Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation rates than CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI.

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.

Hematopoietic stem cells (HSCs) require highly regulated rates of protein synthesis, but it is unclear if they or lineage-committed progenitors preferentially recruit transcripts to translating ribosomes. We utilized polysome profiling, RNA sequencing, and whole-proteomic approaches to examine the translatome in LSK (Lin-Sca-1+c-Kit+) and myeloid progenitor (MP; Lin-Sca-1-c-Kit+) cells. Our studies show that LSKs exhibit low global translation but high translational efficiencies (TEs) of mRNAs required for HSC maintenance. In contrast, MPs activate translation in an mTOR-independent manner due, at least in part, to proteasomal degradation of mTOR by the E3 ubiquitin ligase c-Cbl. In the near absence of mTOR, CDK1 activates eIF4E-dependent translation in MPs through phosphorylation of 4E-BP1. Aberrant activation of mTOR expression and signaling in c-Cbl-deficient MPs results in increased mature myeloid lineage output. Overall, our data demonstrate that hematopoietic stem and progenitor cells (HSPCs) undergo translational reprogramming mediated by previously uncharacterized mechanisms of translational regulation.

The genetic programs that promote retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML) are not known. In a mouse model of human AML, LSCs exhibit variable frequencies that correlate with the initiating MLL oncogene and are maintained in a self-renewing state by a transcriptional subprogram more akin to that of embryonic stem cells (ESCs) than to that of adult stem cells. The transcription/chromatin regulatory factors Myb, Hmgb3, and Cbx5 are critical components of the program and suffice for Hoxa/Meis-independent immortalization of myeloid progenitors when coexpressed, establishing the cooperative and essential role of an ESC-like LSC maintenance program ancillary to the leukemia-initiating MLL/Hox/Meis program. Enriched expression of LSC maintenance and ESC-like program genes in normal myeloid progenitors and poor-prognosis human malignancies links the frequency of aberrantly self-renewing progenitor-like cancer stem cells (CSCs) to prognosis in human cancer.

Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34+ human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.

Inflammation coordinates tissue regeneration via damaged cell removal and stem cell activation. Hematopoietic stem cells (HSCs) survive inflammatory stress that kills other blood cells, but the mechanisms underlying this effect remain poorly understood. Here, we find that tumor necrosis factor α (TNF-α) acts differently on HSCs and progenitors, thus facilitating hematopoietic clearance and promoting regeneration. We show that while inducing myeloid progenitor apoptosis, TNF-α promotes HSC survival and myeloid differentiation by activating a strong and specific p65-nuclear factor κB (NF-κB)-dependent gene program that primarily prevents necroptosis rather than apoptosis, induces immunomodulatory functions, and poises HSCs for myeloid cell production. These TNF-α-driven mechanisms are critical for HSC response to inflammatory stress but are also hijacked in aged and malignant HSCs. Our results reveal several TNF-α-mediated pro-survival mechanisms unique to HSCs, highlight an important role for necroptosis in HSC killing, and establish TNF-α as a major pro-survival and pro-regeneration factor for HSCs.

N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNAs (mRNAs), plays critical roles in many bioprocesses. However, its functions in normal and malignant hematopoiesis remain elusive. Here, we report that METTL14, a key component of the m6A methyltransferase complex, is highly expressed in normal hematopoietic stem/progenitor cells (HSPCs) and acute myeloid leukemia (AML) cells carrying t(11q23), t(15;17), or t(8;21) and is downregulated during myeloid differentiation. Silencing of METTL14 promotes terminal myeloid differentiation of normal HSPCs and AML cells and inhibits AML cell survival/proliferation. METTL14 is required for development and maintenance of AML and self-renewal of leukemia stem/initiation cells (LSCs/LICs). Mechanistically, METTL14 exerts its oncogenic role by regulating its mRNA targets (e.g., MYB and MYC) through m6A modification, while the protein itself is negatively regulated by SPI1. Collectively, our results reveal the SPI1-METTL14-MYB/MYC signaling axis in myelopoiesis and leukemogenesis and highlight the critical roles of METTL14 and m6A modification in normal and malignant hematopoiesis.

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.

Recurrent mutations in cohesin complex proteins have been identified in pre-leukemic hematopoietic stem cells and during the early development of acute myeloid leukemia and other myeloid malignancies. Although cohesins are involved in chromosome separation and DNA damage repair, cohesin complex functions during hematopoiesis and leukemic development are unclear. Here, we show that mutant cohesin proteins block differentiation of human hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo and enforce stem cell programs. These effects are restricted to immature HSPC populations, where cohesin mutants show increased chromatin accessibility and likelihood of transcription factor binding site occupancy by HSPC regulators including ERG, GATA2, and RUNX1, as measured by ATAC-seq and ChIP-seq. Epistasis experiments show that silencing these transcription factors rescues the differentiation block caused by cohesin mutants. Together, these results show that mutant cohesins impair HSPC differentiation by controlling chromatin accessibility and transcription factor activity, possibly contributing to leukemic disease.

Abnormal nuclear morphology is a hallmark of malignant cells widely used in cancer diagnosis. Pelger-Huët anomaly (PHA) is a common abnormality of neutrophil nuclear morphology of unknown molecular etiology in myeloid neoplasms (MNs). We show that loss of nuclear lamin B1 (LMNB1) encoded on chromosome 5q, which is frequently deleted in MNs, induces defects in nuclear morphology and human hematopoietic stem cell (HSC) function associated with malignancy. LMNB1 deficiency alters genome organization inducing in vitro and in vivo expansion of HSCs, myeloid-biased differentiation with impaired lymphoid commitment, and genome instability due to defective DNA damage repair. Nuclear dysmorphology of neutrophils in patients with MNs is associated with 5q deletions spanning the LMNB1 locus, and lamin B1 loss is both necessary and sufficient to cause PHA in normal and 5q-deleted neutrophils. LMNB1 loss thus causes acquired PHA and links abnormal nuclear morphology with HSCs and progenitor cell fate determination via genome organization.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.

Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2(V617F)-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2(V617F+) HSPCs in a xenograft model, and sensitized MLL-AF9(+) leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML.

Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress, which results in enhanced production of inflammatory cytokines, including interleukin-6 (IL-6), and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic long non-coding RNA (lncRNAs), Morrbid, in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2 mutant mice rescues inflammatory-stress-induced abnormalities in HSPCs and mature myeloid cells, including clonal hematopoiesis.

A hierarchical hematopoietic development with myeloid versus lymphoid bifurcation has been proposed downstream of the multipotent progenitor (MPP) stage, based on prospective isolation of progenitors capable of generating only myeloerythroid cells (common myeloid progenitor, CMP) or only lymphocytes (common lymphoid progenitor, CLP). By utilizing GATA-1 and PU.1 transcription factor reporters, here we identified progenitor populations that are precursors for either CMPs or CLPs. Two independent populations expressing either GATA-1 or PU.1 resided within the CD34(+)Sca-1(+)c-Kit(+) MPP fraction. The GATA-1(+) MPP displayed potent myeloerythroid potential without giving rise to lymphocytes, whereas the PU.1(+) MPP showed granulocyte/monocyte/lymphoid-restricted progenitor activity without megakaryocyte/erythroid differentiation. Furthermore, GATA-1(+) and PU.1(+) MPPs possessed huge expansion potential and differentiated into the original CMPs and CLPs, respectively. Thus, the reciprocal activation of GATA-1 and PU.1 primarily organizes the hematopoietic lineage fate decision to form the earliest hematopoietic branchpoint that comprises isolatable myeloerythroid and myelolymphoid progenitor populations.

Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells (HSPCs) has limited their characterization to in vitro assays. We report a strategy to respecify lineage-restricted CD34(+)CD45(+) myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engrafted in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34(+)CD38(-) cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, conferred engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription-factor-mediated engraftment of blood progenitors from human pluripotent cells.

Bacterial infection leads to consumption of short-lived innate immune effector cells, which then need to be replenished from hematopoietic stem and progenitor cells (HSPCs). HSPCs express pattern recognition receptors, such as Toll-like receptors (TLRs), and ligation of these receptors induces HSPC mobilization, cytokine production, and myeloid differentiation. The underlying mechanisms involved in pathogen signal transduction in HSCs and the resulting biological consequences remain poorly defined. Here, we show that in vivo lipopolysaccharide (LPS) application induces proliferation of dormant HSCs directly via TLR4 and that sustained LPS exposure impairs HSC self-renewal and competitive repopulation activity. This process is mediated via TLR4-TRIF-ROS-p38, but not MyD88 signaling, and can be inhibited pharmacologically without preventing emergency granulopoiesis. Live Salmonella Typhimurium infection similarly induces proliferative stress in HSCs, in part via TLR4-TRIF signals. Thus, while direct TLR4 activation in HSCs might be beneficial for controlling systemic infection, prolonged TLR4 signaling has detrimental effects and may contribute to inflammation-associated HSPC dysfunction.

The lineage restriction of prospectively isolated hematopoietic progenitors has been traditionally assessed by bulk in vitro culture and transplantation of large number of cells in vivo. These methods, however, cannot distinguish between homogenous multipotent or heterogeneous lineage-restricted populations. Using clonal assays of 1 or 5 cells in vitro, single-cell quantitative gene expression analyses, and transplantation of mice with low numbers of cells, we show that a common myeloid progenitor (CMP) is Sca-1(lo)lin(-)c-Kit(+)CD27(+)Flk-2(-) (SL-CMP; Sca-1(lo) CMP) and a granulocyte/macrophage progenitor (GMP) is Sca-1(lo)lin(-)c-Kit(+)CD27(+)Flk-2(+)CD150(-/lo) (SL-GMP; Sca-1(lo) GMP). We found that mast cell progenitor potential is present in the SL-CMP fraction, but not in the more differentiated SL-GMP population, and is more closely related to megakaryocyte/erythrocyte specification. Our data provide criteria for the prospective isolation of SL-CMP and SL-GMP and support the conclusion that mast cells are specified during hematopoiesis earlier than and independently from granulocytes.

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.