rearranged Acute Myeloid Leukemia

EIF4A3-dependent nonsense-mediated decay buffers AML1-ETO9a dosage and modulates outcome in t(8;21) acute myeloid leukemia

Functional characterization of snoRNA-derived RNA (sdRNA) expression in healthy hematopoiesis and acute myeloid leukemia.

SnoRNAs are highly expressed in AML and have implications in leukemogenesis and leukemic maintenance. SnoRNAs can be further processed into snoRNA-derived RNAs (sdRNAs). The role of sdRNAs in AML and healthy hematopoiesis remains largely elusive. We characterized sdRNA and snoRNA levels in hematopoietic stem and progenitor cells (HSPCs), healthy WBCs, and 159 intensively treated AML patient samples at initial diagnosis. HSPCs, healthy WBCs, and AML blasts could be differentiated by their sdRNA expression pattern in a cell-type-specific manner. In AML, high sd3'-RNA/snoRNA-host gene ratios were associated with an inverse patient outcome. Particularly, in NPM1-mutated patients with favorable risk stratification and good initial therapy response, high sd3'-RNA ratios identified a subgroup with inferior outcome. High sd3'-RNA ratios were associated with altered oncogenic, inflammatory, and immune response signaling. Forced expression of single sdRNAs, such as sd3'-SNORD78, sd3'-SNORD76, and sd5'-SNORD93, enhanced clonogenic potential in AML and drove sdRNA-specific gene expression signatures in both AML and healthy HSPCs. Exemplarily, we propose and characterize NUDT21, an important regulator of alternative polyadenylation and oncogenic gene expression, as a downstream target of sd3'-SNORD78 in AML. Our data introduce sdRNAs as standalone regulatory effector molecules in healthy hematopoiesis and AML.

Single-cell RNA sequencing uncovers m6A regulator-defined T-cell subpopulations and YTHDF1-HSP90AB1-MYC therapeutic axis in T-cell acute lymphoblastic leukemia.

BACKGROUND N6-methyladenosine (m6A) drives T-cell acute lymphoblastic leukemia (T-ALL); however, its roles in leukemic heterogeneity and tumor microenvironment remodeling unclear. This study characterized N6-methyladenosine regulator-defined subpopulations and YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in leukemogenesis. METHODS Integrated single-cell and bulk RNA sequencing from 17 pediatric T-cell acute lymphoblastic leukemia patients and 3 healthy donors were analyzed. Non-negative matrix factorization based on m6A regulator expression identified distinct T-cell subpopulations. CellChat, Monocle, and single-cell regulatory network inference and clustering analyses revealed intercellular communication patterns, developmental trajectories, and transcriptional regulatory programs. Functional validations included lentiviral-mediated YTHDF1 knockdown in Jurkat and Molt4 cells for proliferation, apoptosis, and migration assays, and a non-obese diabetic severe combined immunodeficiency mouse xenograft model. Methylated RNA immunoprecipitation quantitative polymerase chain reaction, RNA immunoprecipitation quantitative polymerase chain reaction, RNA stability assays, and Western blotting characterized YTHDF1 interactions with heat shock protein 90 alpha family class B member 1 (HSP90AB1). RESULTS Single-cell transcriptomics identified m6A-defined T-cell subpopulations in T-ALL. YTHDF1-enriched cells exhibited hypoxia and cell-cycle programs, occupied early developmental states, and communicated with macrophages. Knockdown suppressed proliferation and migration, increased apoptosis, and reduced leukemic burden in vivo. The protein bound and stabilized m6A-modified HSP90AB1 to sustain MYC proto-oncogene signaling. CONCLUSION This study identified a YTHDF1-high m6A-associated T-cell subpopulation in T-ALL. YTHDF1 promoted leukemic progression through regulation of the HSP90AB1-MYC axis and was associated with altered macrophage-related intercellular signaling.

MBNL1 hijacks a structured single-stranded distal DNA element to sustain FLT3 expression in KMT2A-rearranged leukemias

The molecular mechanisms by which KMT2A-rearranged (KMT2A-r) leukemias maintain the oncogenic FLT3 expression remain largely unclear, limiting therapeutic opportunities. Here, we identify the RNA binding protein MBNL1 as an unexpected positive regulator of FLT3 by DepMap dataset exploration and combinatorial CRISPR screens. MBNL1 promotes leukemia cell survival in cell lines and primary tumors by sustaining FLT3 expression in a KMT2A-r context-dependent manner. Mechanistically, we discover that MBNL1 recognizes a structured single-stranded DNA (ssDNA) element containing five consecutive guanines within the FLT3 enhancer, through MBNL1’s zinc finger domains and the carboxyl-terminal unstructured region. Such MBNL1 protein/ssDNA interaction was evident in KMT2A-r leukemia using ChIP-seq and KAS-seq. Mutations of key amino acids of MBNL1’s ssDNA binding surface or the critical guanines in ssDNA markedly abrogate the protein-ssDNA interactions. These findings implicate MBNL1 as a distinct FLT3 activator by recognizing a structured enhancer ssDNA element, highlighting an unexpected role for RNA binding proteins in transcriptional regulation through direct ssDNA recognition.

DHX15 affects AML1-ETO9a splicing together with HNRNPL, RBM33 in AML1-ETO-positive acute myeloid leukaemia.

The AML1-ETO (AE) fusion gene, resulting from t(8;21)(q22;q22), represents a prevalent subtype of acute myeloid leukaemia (AML). Retention of exon 9a between exon 8 and 9 of the ETO (RUNX1 Partner Transcriptional Co-Repressor 1, also name RUNX1T1) gene generates the oncogenic AML1-ETO9a (AE9a) splice variant. Here, we uncover the potential involvement of RNA (ribonucleic acid) helicase DHX15 (DEAH-box helicase 15) in AE9a splicing and AML progression. Clinically, higher expressed DHX15 is associated with increased AE9a/AE abundance and poor outcomes in AE-positive (AE+) AML patients. DHX15 knockdown impaired cell proliferation, induced S/G2 cell-cycle arrest in vitro and prolonged overall survival in AE+ leukaemia-bearing mice in vivo. In addition, DHX15 knockdown reduced AE and AE9a expression, and RNA immunoprecipitation revealed DHX15 bound to AE and AE9a transcripts and RNA pull-down assay showed a selective interaction of HNRNPL and RBM33 with AE9a transcripts. Three-dimensional structure prediction suggests a sophisticated regulatory complex formation involving DHX15, HNRNPL (heterogeneous nuclear ribonucleoprotein L) and RBM33 (RNA binding motif protein 33). Further, HNRNPL and RBM33 gene expression positively correlated with AE9a levels in AE+ AML samples. Downregulation of HNRNPL and RBM33 reduced AE9a expression in Kasumi-1 cells. Collectively, our findings support an association between DHX15, HNRNPL, RBM33 and AE9a splicing and suggest their contribution to leukemogenic progression in AE+ AML.

Methylome profiling of SetDB1-deficient ESCs reveals coordinated epigenetic cross-talk during pluripotency.

SetDB1 is best known for catalyzing H3K9me3, but it also influences H3K27me3 deposition, CTCF-binding, and DNA methylation (DNAme). Given the interplay between DNAme and the other epigenetic features, we profiled DNAme following Setdb1 knockout (KO) in ground-state and serum-grown mouse embryonic stem cells (ESCs) to illuminate DNAme-dependent and -independent functions of SetDB1. Time-course whole-genome bisulfite sequencing of serum-grown ESCs shows that nearly half of SetDB1 binding sites are enriched with DNAme and H3K9me3, primarily at retrotransposons. Upon Setdb1 KO, both H3K9me3 and DNAme are reduced, with DNAme rapidly removed at many sites by TET enzymes. Some retrotransposons, primarily IAPs, are TET-resistant and lose DNAme slowly via passive dilution. Notably, SetDB1-mediated regulation of H3K27me3, CTCF-binding, and SMAD3 are uncoupled from the DNAme-H3K9me3 axis, and from each other. AlphaFold modeling and co-immunoprecipitation mass spectrometry suggest this uncoupling involves competitive binding to distinct SetDB1 protein domains, highlighting the complex coordination underlying SetDB1 functions.