m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development.

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

Fosl2 Deficiency Predisposes Mice to Osteopetrosis, Leading to Bone Marrow Failure.

Arthritis causes Fos-like 2 (Fosl2) inactivation, and various immune cells contribute to its pathogenesis. However, little is known about the role of Fosl2 in hematopoiesis and the possible pathological role of Fosl2 inactivation in the hematopoietic system in arthritis. In this study, we show that Fosl2 maintains hematopoietic stem cell (HSC) quiescence and differentiation while controlling the inflammatory response via macrophages. Fosl2-specific deletion in the hematopoietic system caused the expansion of HSCs and myeloid cell growth while affecting erythroid and B cell differentiation. Fosl2 inactivation enhanced macrophage M1 polarization and stimulated proinflammatory cytokines and myeloid growth factors, skewing HSCs toward myeloid cell differentiation, similar to hematopoietic alterations in arthritic mice. Loss of Fosl2 mediated by Vav-iCre also displays an unexpected deletion in embryonic erythro-myeloid progenitor-derived osteoclasts, leading to osteopetrosis and anemia. The reduced bone marrow cellularity in Vav-iCreFosl2f/f mice is a consequence of the reduced bone marrow space in osteopetrotic mice rather than a direct role of Fosl2 in hematopoiesis. Thus, Fosl2 is indispensable for erythro-myeloid progenitor-derived osteoclasts to maintain the medullary cavity to ensure normal hematopoiesis. These findings improve our understanding of the pathogenesis of bone-destructive diseases and provide important implications for developing therapeutic approaches for these diseases.

Efficacy of JAK1/2 inhibition in murine immune bone marrow failure.

Immune aplastic anemia (AA) is a severe blood disease characterized by T-lymphocyte- mediated stem cell destruction. Hematopoietic stem cell transplantation and immunosuppression are effective, but they entail costs and risks, and are not always successful. The Janus kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T-cell activation and inhibits cytokine production in models of graft-versus-host disease. We tested RUX in murine immune AA for potential therapeutic benefit. After infusion of lymph node (LN) cells mismatched at the major histocompatibility complex [C67BL/6 (B6)⇒CByB6F1], RUX, administered as a food additive (Rux-chow), attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, preserved hematopoietic progenitors, and prevented mortality, when used either prophylactically or therapeutically. RUX suppressed the infiltration, proliferation, and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. Similar effects were obtained when Rux-chow was fed to C.B10 mice in a minor histocompatibility antigen mismatched (B6⇒C.B10) AA model. RUX only modestly suppressed lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice. Our data support clinical trials of JAK/STAT inhibitors in human AA and other immune bone marrow failure syndromes.

Gain-of-function mutations in RPA1 cause a syndrome with short telomeres and somatic genetic rescue.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

Immunodeficiency and bone marrow failure with mosaic and germline TLR8 gain of function.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with <30% mosaicism, suggesting a dominant mechanism responsible for the clinical phenotype. Mosaicism was also detected in skin-derived fibroblasts in 3 patients, demonstrating that mutations were not limited to the hematopoietic compartment. All patients had refractory chronic neutropenia, and 3 patients underwent allogeneic hematopoietic cell transplantation. All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. Differentiation of myeloid cells from patient-derived induced pluripotent stem cells demonstrated increased responsiveness to TLR8. Together, these findings demonstrate that gain-of-function variants in TLR8 lead to a novel childhood-onset IEI with lymphoproliferation, neutropenia, infectious susceptibility, B- and T-cell defects, and in some cases, bone marrow failure. Somatic mosaicism is a prominent molecular mechanism of this new disease.

The Fanconi Anemia Pathway and Fertility.

Fanconi anemia (FA) is a life-threatening syndrome characterized by bone marrow failure and cancer predispositions. The past two decades have seen an explosion of data in the FA field, both in humans and other organisms, following the cloning of 22 FA genes. A common but notably understudied clinical feature of the disease is the reduced fertility of individuals with FA. This review focuses on the known causes of reduced fertility in FA, and integrates them with the emerging role of the FA pathway in double-strand break (DSB) repair at meiosis in a variety of organisms, as well as providing future directions for research and diagnostics.

Hallmarks of ribosomopathies.

Ribosomopathies are diseases caused by defects in ribosomal constituents or in factors with a role in ribosome assembly. Intriguingly, congenital ribosomopathies display a paradoxical transition from early symptoms due to cellular hypo-proliferation to an elevated cancer risk later in life. Another association between ribosome defects and cancer came into view after the recent discovery of somatic mutations in ribosomal proteins and rDNA copy number changes in a variety of tumor types, giving rise to somatic ribosomopathies. Despite these clear connections between ribosome defects and cancer, the molecular mechanisms by which defects in this essential cellular machinery are oncogenic only start to emerge. In this review, the impact of ribosomal defects on the cellular function and their mechanisms of promoting oncogenesis are described. In particular, we discuss the emerging hallmarks of ribosomopathies such as the appearance of 'onco-ribosomes' that are specialized in translating oncoproteins, dysregulation of translation-independent extra-ribosomal functions of ribosomal proteins, rewired cellular protein and energy metabolism, and extensive oxidative stress leading to DNA damage. We end by integrating these findings in a model that can provide an explanation how ribosomopathies could lead to the transition from hypo- to hyper-proliferation in bone marrow failure syndromes with elevated cancer risk.

High-throughput STELA provides a rapid test for the diagnosis of telomere biology disorders.

Telomere biology disorders are complex clinical conditions that arise due to mutations in genes required for telomere maintenance. Telomere length has been utilised as part of the diagnostic work-up of patients with these diseases; here, we have tested the utility of high-throughput STELA (HT-STELA) for this purpose. HT-STELA was applied to a cohort of unaffected individuals (n = 171) and a retrospective cohort of mutation carriers (n = 172). HT-STELA displayed a low measurement error with inter- and intra-assay coefficient of variance of 2.3% and 1.8%, respectively. Whilst telomere length in unaffected individuals declined as a function of age, telomere length in mutation carriers appeared to increase due to a preponderance of shorter telomeres detected in younger individuals (< 20 years of age). These individuals were more severely affected, and age-adjusted telomere length differentials could be used to stratify the cohort for overall survival (Hazard Ratio = 5.6 (1.5-20.5); p < 0.0001). Telomere lengths of asymptomatic mutation carriers were shorter than controls (p < 0.0001), but longer than symptomatic mutation carriers (p < 0.0001) and telomere length heterogeneity was dependent on the diagnosis and mutational status. Our data show that the ability of HT-STELA to detect short telomere lengths, that are not readily detected with other methods, means it can provide powerful diagnostic discrimination and prognostic information. The rapid format, with a low measurement error, demonstrates that HT-STELA is a new high-quality laboratory test for the clinical diagnosis of an underlying telomeropathy.

Pediatric bone marrow failure: Clinical, hematological and targeted next generation sequencing data.

OBJECTIVE: In this study, clinico-hematological, genetic and outcome profile of children with BMF was evaluated to delineate the underlying genotype and phenotype. DESIGN: Cases were evaluated as two groups: Group 1 (n = 56; DBA-23, FA-18, DC-2, UBMFS-13) included children with suspected IBMFS based on clinical phenotype and accessible lab investigations and Group 2 (n = 53) included children with IAA treated with IST. Targeted NGS was carried out in a subset of these children (n = 42) and supplemented with WES wherever required. RESULTS: We identified causative mutation in overall 15 of 27 tested children (55.5%) in group 1 and 2 of 15 tested children (13.3%) in group 2. In DBA, a mutation was noted in 50% cases with involvement of RPS 19 (75%) and RPL5 (25%) genes. Phenotypic abnormalities were present in 69.5% and response to steroids in 68.4% of cases at a median follow up of 33 months. In children with IAA, overall response (complete + partial) was present in 51% at a median follow up of 23 months. The 3-year OS and FFS for the cohort of IAA were 68% and 48% respectively. Targeted sequencing could also pick up germline mutations in 50% of UBMFS cases and nearly 19% of IAA cases.

The clinical significance of PNH-phenotype cells accounting for < 0.01% of total granulocytes detected by the Clinical and Laboratory Standards Institute methods in patients with bone marrow failure.

Small populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells accounting for up to 0.01% of total granulocytes can be accurately detected by a high-sensitivity flow cytometry (FCM) assay established by the Clinical and Laboratory Standards Institute (CLSI method) and have a prognostic value in bone marrow failure (BMF); however, the significance of GPI(-) granulocytes accounting for 0.001-0.009% of granulocytes remains unclear. To clarify this issue, we examined the peripheral blood of 21 BMF patients in whom minor (around 0.01%) populations of GPI(-) granulocytes had been previously detected by a different high-resolution FCM method (OPTIMA method, which defines ≥ 0.003% GPI(-) granulocytes as an abnormal increase) using both the CLSI and OPTIMA methods simultaneously. These two methods detected an "abnormal increase" in GPI(-) granulocytes in 10 patients (48%) and 17 patients (81%), respectively. CLSI detected 0.002-0.005% (median, 0.004%) GPI(-) granulocytes in 7 patients who were deemed positive for PNH-type cells according to the OPTIMA method, which detected 0.003-0.012% (median 0.006%) GPI(-) granulocytes. The clone sizes of GPI(-) cells detected by each assay were positively correlated (r = 0.994, p < 0.001). Of the seven patients who were judged positive for PNH-type cells by OPTIMA alone, five received immunosuppressive therapy, and all of them achieved a partial or complete response. GPI(-) granulocytes detected in BMF patients by the CLSI method should thus be considered significant, even at percentages of < 0.01%.

Genetic screening of children with marrow failure. The role of primary Immunodeficiencies.

The differential diagnosis of marrow failure (MF) is crucial in the diagnostic work-up, since genetic forms require specific care. We retrospectively studied all patients with single/multi-lineage MF evaluated in a single-center to identify the type and incidence of underlying molecular defects. The diepoxybutane test was used to screen Fanconi Anemia. Other congenital MFs have been searched using Sanger and/or Next Generation Sequencing analysis, depending on the available tools over the years. Between 2009-2019, 97 patients (aged 0-32 years-median 5) with single-lineage (29%) or multilineage (68%) MF were evaluated. Fifty-three (54%) and 28 (29%) were diagnosed with acquired and congenital MF, respectively. The remaining 16 (17%), with trilinear (n=9) and monolinear (n=7) MF, were found to have an underlying primary immunodeficiency (PID) and showed clinical and biochemical signs of immune-dysregulation in 10/16 (62%) and in 14/16 (87%) of cases, respectively. Clinical signs were also found in 22/53 (41%) and 8/28 (28%) patients with idiopathic and classical cMF, respectively. Eight out of 16 PIDs patients were successfully transplanted, four received immunosuppression, two did not require treatment, and the remaining two died. We show that patients with single/multi-lineage MF may have underlying PIDs in a considerable number of cases and that MF may represent a relevant clinical sign in patients with PIDs, thus widening their clinical phenotype. An accurate immunological work-up should be performed in all patients with MF, and PID-related genes should be considered when screening MF in order to identify disorders that may receive targeted treatments and/or appropriate conditioning regimens before transplant.

Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure.

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER-Grem1 flx/flx ) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7-13 days). Post-mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Bone Marrow Failure Syndromes, Overlapping Diseases with a Common Cytokine Signature.

Bone marrow failure (BMF) syndromes are a heterogenous group of non-malignant hematologic diseases characterized by single- or multi-lineage cytopenia(s) with either inherited or acquired pathogenesis. Aberrant T or B cells or innate immune responses are variously involved in the pathophysiology of BMF, and hematological improvement after standard immunosuppressive or anti-complement therapies is the main indirect evidence of the central role of the immune system in BMF development. As part of this immune derangement, pro-inflammatory cytokines play an important role in shaping the immune responses and in sustaining inflammation during marrow failure. In this review, we summarize current knowledge of cytokine signatures in BMF syndromes.

Impact of germline CTC1 alterations on telomere length in acquired bone marrow failure.

Compound heterozygous germline mutations in CTC1 gene have been found in patients with atypical dyskeratosis congenita (DC), whereas heterozygous carriers are unaffected. Through screening of a large cohort of adult patients with acquired bone marrow failure syndromes, in addition to a DC case, we have also found extremely rare or novel heterozygous deleterious germline variants of CTC1 in patients with aplastic anaemia (AA; n = 5), paroxysmal nocturnal haemoglobinuria (PNH; n = 3) and myelodysplastic syndrome (MDS; n = 2). A compound heterozygous case of AA showed clonal evolution. Our results suggest that some of the inherited CTC1 variants may represent predisposition factors for acquired bone marrow failure.

Peering through zebrafish to understand inherited bone marrow failure syndromes.

Inherited bone marrow failure syndromes are experiments of nature characterized by impaired hematopoiesis with cancer and leukemia predisposition. The mutations associated with inherited bone marrow failure syndromes affect fundamental cellular pathways, such as DNA repair, telomere maintenance, or proteostasis. How these disturbed pathways fail to produce sufficient blood cells and lead to leukemogenesis are not understood. The rarity of inherited cytopenias, the paucity of affected primary human hematopoietic cells, and the sometime inadequacy of murine or induced pluripotential stem cell models mean it is difficult to acquire a greater understanding of them. Zebrafish offer a model organism to study gene functions. As vertebrates, zebrafish share with humans many orthologous genes involved in blood disorders. As a model organism, zebrafish provide advantages that include rapid development of transparent embryos, high fecundity (providing large numbers of mutant and normal siblings), and a large collection of mutant and transgenic lines useful for investigating the blood system and other tissues during development. Importantly, recent advances in genomic editing in zebrafish can speedily validate the new genes or novel variants discovered in clinical investigation as causes for marrow failure. Here we review zebrafish as a model organism that phenocopies Fanconi anemia, Diamond-Blackfan anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, congenital amegakaryocytic thrombocytopenia, and severe congenital neutropenia. Two important insights, provided by modeling inherited cytopenias in zebrafish, widen understanding of ribosome biogenesis and TP53 in mediating marrow failure and non-hematologic defects. They suggest that TP53-independent pathways contribute to marrow failure. In addition, zebrafish provide an attractive model organism for drug development.

Integrated proteogenomic analysis for inherited bone marrow failure syndrome.

Recent advances in in-depth data-independent acquisition proteomic analysis have enabled comprehensive quantitative analysis of >10,000 proteins. Herein, an integrated proteogenomic analysis for inherited bone marrow failure syndrome (IBMFS) was performed to reveal their biological features and to develop a proteomic-based diagnostic assay in the discovery cohort; dyskeratosis congenita (n = 12), Fanconi anemia (n = 11), Diamond-Blackfan anemia (DBA, n = 9), Shwachman-Diamond syndrome (SDS, n = 6), ADH5/ALDH2 deficiency (n = 4), and other IBMFS (n = 18). Unsupervised proteomic clustering identified eight independent clusters (C1-C8), with the ribosomal pathway specifically downregulated in C1 and C2, enriched for DBA and SDS, respectively. Six patients with SDS had significantly decreased SBDS protein expression, with two of these not diagnosed by DNA sequencing alone. Four patients with ADH5/ALDH2 deficiency showed significantly reduced ADH5 protein expression. To perform a large-scale rapid IBMFS screening, targeted proteomic analysis was performed on 417 samples from patients with IBMFS-related hematological disorders (n = 390) and healthy controls (n = 27). SBDS and ADH5 protein expressions were significantly reduced in SDS and ADH5/ALDH2 deficiency, respectively. The clinical application of this first integrated proteogenomic analysis would be useful for the diagnosis and screening of IBMFS, where appropriate clinical screening tests are lacking.

AML-derived extracellular vesicles negatively regulate stem cell pool size: A step toward bone marrow failure.

PURPOSE OF THE STUDY: Long-term repopulating hematopoietic stem cells (LTR-HSCs) have been previously shown to reside in close proximity to osteoblasts, where they take shelter in the bone marrow (BM) microenvironment against cytotoxic and apoptotic stimuli. Nevertheless, the function of the HSC niche is believed to undergo an adaptive evolutionary modification during leukemogenesis. Recent studies have demonstrated that leukemic clones can impact BM homing through extracellular vesicle (EV) secretion. However, the exact mechanism driving BM conversion is still unclear. In the present study, the human osteoblast cell line (MG-63) were subjected to various concentration of sera-derived EVs of patients with acute myeloid leukemia (AML) and healthy volunteers to assess if they are associated strongly enough to alter the expression pattern of cross-talk molecules involved in niche interactions. METHOD: To gain a brief insight into the EVs secretion criteria, we first conducted a comparative analysis of sera-derived EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM), and Bradford assay. After incubating MG-63 cell lines with increasing concentrations of the EVs, Trypan-blue and microculture tetrazolium test (MTT) assays were used to evaluate the cell survival, logarithmic growth, and metabolic activity. Finally, the expression levels of OPN, ANGPT-1, and JAG-1 transcripts were evaluated through the qRT-PCR technique. RESULTS: Here, we report that AML-derived EVs can affect the viability, cell growth, and metabolic activity of the human osteoblasts cell line (MG-63) compared to those that received healthy-derived EVs. We also found that leukemic EVs tend to induce overexpression of OPN but reduce the expression of ANGPT-1 and JAG-1 genes in the osteoblast transcriptome, which may provide a potential context imposing selective suppression of HSC pool size. CONCLUSION: These findings extend the general concept of a novel mechanism in which leukemic EVs would make it possible to create a specialized pre-metastatic microenvironment in the interest of tumor expansion, allowing leukemic clones to overcome their HSCs counterparts.

TIRAP drives myelosuppression through an Ifnγ-Hmgb1 axis that disrupts the endothelial niche in mice.

Inflammation is associated with bone marrow failure syndromes, but how specific molecules impact the bone marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS) and suppresses all three major hematopoietic lineages. TIRAP expression promotes up-regulation of Ifnγ, leading to myelosuppression through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the bone marrow endothelial niche. Deletion of Ifnγ blocks Hmgb1 release and is sufficient to reverse the endothelial defect and restore myelopoiesis. Contrary to current dogma, TIRAP-activated Ifnγ-driven bone marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of MDS to acute leukemia. These findings reveal novel, noncanonical roles of TIRAP, Hmgb1, and Ifnγ in the bone marrow microenvironment and provide insight into the pathophysiology of preleukemic syndromes.

U2af1 is required for survival and function of hematopoietic stem/progenitor cells.

U2AF1 is involved in the recognition of the 3' splice site during pre-mRNA splicing. Mutations in U2AF1 are frequently observed in myelodysplastic syndromes. However, the role of wild-type U2AF1 in normal hematopoiesis has remained elusive. Using a novel conditional U2af1 knockout allele, we have found that deletion of U2af1 results in profound defects in hematopoiesis characterized by pancytopenia, ablation of hematopoietic stem/progenitor cells (HSPC) leading to bone marrow failure and early lethality in mice. U2af1 deletion impairs HSPC function and repopulation capacity. U2af1 deletion also causes increased DNA damage and reduced survival in hematopoietic progenitors. RNA sequencing analysis reveals significant alterations in the expression of genes related to HSC maintenance, cell proliferation, and DNA damage response-related pathways in U2af1-deficient HSPC. U2af1 deficiency also induces splicing alterations in genes important for HSPC function. This includes altered splicing and perturbed expression of Nfya and Pbx1 transcription factors in U2af1-deficient HSPC. Collectively, these results suggest an important role for U2af1 in the maintenance and function of HSPC in normal hematopoiesis. A better understanding of the normal function of U2AF1 in hematopoiesis is important for development of appropriate therapeutic approaches for U2AF1 mutant induced hematologic malignancies.