Aberrant pre-mRNA splicing is a pervasive feature of cancer and an emerging therapeutic vulnerability. Recurrent mutations in core spliceosomal components, including SF3B1, SRSF2, U2AF1, and ZRSR2, are common in myeloid malignancies, while dysregulated splicing regulators and cis-acting splice-site alterations shape cancer-relevant isoform programs across solid tumors. Together with high transcriptional output, rapid proliferation, and oncogene-driven RNA-processing demand, these alterations can reduce the capacity of cancer cells to tolerate additional splicing perturbation, creating a therapeutic window for pharmacological splicing modulation. Multiple strategies are under investigation, including SF3B complex modulators, splicing kinase inhibitors, RBM39-directed molecular glues, PRMT/arginine-methylation-directed approaches, and selected splice-switching strategies. Early clinical experience indicates that pharmacodynamic modulation of splicing is achievable in patients, yet objective clinical benefit has been inconsistent. This reflects narrow therapeutic windows, incomplete concordance between peripheral-blood pharmacodynamic markers and tumor-tissue splicing perturbation, and the limited predictive value of mutation status alone. Rational combinations with apoptosis-targeted agents, oncogene-directed therapies, DNA-damaging agents, PARP inhibitors, and immunotherapies may offer a more effective route to clinical translation than maximal single-agent splicing inhibition. Continued progress will require more selective splicing-directed modalities, pharmacodynamic biomarkers that measure splicing perturbation in the relevant tumor or blood compartment, longitudinal mapping of genetic and tumor cell-state plasticity-driven resistance, and biomarker-defined combination trials to support expansion from hematologic malignancies into solid tumors.

Acute myeloid leukaemia (AML) is an aggressive blood cancer characterized by the unregulated proliferation of immature myeloblasts. Gene mutations have been shown to have a large effect on pathogenesis, inter-tumour heterogeneity and clinical outcomes in AML1-8; however, the role of epigenetic alterations in these respects has been investigated less extensively. Here we use ATAC-seq (assay for transposase-accessible chromatin with sequencing) in a cohort of 1,563 individuals with a recent diagnosis of AML (the 'eCHROMA' cohort) to show that AML can be classified into 16 subgroups on the basis of chromatin accessibility profiles. Multiomics analyses of gene mutations, the transcriptome, DNA methylation and histone marks show that these ATAC subgroups exhibit distinct driver mutations, differentiation states, gene expression, DNA methylation and super-enhancer profiles, and are also associated with clinical outcomes. These findings were validated in independent cohorts. Single-cell ATAC sequencing reveals that all leukaemic cells in each subgroup share a common chromatin accessibility profile, which suggests that subgroup-specific epigenomic fingerprints underlie the ATAC-based classification. Mechanistically, the subgroups have distinct gene-regulatory networks that are driven by the activities of key transcription factors in haematopoiesis, and in which subgroup-specific super-enhancers have a pivotal role. Multiomics single-cell analysis further reveals deregulated trajectories of differentiation coupled with chromatin accessibility and gene expression. Notably, ATAC subgroups have an independent prognostic effect, compared with genomic classification, and are associated with particular drug sensitivities. In summary, ATAC-based chromatin profiling, combined with multiomics data, provides insights into AML pathogenesis beyond genomics and constitutes a valuable resource for AML research.

Splicing factor (SF) mutations are recurrent driver alterations in myeloid neoplasms and also represent a distinctive class of mutations in clonal hematopoiesis (CH), an age-associated expansion of hematopoietic clones carrying somatic driver mutations. SF-mutant CH is characterized by relatively late emergence and accelerated clonal expansion in older individuals. This review summarizes current knowledge of the genetic features and clonal dynamics of SF-mutant CH. We discuss disease-associated molecular consequences of SF mutations, including dysregulation of innate immune and inflammatory signaling, R-loop accumulation associated with replication stress, altered stress granule dynamics, impaired minor intron splicing, and cooperative genetic interactions. We further consider candidate mechanisms that may contribute to age-dependent selection of SF-mutant clones during CH, including attenuated interferon responsiveness and telomere-dependent changes in replicative fitness. Together, current evidence highlights the unique biology of SF-mutant CH and the unresolved mechanisms underlying its preferential expansion in older individuals. Clarifying these mechanisms may improve risk stratification, inform early-intervention strategies, and advance our understanding of age-associated clonal evolution and leukemogenesis.

Children with Down syndrome (DS) have an elevated risk of developing myeloid leukemia in DS (ML-DS). In addition to mutations in GATA1, which generate the truncated isoform GATA1-short (GATA1s), ML-DS requires additional somatic gene mutations, most frequently in cohesion and polycomb repressive complex 2 (PRC2) genes. Here, we show that PRC2 insufficiency underlies ML-DS pathogenesis. Transplantation of Gata1s fetal liver cells followed by deletion of the cohesion subunit Stag2 and/or the PRC2 component Ezh2 induced megakaryocyte-biased differentiation and expansion of megakaryocytic progenitors, culminating in lethal myelofibrosis. Mechanistically, loss of Stag2 or Ezh2 reinforced Gata1s-driven reduced chromatin accessibility at erythroid transcription factor target loci in pre-megakaryocyte/erythroid progenitors (pre-MegEs), thereby promoting megakaryocytic skewing. Ezh2 loss attenuated the Gata1s-mediated global elevation of H3K27me3 in pre-MegEs, resulting in derepression of a broad set of PRC2 target genes and establishing a functionally PRC2-insufficient state. Similarly, Stag2 loss induced a moderate but significant degree of PRC2-insufficient state in Gata1s progenitors. Furthermore, chromosome 21-encoded miR-125b blocked megakaryocytic differentiation of Gata1s progenitors lacking either Stag2 or Ezh2 alone, but drove full transformation and expansion of CD150+Sca-1+c-Kit+ leukemic stem cell-like populations only upon concurrent loss of both Stag2 and Ezh2, leading to acute megakaryoblastic leukemia in mice. These findings reveal that cohesin and PRC2 insufficiencies converge on PRC2 dysfunction while exerting distinct epigenetic effects, and synergize with trisomy 21 and GATA1s to remodel the epigenetic landscape, driving progression from a preleukemic state to overt leukemia.

Background Age-related clonal hematopoiesis (CH) is common in the elderly and associated with increased risk of hematologic malignancies and other age-related non-neoplastic disorders. Despite its clinical relevance, the mechanisms underlying the selection and expansion of mutant CH clones in aging remain unclear. In particular, studies using human bone marrow (BM) are limited by the small size of mutant clones and the lack of robust platforms to distinguish mutant from wild-type (WT) cells. Methods To address these questions, we established a single-cell multi-omics platform enabling simultaneous detection of somatic mutations and gene expression in human hematopoietic stem and progenitor cells (HSPCs). Using this platform, we analyzed Lineage⁻ CD34⁺ HSPCs from CH(+) individuals (7,116 cells from 16 cases) and age-matched CH(−) controls (3,898 cells from 16 cases). In parallel, we also analyzed whole BM cells from CH(+) (97,459 cells from 11 cases) and CH(−) cases (145,283 cells from 18 cases) using Chromium-based single-cell RNA sequencing to characterize BM microenvironment, including stromal cells. To investigate the functional impact of mutant cells on WT cells, we performed competitive BM transplantation (BMT) assays in mice. BM from Ly5.2/Ly5.2 Tet2⁺/− Mx1-Cre conditional heterozygous knockout (cKO) mice or their WT littermates was mixed with Ly5.1/Ly5.2 WT competitor BM (1–5% mutant to 95–99% competitor), modeling the small clone sizes observed in human CH. Following Cre induction by pIpC, BM cells were harvested for single-cell RNA sequencing and surface protein profiling. Donor-derived cKO and WT cells were distinguished based on CD45.1/CD45.2 expression. Results Single-cell multi-omics of CH(+) HSPCs carrying TET2, DNMT3A, SF3B1, SRSF2, IDH1, and IDH2 mutations revealed consistent upregulation of proliferation-associated genes compared with WT cells from the same individuals. Mutant cells also exhibited markedly attenuated transcriptional responses to proinflammatory cytokines such as TNFA, in contrast to strongly upregulated proinflammatory cytokine signaling in both hematopoietic and stromal cells in aged CH(−) BM. These results suggest that attenuated response to age-associated proinflammatory stimuli in the aged BM may contribute to the selective expansion of CH clones. Further analysis of the BM microenvironment in CH(+) individuals revealed broadly remodeled BM immune microenvironment, which may support clonal expansion. Compared with WT cells from CH(−) individuals, those from CH(+) marrow showed upregulation of cell cycle- and interferon-related genes. CH(+) BM also exhibited an altered immune composition, characterized by increased T cells and decreased B cells, together with shifts in myeloid and erythroid progenitors. Consistently, even older TET2-CH(+) cases with relatively small clone sizes (variant allele frequency [VAF] 4.6–8.1%) showed marked upregulation of interferon response genes in endogenous WT cells, suggesting that mutant clones may influence the surrounding BM environment even at low levels. To directly test whether these BM environmental changes could be attributed to the presence of mutant cells, we performed competitive BMT assays using 1–5% Tet2 heterozygous mutant donor cells mixed with WT competitors to model the low-level chimerism observed in human TET2-mutated CH. Strikingly, WT HSPCs co-transplanted with Tet2-mutant cells exhibited a gene expression profile closely resembling that of WT cells from human TET2-CH(+) marrow, including enhanced interferon signaling. By contrast, WT cells co-transplanted with Tet2-WT competitors did not. These results provide direct evidence that mutant cells in CH(+) marrow can exert non–cell-autonomous effects on WT cells, thereby altering their transcriptional state. Conclusions CH-associated mutant cells not only display an intrinsically proliferative phenotype but also induce non-cell-autonomous effects on WT cells within the BM environment. In the context of the proinflammatory milieu associated with aging, such environmental alterations may promote the positive selection and expansion of mutant clones, thereby shaping the pathogenesis of CH.

Somatic mutations in SET binding protein 1 (SETBP1), a putative chromatin regulator, occur in ~30% of high-risk myeloid malignancies, including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and secondary acute myeloid leukemia (AML), and strongly correlate with adverse clinical outcomes. Despite this significance, the precise roles of SETBP1 mutations in blood cancer remain enigmatic, primarily due to the lack of physiologically relevant cell lines or animal models that recapitulate the clonal evolution. Indeed, our 23 attempts to establish SETBP1-mutated PDX models failed, as the clones did not show advantageous effects. While conventional wisdom suggests that SETBP1 mutations escape proteasomal degradation to drive oncogenesis, our Setbp1 knockout mice revealed that SETBP1 is dispensable for normal and malignant hematopoiesis (Tanaka et al. Leukemia 2023), challenging prevailing paradigms. To resolve these contradictions, we developed a blood-specific Setbp1 mutant locus knock-in (KI) mouse model using Vav1-iCre, corresponding to the most prevalent D868N mutation. Unlike retroviral overexpression that immortalizes murine hematopoietic stem and progenitor cells (HSPCs), our KI model did not perturb HSPC function or transcriptome in vivo, indicating that SETBP1 acts as a cooperative rather than an initiating driver. As ASXL1 mutations are the most frequent founder mutations in SETBP1-mutated MDS/AML, we created double KI mice with Asxl1/Setbp1 physiologic mutations for transplant studies. These mice exhibited striking monocytosis (~40% of peripheral white blood cells) and HSPC expansion, rescuing stemness defects imposed by ASXL1 mutations. scRNA-seq/ATAC-seq revealed unique transcriptional clusters enriched for Hoxa9/10, Eya1, Myb, and Myc pathway alongside increased chromatin accessibility, suggesting that both mutants cooperate to remodel chromatin and amplify oncogenic transcriptional programs. Over time, the transplant model with double KI developed a leukemia phenotype, including serially transplantable AML and CMML-like diseases, all spontaneously acquired RAS pathway-activating mutations in Nras, Flt3, and Ptpn11. This reflects findings from over 7,000 patient samples, showing a significant correlation between SETBP1, ASXL1, and RAS pathway mutations. Indeed, the presence of RAS mutations predicts poor outcomes in SETBP1-mutated cases. Functional validation confirmed that all three alterations are required for complete transformation toward AML, establishing a three-step model: ASXL1mutations initiate clonal evolution, SETBP1 mutations enhance oncogenic programs, and RAS pathway activation completes the transformation. Leveraging these unprecedented in vivo models and cell lines with physiologically relevant SETBP1 mutations, we conducted comprehensive therapeutic screens using whole-genome CRISPR/sgRNA and pan-cancer drug libraries. The integrated results revealed a critical dependency on the druggable Exportin 1 (XPO1), shown by a 20.8-fold reduction (p = 0.00004) in the 7-day CRISPR screen and a 0.0003-fold growth under Leptomycin B treatment compared to DMSO at day 4. This unique dependency was robustly validated through genetic and pharmacological approaches using shXpo1, Selinexor, and Leptomycin B, resulting in monocytic differentiation and apoptosis in Setbp1-mutated AML cells. Notably, Selinexor monotherapy achieved sustained remissions exceeding 110 days in vivo, profoundly extending survival compared to controls, which succumbed to leukemia with a median survival of 59.5 days (p = 0.0004), demonstrating its potential to eradicate SETBP1-mutated leukemia. Mechanistically, XPO1 physically interacts with SETBP1 through the nuclear export signal groove and acts as a chromatin regulator beyond its canonical nuclear-cytoplasmic transport role. XPO1 inhibition suppressed the transcription of leukemogenic genes, including Hoxacluster genes, Myb, Eya1, and, critically, Myc pathway, with altered XPO1-mediated chromatin accessibility, as shown by integrated ChIP/RNA/ATAC-seq. These results uncover a critical epigenetic vulnerability that represents a promising target for therapeutic intervention in SETBP1-mutated leukemia. In summary, our study elucidates the cooperative leukemogenic roles of SETBP1, ASXL1, and RAS pathway mutations and identifies XPO1 as a targetable epigenetic vulnerability. These findings support precision treatment strategies for aggressive myeloid malignancies.

Gene expressions are regulated by an interplay between epigenetics and spatial genome organization, the deregulation of which has been implicated in the development of cancers, including myeloid neoplasms. However, it is unclear how they coordinately contribute to normal and malignant hematopoiesis. Here, we show that simultaneous dysregulations of histone modifications and chromatin structures caused by mutations of the epigenetic modulator Asxl1 and cohesin subunit Stag2 cooperatively induce ectopic interactions between polycomb-regulated promoters and active enhancers, leading to the aberrant upregulation of hematopoietic stem cell related genes and development of myelodysplastic syndromes (MDS). De-repression of polycomb-regulated genes induces their translocation to the transcriptionally active loci, where active promoters and enhancers are assembled, in the absence of Stag2-mediated chromatin organization. Our findings revealed that cohesin counteracts histone modification-driven chromatin conformations, manifesting the coordinate roles of histone modifiers and cohesin in regulating genome architectures and gene expressions to prevent malignant transformation. ### Competing Interest Statement The authors have declared no competing interest. Japan Society for the Promotion of Science, 23K27442 Grant-in-Aid for Scientific Research on Innovative Areas, 19H05745 Grant-in-Aid for Transformative Research Areas, 20H05940 Grant-in-Aid for Scientific Research (S), 20H05686 Grant-in-Aid for Scientific Research (B), 23K24360 Grant-in-Aid for Scientific Research on Innovative Areas, 15H05909 Grant-in-Aid for Scientific Research (S), 19H05656 Grant-in-Aid for Specially Promoted Research, 24H00009 Grant-in-Aid for Scientific Research (A), 20H00537 Grant-in-Aid for Research Activity Start-up, 20K22809 Grant-in-Aid for Early-Career Scientists, 22K16320, 24K19223 Japan Agency for Medical Research and Development, JP24gm1310006, JP21cm0106501h0006, JP19ck0106250h0003 Moonshot Research and Development Program, JP22zf0127008, JP22zf0127009 The Ministry of Education, Culture, Sports, Science and Technology of Japan, hp160219, hp170227, hp180198, hp190158, hp200138, hp210167 The Japanese Society of Hematology Research Grant The Japan Science and Technology Agency, JPMJFR220L Takeda Science Foundation Daiichi Sankyo Foundation of Life Science Princess Takamatsu Cancer Research Fund Kanae Foundation for the Promotion of Medical Science Ichiro Kanehara Foundation for the promotion of Medical Sciences and Medical Care Mochida Memorial Foundation for Medical and Pharmaceutical Research Japan Leukemia Research Fund

Abstract Background Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease. While traditional classification systems such as FAB relied on morphology and immunophenotype, recent classifications (WHO, ICC) emphasize genetic alterations for diagnosis and therapy. However, more directly involved in the phenotypic cellular inheritance, the epigenomic profile may offer an additional dimension for understanding AML heterogeneity. Previously, we performed ATAC-seq of 1,536 AML samples and identified 16 epigenetically defined AML subgroups (subgroups A-P), which were associated with distinct clinical, genetic, and transcriptional features (Ochi et al., ASH 2024). In the present study, we extend these findings by using single-cell RNA and ATAC-seq (scRNA/ATAC-seq) profiling to explore intra- and inter-tumor epigenetic heterogeneity, transcriptional regulation, and hierarchical differentiation trajectories across AML subgroups. Methods We performed multi-platform scRNA/ATAC-seq on 36 AML samples including all the 16 epigenomic subgroups and 4 remission samples (as controls), profiling a total of 281,167 mononuclear cells. Computational analyses included cell clustering, projection onto normal hematopoiesis reference maps, pseudotime inference, transcription factor (TF) activity analysis using SCENIC+, and leukemic stem cell (LSC) signature scoring. Results scATAC-based clustering showed that leukemic cells from individual AML samples formed distinct clusters largely separated from normal cells, typically co-clustered by subgroup, regardless of their differentiation status, indicating that leukemic cells within each subgroup share a distinct intrinsic epigenetic program. Projection onto a normal hematopoiesis reference map revealed divergent differentiation arrest among subgroups, even within genetically similar cases. For instance, all NPM1-mutant subgroups (D–F) were HOX-related but differed in differentiation states: subgroup E was arrested at the HSC stage, D at the GMP stage, and F contained both progenitors and mature cells. Single-cell analysis also refined differentiation states of subgroups F-H: while bulk ATAC-based deconvolution suggested monocyte enrichment in these subgroups, single-cell analysis revealed mature monocytes predominated in H, whereas F and G were enriched for immature promonocytes. Combined with LSC signature analysis based on gene expression, pseudotime analysis demonstrated that cells showing high LSC scores consistently mapped to early stages of the differentiation trajectory across all subgroups. These results indicate the presence of a conserved leukemic hierarchy, with stem-like cells positioned at the apex, irrespective of epigenetic subgroup, thereby underpinning AML pathogenesis. We further analyzed TF activity using the SCENIC+ program based on scRNA/ATAC-seq data, which identified key TFs enriched in each subgroup, many of which overlapped with those found in bulk RNA/ATAC-seq analysis. Pseudotime analysis further showed that TFs exhibited subgroup-specific activation dynamics. For example, HOXA9 was activated throughout the myeloid differentiation trajectory in HOX-related subgroups but peaked at different stages according to subgroups. In the RUNX1-enriched subgroup, IRF8 and BCL11A were both active but peaked at mature and immature stages, respectively. These findings indicate that key TFs exhibit subgroup-specific activation patterns at distinct stages along the myeloid differentiation trajectory. Conclusion Single-cell multi-omics analysis reveals that AML epigenetic subgroups exhibit distinct but hierarchically organized differentiation trajectories, driven by dynamically regulated TF programs. These findings refine our understanding of AML diversity and may inform more precise, differentiation-stage–specific therapeutic strategies.

Abstract Recent genomic analyses have revealed that mutations in RNA splicing factors—including SF3B1, SRSF2, U2AF1, and ZRSR2—are among the most frequent mutations in myelodysplastic syndromes (MDS). Among these, U2AF1 mutations are enriched in MDS without ring sideroblasts and in AML with myelodysplasia-related changes, and are associated with poor clinical outcomes. U2AF1 mutations primarily affect two conserved residues, S34 and Q157, located within the N- and C-terminal zinc finger motifs. Mechanistically, U2AF1 mutant proteins preferentially recognize the ‘UAG’ motif upstream of the 3‘ splice site, causing exon skipping and aberrant 3‘ splice site selection. Among the reported targets of U2AF1 mutations are BCOR and GNAS, both of which are known drivers of MDS, as well as IRAK4, a key mediator of innate immune signaling. However, how U2AF1 mutations affect hematopoietic stem cell (HSC) function and lineage differentiation, and how they shape inflammatory responses or cell-cell interactions, remains incompletely understood. To address these issues, we utilized a conditional knock-in mouse model independently generated to express the U2af1 S34F allele under the control of Vav1-Cre promoters. U2af1 mutant mice exhibited macrocytic anemia, leukopenia, morphological abnormalities, and myeloid-skewed hematopoiesis, resulting in shortened survival. Transplantation experiments revealed defective HSC reconstitution under competitive conditions. Bulk RNA-seq of KSL (Kit⁺Sca-1⁺Lin⁻) cells revealed splicing abnormalities in myeloid malignancy-related genes, including Hnrnpa2b1, Csf3r and Gnas, many of which overlapped with findings from the Srsf2 P95H mutant mouse model (Kon et al., Blood 2018). In contrast, U2af1 mutant KSL cells showed specific exon skipping in genes related to inflammatory signaling and RNA metabolism. Gene set enrichment analysis revealed that both U2af1 and Srsf2 mutations upregulated pathways related to DNA repair, cell cycle regulation, and RNA processing. However, genes related to cell migration and inflammation were uniquely downregulated in U2af1 mutant KSL cells, highlighting distinct pathogenic mechanisms associated with splicing factor mutations in MDS. To investigate the cell type-specific impact of the U2af1 S34F mutation, we performed single-cell RNA sequencing (scRNA-seq) on hematopoietic stem/progenitor cells and analyzed transcriptional changes across lineages. In monocyte-dendritic progenitors (MDPs), genes involved in migration and motility, as well as inflammatory response pathways, were significantly downregulated. Additionally, adhesion-related genes, including Cd34 and Tgfb1, were markedly reduced, suggesting diminished mobilization capacity and altered interactions with the bone marrow microenvironment. Similarly, in megakaryocyte-erythroid progenitors (MEPs), adhesion and migration-related genes, including Spn and Itga4, were downregulated, while tumor-related pathways were activated. In contrast, multipotent lymphoid progenitors (MLPs) exhibited upregulation of inflammatory genes. These findings suggest that lymphoid lineage cells, such as MLPs, contribute to the activation of immune responses and inflammatory pathways, while myeloid lineage progenitors, including MDPs and MEPs, showed impaired migration and adhesion, alongside reduced immune response capabilities, contributing to MDS pathogenesis. Based on scRNA-seq data, we analyzed known ligand-receptor interactions and predicted that interactions related to cell migration pathways, such as Cd34–Selp, Sell–Cd34, and Icam1–Spn, were significantly altered. Notably, while a subset of inflammation-related genes exhibited splicing alterations, the majority of differentially expressed genes associated with motility and inflammation did not, suggesting that these changes are largely driven by transcriptional dysregulation rather than splicing defects. Importantly, these transcriptional changes were consistently observed in bulk RNA-seq data from U2AF1-mutant MDS patients, as well as in CRISPR-engineered MOLM13 human leukemia cells harboring the U2AF1 S34F mutation. In conclusion, our findings demonstrate that U2AF1 mutations perturb hematopoiesis through lineage-specific transcriptional reprogramming, particularly impairing inflammatory and migratory pathways. These results highlight the mechanistic divergence among splicing factor mutations and underscore the therapeutic potential of targeting dysregulated inflammation and cell migration in MDS.

Background Recent genetic studies have identified frequent mutations affecting a number of genes encoding RNA splicing factors (SDs) in myelodysplastic syndromes (MDS) and related myeloid neoplasms (MNs). Among these, most frequently mutation are SF3B1, SRSF2, U2AF1, and ZRSR2, whose functions have been intensively studied. By contrast, U2AF2, another SF, is also recurrently mutated in MNs. U2AF2 is a component of the U2 auxiliary factor that forms a heterodimer with U2AF1 for the recognition of the 3’ splice site (3'SS). U2AF2 contains a sequence-specific RNA-binding region with two RNA recognition motifs and identifies polypyrimidine (Py) tract signals of nascent transcripts. However, compared to other splicing factor mutations, U2AF2 mutations are much rare, preventing the detailed analysis of their role in leukemogenesis. Methods To characterize the role of U2AF2 mutations, we systematically analyzed mutation spectrum in 6,369 with different MNs using targeted-capture sequencing. We also performed RNA sequencing of cKit(+) bone marrow cells from 6 U2AF2 mutated MDS cases along with 52 MDS patients without common splicing factor mutations and 25 healthy individuals. THP1 and HL60 leukemia cells with exogenous expression of wildtype or mutant U2AF2 were established to evaluate the splicing response. We also used clustered regularly interspaced short palindromic repeats /CRISPR-associated protein-9 nuclease (CRISPR/cas9) to introduce the p.190_195del mutation to U2AF2 in K562 and MOLM-13 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant U2AF2. Results In total, U2AF2 mutations were found in 34 (0.53%) of 6,369 MN cases, of which 29 had the recurrent p.190_195del mutation, the median age of U2AF2 mutations was 55 years (range 31-79) and 55% were diagnosed with MDS, 31% with CMML and 7% with AML. U2AF2-mutated patients showed a male predominance (90%). The most frequently co-mutated gene was ASXL1 with 52%, followed by SETBP1 (35%) STAG2 (21%),RUNX1 (21%), CSF3R, NRAS, DNMT3A, NF1, PTPN11, ETV6, and STAT3. To investigate the effects of the U2AF2 p.190_195del mutation on RNA splicing, we analyzed transcriptome sequencing, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events . Various types of AS events were identified, including skipped exons (SEs), alternative 5’ ss exons (A5SSs), alternative 3’ ss exons (A3SSs), retained introns (RIs), and mutually exclusive exons (MXEs). Among these, the skipping exons are the most frequent in both U2AF2-mutant primary MDS cases and cell lines. To explore these alternative splicing results in more detail, we next examined differentially skipped exons. Unsupervised uniform manifold approximation and projection (UMAP) analysis based on the standardized splicing ratio of skipping exons segregated U2AF2-mutated patients from other MDS cases without common splicing factor mutations and healthy individuals. To prioritize mutant U2AF2-induced alterative splicing events, we intersected significant skipping exons across 2 datasets: MDS patient samples with and without U2AF2 p.190_195del mutations, and MDS patient samples with U2AF2 p.190_195del mutations and healthy individuals. The differential skipping exon usages and differential gene expression profiles enables both consensus sequence analysis and pathway/gene-set enrichment analysis. Conclusions Our study revealed that the U2AF2 p.190_195del regulated aberrant alternative splicing facilitated MDS progression through perturbations in splice isoforms. A better understanding of the U2AF2 p.190_195del and its critical specific changes will provide novel insights into disease pathophysiology and potentially inform future treatment decisions.

IntroductionDespite the significant impact of clonal hematopoiesis (CH) on leukemogenesis, the pathogenesis of CH is still not fully understood.MethodsUtilizing a novel single-cell sequencing platform that allows for simultaneous detection of mutations and gene expression, we examined the gene expression profiles of hematopoietic stem and progenitor cells (HSPCs) harboring CH-related mutations from CH(+) cases, which was compared with that of wild-type (WT) cells from both CH(+) and CH(−) cases. Age-related changes in the bone marrow (BM) environment were also assessed using CH(−) cases.ResultsIn 12 patients with CH, genes associated with cell proliferation were upregulated in mutant cells. Significantly, mutant cells showed decreased expression of genes related to inflammatory responses, which were enhanced in BM cells from aged CH(−) cases, indicating the potential contribution of aged BM environment to the positive selection of mutant cells. Unexpectedly, WT cells from 3 TET2-CH(+) cases demonstrated significant upregulation of genes related to interferon response and cell proliferation, compared with those from age-matched CH(−) cases, suggesting the altered BM environments. Notably, when competitively transplanted with Tet2-knockout (KO) cells, WT HSPCs displayed enhanced expression of genes associated with cell proliferation and interferon signalling, compared with those transplanted with WT cells, implying non-cell autonomous effects of mutant cells.ConclusionsThese results suggest that mutant cells in CH(+) BM may exert non-cell autonomous effects on WT cells. Alongside aged BM environments, these effects may contribute to the positive selection of CH clones, playing a pivotal role in the pathogenesis of CH.

DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms have not fully been elucidated. To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in alleles. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 (Ddx41+/+), heterozygous Ddx41 KO (Ddx41+/-), homozygous Ddx41 KO (Ddx41-/-), heterozygous for the Ddx41 R525H mutation (Ddx41R525H/+), or hemizygous for the Ddx41 R525H mutation (Ddx41R525H/-), in which expression of the mutant allele was induced by tamoxifen administration. In noncompetitive BM transplantation, most of the recipient mice that were transplanted with BM from Ddx41-/- or Ddx41R525H/- mice died within a month after CreERT2 induction due to severe BM failure, which was not observed in mice transplanted with BM from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice. Transcriptome analysis revealed that stem cells (Kit+Sca-1-Linlow cells) derived from Ddx41R525H/- BM-transplanted mice exhibited a significant upregulation of ribosomal genes and several snoRNA genes compared with those derived from Ddx41+/+ BM-transplanted mice, which could result in abnormal ribosome biogenesis. In addition, Ddx41R525H/--derived Gr1+CD11b+ myeloid cells showed a significant upregulation of genes involved in cGAS-STING signaling pathways compared with Ddx41+/+-derived myeloid cells. However, the survival and cytopenia and the ribosome biogenesis in the stem cells in Ddx41R525H/- BM-transplanted mice were improved only marginal when the intact Sting alleles were deleted. These results suggest that the impact of the cGAS-Sting signaling on these phenotypes was, if ever, very small, highlighting the role of Sting-independent mechanisms. In long-term observations, mice transplanted with Ddx41+/- or Ddx41R525H/+ BM exhibited significantly lower white blood cell counts and anemia, both in primary and subsequent transplantations, although they did not exhibit significant transcriptional changes relative to the wild-type control animals until just before the onset of disease. Some of these mice developed MDS-like phenotypes, including ineffective hematopoiesis and erythroid dysplasia. Stem cells from these mice showed abnormal ribosome biogenesis and reduced expression of interferon response genes. It may be that clones that adapted to the inflammatory environment were gradually selected and contribute to their clonal advantage. Given that the MDS clones with the DDX41 R525H somatic allele are commonly observed as a small subclone in patients, we next co-transplanted Ddx41+/+- and Ddx41525H/--derived BM cells with Ddx41+/+- or Ddx41+/--derived BM cells at the ratio of 1:9. The recipient mice showed significantly reduced WBC counts when Ddx41+/+- or Ddx41+/- were co-transplanted with Ddx41525H/- -derived BM, suggesting that Ddx41525H/--derived hematopoietic cells have negative effect on normal hematopoiesis. In order to assess the crosstalk between biallelic Ddx41 mutant cells and their microenvironment cells harboring monoallelic Ddx41 mutations, we co-transplanted Ddx41+/+- or Ddx41525H/--derived BM cells with Ddx41+/--derived BM cells into the constitutive heterozygous Ddx41 KO mice. The phenotypic and molecular characteristics of these mice are under investigation. In summary, conditionally introduced compound loss-of function and R525 alleles caused severe BM failure, whereas heterozygous Ddx41 loss-of function and R525H knock-in alleles are compatible with hematopoiesis, although associated with impaired hematopoiesis and the development of MDS with aging, where an attenuated inflammatory response and ribosome functions may play important roles.

Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained-80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.

MDS and its related disorders frequently carry mutations in multiple genes implicated in RNA splicing and epigenetic regulation. Among these, mutations in SRSF2 and STAG2 co-occur more frequently than just by chance, suggesting a functional interaction between both mutations during the development of MDS. However, the leukemogenic mechanism of this combination remains unclear. To elucidate the functional relevance of this combination of mutations, we first analyzed the mutational profile of MDS/AML harboring SRSF2 and STAG2 mutations using published data of MDS or secondary AML patients (N=3,047). In total, SRSF2 and STAG2 mutations were found in 12.0% and 6.8%, respectively, and 86 patients (2.8%) had both mutations. SRSF2 and STAG2 mutations were significantly co-mutated, with frequent additional mutations in ASXL1, RUNX1, BCOR,IDH2 and CEBPA. Analysis of variant allele frequencies suggests that in typical cases, SRSF2 mutations are likely to be acquired first, followed by STAG2 mutations, where multiple independent STAG2 mutations evolved in many cases. Higher-risk MDS and secondary AML accounted for 59.3% and 26.7% of SRSF2 and STAG2 double-mutated patients, respectively. Among MDS patients, those with both mutations tended to have lower WBC and platelet counts, higher blast counts in peripheral blood, higher IPSS-R scores and a poorer overall survival. These results suggest that SRSF2 and STAG2 mutations co-occurred in MDS/AML, promoting disease progression of MDS to secondary AML. Next, to reveal the functional mechanisms by which this combination cooperates to develop MDS in vivo, we crossed mice carrying Srsf2-P95H conditional knock-in and Stag2 conditional knockout alleles with an Mx1-Cre transgene to generate double mutant mice. Their phenotype was evaluated in the transplant setting, in which bone marrow derived from double mutant mice, as well as WT or single mutant mice, was transplanted into the lethally irradiated mice and pIpC was injected after engraftment. Compared to single mutant- or mock-transplanted mice, double-mutant transplanted mice showed lower WBC counts at 8-16 weeks after pIpC administration, with increased granulocytes/monocyte and decreased B lymphocyte subfractions. Moreover, double mutant mice showed a lower hemoglobin level and larger RDW and MCV. These results suggest that the combination of Srsf2 and Stag2 mutations leads to more severe leukopenia and macrocytic anemia than single mutations. In bone marrow, Lin−Sca-1+Kit+(LSK) cells were increased in Stag2 single mutant mice and double mutant mice, compared with wild-type mice and Srsf2 single mutant mice. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) in Stag2 single and double mutant mice, compared to wild-type or Srsf2 single mutant mice. By contrast, megakaryocyte/erythroid progenitors (MEPs) and CD71+ Ter119+ erythroblasts strikingly decreased in double mutant mice. Double mutant mice also showed enlarged spleen, in which an increase MEP as well as CMP and GMP fractions were observed. These results suggest that the combination of Srsf2 and Stag2 mutations skews erythroid lineage toward myeloid lineage in bone marrow and causes extramedullary hematopoiesis in the spleen. Given that Srsf2 gene is a key component of spliceosome machinery, we next assessed different splicing changes in double mutant mice. RNA sequencing of samples obtained from LSK cells showed increased aberrant splicing events of cassette exons in double mutant mice, indicating that the combination of Srsf2/ Stag2 mutations leads to the progression of MDS through aberrant splicing changes. In conclusion, our findings suggest that SRSF2 and STAG2 mutations cooperatively induced more impaired hematopoietic differentiation compared to single mutations alone and contribute to the progression of MDS, possibly thorough aberrant splicing changes.

SETBP1 is a potential epigenetic regulator whose hotspot mutations preventing proteasomal degradation are recurrently detected in myeloid malignancies with poor prognosis. It is believed that the mutant SETBP1 exerts amplified effects of wild-type SETBP1 rather than neomorphic functions. This indicates that dysregulated quantitative control of SETBP1 would result in the transformation of hematopoietic cells. However, little is known about the roles of endogenous SETBP1 in malignant and normal hematopoiesis. Thus, we integrated the analyses of primary AML and healthy samples, cancer cell lines, and a newly generated murine model, Vav1-iCre;Setbp1(fl/fl). Despite the expression in long-term hematopoietic stem cells, SETBP1 depletion in normal hematopoiesis minimally alters self-renewal, differentiation, or reconstitution in vivo. Indeed, its loss does not profoundly alter transcription or chromatin accessibilities. Furthermore, although AML with high SETBP1 mRNA is associated with genetic and clinical characteristics for dismal outcomes, SETBP1 is dispensable for the development or maintenance of AML. Contrary to the evidence that SETBP1 mutations are restricted to myeloid malignancies, dependency on SETBP1 mRNA expression is not observed in AML. These unexpected results shed light on the unrecognized idea that a physiologically nonessential gene can act as an oncogene when the machinery of protein degradation is damaged.