Myeloid Fate Research Articles

Abstract PR07: Connecting acetate and citrate metabolism with epigenetic regulation of hematopoiesis

Abstract Here we hypothesize that acetate metabolism and citrate metabolism play integral roles to balance the fate decision of HSCs and that targeting Acly-Coa Synthetase Short-Chain 2 (ACSS2) and ATP-Citrate Lyase (ACLY) will impact epigenetic modifications and gene expression to affect cell fate. Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to generate a variety of myeloid, erythroid, and lymphoid cells through hematopoiesis. Hematopoiesis precisely balances many stimuli to maintain HSC self-renewal and differentiation. These stimuli classically consist of chemokines, cytokines, and transcription factors acting at key precursor stages to influence cell fate. Metabolic molecules can directly influence epigenetic state, chromatin accessibility, and cell lineage maturation via regulation of epigenetic modifying enzymes. However, understanding remains limited of how HSCs balance metabolic and other microenvironmental stimuli to regulate self-renewal and differentiation. Acetate and citrate metabolism regulate methylation and acetylation, respectively, and we investigated how these pathways may impact hematopoiesis. The Rathmell lab has previously shown that metabolism is a key regulator of T effector cells, as we discovered that Glutaminase deficiency promotes Th1 effector differentiation. We have now shown that ACLY inhibition can drive myeloid differentiation. To test our hypothesis, we treated methylcellulose-cultured HSCs with small-molecule inhibitors of ACLY and ACSS2 for two weeks to determine if inhibition of these enzymes alters lineage commitment of HSCs to myeloid vs. erythroid fate decisions. We observed that inhibition of ACLY using SB-204990 drives HSC differentiation into myeloid fates, indicated by increased CD11b expression. Further, treatment with SB-204990 decreased population of the conventional stem compartment Sca-1+ c-Kit+ Lin-. We will now use conditionally-deficient models of ACLY to directly and genetically establish the roles of acetate and citrate metabolism in HSC self-renewal and differentiation. Single-cell RNA-sequencing and chromatin accessibility assays will be employed to identify metabolically sensitive gene regulatory networks within heterogeneous hematopoietic cell populations. Unraveling the link between acetate metabolism, citrate metabolism, and stem cell function presents an opportunity to develop a more nuanced picture of metabolism’s increasingly discussed role as a regulator of cellular development. Ultimately, elucidating possible mechanisms to promote hematopoietic lineage differentiation presents a novel therapeutic opportunity for various hematologic malignancies and disorders. Citation Format: Dalton L. Greenwood, Jeffrey C. Rathmell. Connecting acetate and citrate metabolism with epigenetic regulation of hematopoiesis [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR07.

Loss of Canonical Notch Signaling Affects Multiple Steps in NK Cell Development in Mice.

Within the hematopoietic system, the Notch pathway is critical for promoting thymic T cell development and suppressing the B and myeloid lineage fates; however, its impact on NK lymphopoiesis is less understood. To study the role of Notch during NK cell development in vivo, we investigated different NK cell compartments and function in Rbp-Jkfl/flVav-Cretg/+ mice, in which Rbp-Jk, the major transcriptional effector of canonical Notch signaling, was specifically deleted in all hematopoietic cells. Peripheral conventional cytotoxic NK cells in Rbp-Jk-deleted mice were significantly reduced and had an activated phenotype. Furthermore, the pool of early NK cell progenitors in the bone marrow was decreased, whereas immature NK cells were increased, leading to a block in NK cell maturation. These changes were cell intrinsic as the hematopoietic chimeras generated after transplantation of Rbp-Jk-deficient bone marrow cells had the same NK cell phenotype as the Rbp-Jk-deleted donor mice, whereas the wild-type competitors did not. The expression of several crucial NK cell regulatory pathways was significantly altered after Rbp-Jk deletion. Together, these results demonstrate the involvement of canonical Notch signaling in regulation of multiple stages of NK cell development.

Metabolic Reprogramming of Myeloid Cells in Response to Factors of “Emergency” Myelopoiesis By Myeloid-Specific PD-1 Ablation, Regulates Myeloid Lineage Fate Commitment and Anti-Tumor Immunity

PD-1 is a T cell inhibitor for which blocking agents have achieved success as anti-cancer therapeutics. The current view is that cancer limits host immune responses by upregulating PD-L1 in the tumor microenvironment (TME) thereby causing PD-1 ligation and inactivation of CD8+ Teff cells. However, PD-L1 expression in the TME does not always correlate with therapeutic response. Thus, the mechanism(s) by which PD-1 blockade reverses compromised anti-tumor immunity are poorly understood. The rapid increase in hematopoietic cell output that occurs in response to immunologic stress is known as emergency myelopoiesis. Low-level stimulation by cancer-generated factors induces modest but continuous expansion of myeloid progenitors (MP) (common myeloid progenitors (CMP) and granulocyte/macrophage progenitors (GMP)) albeit with hindered differentiation, leading to output of tumor-promoting myeloid-derived suppressor cells (MDSCs). We determined that myeloid cells expanding during cancer-driven emergency myelopoiesis in tumor-bearing mice express PD-1 and PD-L1. Using PD-1 KO mice we found that PD-1 deletion prevented the accumulation of GMP and stimulated the output of Ly6Chi effector monocytes, macrophages and dendritic cells (DC). To determine whether these outcomes were mediated by a myeloid-intrinsic impact of PD-1 ablation or by the effects of PD-1neg T cells on myeloid cells, we generated mice with conditional targeting of the Pdcd1 gene (PD-1f/f) and selectively eliminated PD-1 in myeloid cells (PD-1f/fLysMcre) or T cells (PD-1f/fCD4cre). Myeloid-specific, but not T cell-specific PD-1 ablation, prevented the accumulation of GMP while promoting the output of effector-like myeloid cells expressing CD80, CD86, CD16/32 (FcRII/III) and CD88 (C5aR). Myeloid cells with PD-1 ablation had elevated expression of IRF8 that drives monocyte and DC differentiation and decreased expression of the MDSC hallmark markers IL-4R, CD206, ARG1 and CD38. Nutrient utilization has a decisive role on the fate of hematopoietic progenitors (HP) and MP. Stemness and pluripotency are regulated by maintenance of glycolysis whereas switch to mitochondrial metabolism is associated with differentiation. To examine whether PD-1 ablation affected these metabolic proceces, bone marrow (BM) from PD-1f/f and PD-1f/fLysMcre mice was cultured with G-CSF/GM-CSF/IL-6, key drivers of emergency myelopoiesis. MP differentiation was documented by decrease of Linneg and increase of Linpos cells, which was more prominent in PD-1f/fLysMcre BM cultures. This coincided with increase of CD45+CD11b+ and dominance of Ly6C+ monocytic cells consistent with a cell-intrinsic mechanism of monocytic lineage commitment. PD-1f/fLysMcre MP had elevated mTORC1, Erk1/2 and Stat1 activation, and enhanced glucose uptake and mitochondrial biogenesis. Bioenergetics studies showed robust development of a mitochondrial-dominant profile, consistent with metabolism-driven enhanced differentiation of MP. Mass spectrometry revealed enhanced intermediates of glycolysis, PPP and TCA cycle, but the most prominent difference was the increased cholesterol. Because mTORC1 signaling, which was enhanced in PD-1f/fLysMcre MP, activates de novo lipid and cholesterol synthesis via SREBP1, we examined the mevalonate pathway of cholesterol synthesis. mRNA for genes mediating cholesterol synthesis and uptake was increased whereas mRNA for genes mediating cholesterol metabolism was decreased. Cholesterol induces a proinflammatory program in myeloid cells, drives differentiation of monocytes, macrophages and DC and promotes antigen-presenting function. We examined how such changes in myeloid cells might affect the function of T cells, which are key anti-tumor mediators. Compared to tumor-bearing PD-1f/f mice, PD-1f/fLysMcre tumor-bearers had no quantitative T cell differences but had an increase in IFNγ- IL-17-, and IL-10-expressing CD8+ Teff-mem and IL-2-expressing Tcentral-mem cells, consistent with superior functionality. These changes correlated with enhanced anti-tumor protection despite preserved PD-1 expression in T cells. Our findings reveal a previously unidentified role of PD-1 in metabolism-driven myeloid cell lineage fate commitment and differentiation and suggest that switch to effector myeloid cells might be a key mechanism by which PD-1 blockade mediates systemic anti-tumor immunity. DisclosuresNo relevant conflicts of interest to declare.

Sphingosine-1-Phosphate Receptor 3 (S1PR3) Promotes Myeloid Commitment of Human Hematopoietic and Leukemic Stem Cells

Understanding the mechanisms underlying the abnormal differentiation of human acute myeloid leukemia (AML) may reveal novel therapies to eradicate leukemic stem cells (LSC), which are often resistant to standard treatments and contribute to relapse. Cellular metabolism is recognized as a hallmark of cancer and is known to be distinct between hematopoietic stem cells (HSC) and downstream progenitors. In particular, sphingosine-1-phosphate (S1P) is a bioactive lipid produced from sphingolipid metabolism that regulates proliferation and survival and is implicated in HSC egress, lymphocyte trafficking and mouse lymphoid lineage determination primarily through binding to S1PR1, one of five S1P G-protein coupled receptors. However, sphingolipids are understudied in human LSC and HSC. We recently found that sphingolipid perturbation governs HSC self-renewal and influences lineage outcome. Here, we show that S1PR3 governs myeloid commitment of LSC in a subset of human AML and is thus an attractive therapeutic target.Lipidomic analysis of LSC+ and LSC‒ fractions derived from AML patient samples and validated by xenotransplantation assays showed distinct sphingolipid alterations when compared to each other and to normal human cord blood (CB) CD34+CD38‒ stem cells and CD34+CD38+ progenitor populations. Interestingly, LSC+ fractions have increased S1P levels, prompting us to wonder if S1P signaling contributes to LSC maintenance. To gain a better understanding of the role of S1P in stemness, we examined S1P signaling in normal CB. Analysis of gene expression of S1PR1-5 and S1P transporters (SPNS2 and MFSD2B) within a comprehensive transcriptional roadmap of human hematopoiesis comprising thirteen normal populations from HSC to mature blood lineages demonstrated distinct expression patterns in specific blood lineages. S1PR1 and MFSD2B were most highly expressed in lymphoid and erythroid lineages, respectively, consistent with murine data. Notably, S1PR3 expression is specific to mature monocytes and granulocytes in human CB. S1PR3 protein was absent from the surface of long-term (LT) and short-term (ST)-HSC as determined by flow cytometry. Remarkably, lentiviral overexpression (OE) of S1PR3 was sufficient to promote myelopoiesis at the expense of erythropoiesis from LT- and ST-HSC in vitro in liquid culture, single cell assays and colony forming assays. To ascertain the mechanisms promoting myeloid commitment, we performed gene expression profiling by RNA-seq of LT- and ST-HSC following S1PR3 OE. This yielded a subset of shared genes similarly upregulated following S1PR3 OE relative to controls, including the known myeloid differentiation and AML-associated factors Early Growth Response 1 and 2 (EGR1/2) and Tribbles 1 (TRIB1). Thus, promiscuous expression of S1PR3 promotes a myeloid fate program in human HSC.S1PR3 protein expression was higher in AML patient samples relative to human CB and bone marrow cells by flow cytometry. Bioinformatic analysis of three independent AML cohorts revealed that AML patient samples with high S1PR3 gene expression also had high EGR2 and TRIB1 expression. Limiting dilution xenotransplantation assays of LSC-containing fractions sorted based on surface expression of S1PR3 demonstrated lower LSC frequency in S1PR3high versus S1PR3low/- LSC fractions. Moreover, S1PR3 OE in LSC+ fractions virtually abolished leukemic engraftment in xenotranplantation assays. These results suggest that higher S1PR3 levels in LSC are associated with a more mature myeloid state and that further increasing S1PR3 levels disrupts LSC maintenance. Treatment of mice bearing primary AML xenografts with FTY720, a S1P mimetic that targets S1P receptors including S1PR3, decreased leukemia burden in a subset of patient samples tested, including those obtained from relapsed and treatment-refractory patients. 70% of AML samples tested showed decreased LSC frequency in serial repopulation assays following FTY720 treatment. Importantly, treatment with FTY720 did not alter normal hematopoietic xenografts, demonstrating the existence of a therapeutic window. Collectively, our results provide the first direct evidence that sphingolipids govern myeloid commitment in human HSC and LSC, and demonstrate the potential of S1PR3 as a novel therapeutic target in AML for eradication of LSC while sparing HSC. DisclosuresNo relevant conflicts of interest to declare.

GM-CSF Quantity Has a Selective Effect on Granulocytic vs. Monocytic Myeloid Development and Function.

GM-CSF promotes myeloid differentiation of cultured bone marrow cells into cells of the granulocytic and monocytic lineage; the latter can further differentiate into monocytes/macrophages and dendritic cells. How GM-CSF selects for these different myeloid fates is unresolved. GM-CSF levels can change either iatrogenically (e.g., augmenting leukopoiesis after radiotherapy) or naturally (e.g., during infection or inflammation) resulting in different immunological outcomes. Therefore, we asked whether the dose of GM-CSF may regulate the development of three types of myeloid cells. Here, we showed that GM-CSF acted as a molecular rheostat where the quantity determined which cell type was favored; moreover, the cellular process by which this was achieved was different for each cell type. Thus, low quantities of GM-CSF promoted the granulocytic lineage, mainly through survival. High quantities promoted the monocytic lineage, mainly through proliferation, whereas moderate quantities promoted moDCs, mainly through differentiation. Finally, we demonstrated that monocytes/macrophages generated with different doses of GM-CSF differed in function. We contend that this selective effect of GM-CSF dose on myeloid differentiation and function should be taken into consideration during pathophysiological states that may alter GM-CSF levels and during GM-CSF agonistic or antagonistic therapy.

Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis.

The dynamics of haematopoietic stem cell differentiation and the hierarchy of oligopotent stem cells in the bone marrow remain controversial. Here we dissect haematopoietic progenitor populations at single cell resolution, deriving an unbiased reference model of transcriptional states in normal and perturbed murine bone marrow. We define the signature of the naive haematopoietic stem cell and find a continuum of core progenitor states. Core cell populations mix transcription of pre-myeloid and pre-lymphoid programs, but do not mix erythroid or megakaryocyte programs with other fates. CRISP-seq perturbation analysis confirms our models and reveals that Cebpa regulates entry into all myeloid fates, while Irf8 and PU.1 deficiency block later differentiation towards monocyte or granulocyte fates. Our transcriptional map defines a reference network model for blood progenitors and their differentiation trajectories during normal and perturbed haematopoiesis.

Enhancer DNA methylation in acute myeloid leukemia and myelodysplastic syndromes.

DNA methylation (CpG methylation) exerts an important role in normal differentiation and proliferation of hematopoietic stem cells and their differentiated progeny, while it has also the ability to regulate myeloid versus lymphoid fate. Mutations of the epigenetic machinery are observed in hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) resulting in hyper- or hypo-methylation affecting several different pathways. Enhancers are cis-regulatory elements which promote transcription activation and are characterized by histone marks including H3K27ac and H3K4me1/2. These gene subunits are target gene expression 'fine-tuners', are differentially used during the hematopoietic differentiation, and, in contrast to promoters, are not shared by the different hematopoietic cell types. Although the interaction between gene promoters and DNA methylation has extensively been studied, much less is known about the interplay between enhancers and DNA methylation. In hematopoiesis, DNA methylation at enhancers has the potential to discriminate between fetal and adult erythropoiesis, and also is a regulatory mechanism in granulopoiesis through repression of neutrophil-specific enhancers in progenitor cells during maturation. The interplay between DNA methylation at enhancers is disrupted in AML and MDS and mainly hyper-methylation at enhancers raising early during myeloid lineage commitment is acquired during malignant transformation. Interactions between mutated epigenetic drivers and other oncogenic mutations also affect enhancers' activity with final result, myeloid differentiation block. In this review, we have assembled recent data regarding DNA methylation and enhancers' activity in normal and mainly myeloid malignancies.

Critical Modulation of Hematopoietic Lineage Fate by Hepatic Leukemia Factor

SummaryA gradual restriction in lineage potential of multipotent stem/progenitor cells is a hallmark of adult hematopoiesis, but the underlying molecular events governing these processes remain incompletely understood. Here, we identified robust expression of the leukemia-associated transcription factor hepatic leukemia factor (Hlf) in normal multipotent hematopoietic progenitors, which was rapidly downregulated upon differentiation. Interference with its normal downregulation revealed Hlf as a strong negative regulator of lymphoid development, while remaining compatible with myeloid fates. Reciprocally, we observed rapid lymphoid commitment upon reduced Hlf activity. The arising phenotypes resulted from Hlf binding to active enhancers of myeloid-competent cells, transcriptional induction of myeloid, and ablation of lymphoid gene programs, with Hlf induction of nuclear factor I C (Nfic) as a functionally relevant target gene. Thereby, our studies establish Hlf as a key regulator of the earliest lineage-commitment events at the transition from multipotency to lineage-restricted progeny, with implications for both normal and malignant hematopoiesis.

Bcl11b and combinatorial resolution of cell fate in the T-cell gene regulatory network

T-cell development from hematopoietic progenitors depends on multiple transcription factors, mobilized and modulated by intrathymic Notch signaling. Key aspects of T-cell specification network architecture have been illuminated through recent reports defining roles of transcription factors PU.1, GATA-3, and E2A, their interactions with Notch signaling, and roles of Runx1, TCF-1, and Hes1, providing bases for a comprehensively updated model of the T-cell specification gene regulatory network presented herein. However, the role of lineage commitment factor Bcl11b has been unclear. We use self-organizing maps on 63 RNA-seq datasets from normal and perturbed T-cell development to identify functional targets of Bcl11b during commitment and relate them to other regulomes. We show that both activation and repression target genes can be bound by Bcl11b in vivo, and that Bcl11b effects overlap with E2A-dependent effects. The newly clarified role of Bcl11b distinguishes discrete components of commitment, resolving how innate lymphoid, myeloid, and dendritic, and B-cell fate alternatives are excluded by different mechanisms.

PD-1 blockade alters fate commitment of myeloid progenitors during tumor-mediated “emergency” myelopoiesis

Abstract PD-1 is an immune inhibitory receptor for which blocking agents have achieved significant success as anti-cancer therapeutics. The mechanism(s) of the how PD-1 compromises anti-tumor function and how such effect is reversed by PD-1 blockade remain poorly understood. The rapid change in hematopoietic output that occurs in response to immunologic stress is known as “emergency” myelopoiesis. This process is co-opted by tumors to enhance the generation of tumor-promoting myeloid cells, mostly undifferentiated myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). We analyzed the myeloid compartment of tumor-bearing mice and determined that myeloid cells express PD-1 and PD-L1. Using PD-1 KO mice or WT mice treated with PD-1 blocking antibody in cancer-driven myelopoiesis experiments we determined a cell-switch fate from MDSCs to effector myeloid cells. PD-1 deletion or blockade, which impaired tumor growth and metastases, stimulated the differentiation of hematopoietic progenitor cells and resulted in increase of Ly6Chimonocytes and monocyte-derived DC (mo-DC). Abrogation of PD-1 signaling enhanced the maturation of MDSCs by inducing the lineage commitment transcription factors IRF8, IRF4 and RORC1. IRF8 and IRF4 promote monocytes/macrophage and hinder granulocyte differentiation, whereas RORC1 is required for expansion of Ly6Chi monocytes and resolution of inflammation. These results reveal a role of the PD-1:PD-L1 pathway in the differentiation of lineage-committed myeloid progenitors to promote undifferentiated MDSCs and suppress effector monocytes/DCs. Switch of myeloid progenitor fate commitment might be a key mechanism by which PD-1 blockade mediates anti-tumor function.

Quo vadis hematology?

For decades, developing hematopoietic cells have been strictly compartmentalized into a small population of multipotent self-renewing hematopoietic stem cells, multipotent hematopoietic progenitor cells that are undergoing commitment to myeloid or lymphoid fates, and unipotent precursor cells that mature towards peripheral blood and immune cells. Recent studies, however, have provided a battery of findings that cannot be explained by this "classical" hierarchical model for the architecture of hematopoiesis. It is emerging that heterogeneous hematopoietic stem cell populations in the bone marrow coexist, each with distinct, preprogrammed differentiation and proliferation behaviors. Three subsets can be distinguished among them: myeloid-biased (α), balanced (β), and lymphoid-biased (γ/δ) hematopoietic stem cells. The ratio of these hematopoietic stem cell subsets is developmentally regulated in the foetal liver and hematopoietic stem cells adult bone marrow, and coordinately gives rise to hematopoiesis. Beta- and γ/δ-hematopoietic stem cells are found predominantly early in the life of an organism, whereas α-hematopoietic stem cells accumulate in aged mice and humans. In addition, new sophisticated genetic experiments in mice have identified a major role of long-lived, committed progenitor cells downstream from hematopoietic stem cells as drivers of normal adult hematopoiesis, and revealed that post-transplantation hematopoiesis differs qualitatively and quantitatively from normal steady-state hematopoiesis. These findings have important implications for understanding in situ the regulation of haematopoiesis in health and disease. Orv. Hetil., 2016, 157(46), 1819-1829.

Zbtb1 prevents default myeloid differentiation of lymphoid-primed multipotent progenitors.

Zbtb1 is a transcription factor that prevents DNA damage and p53-mediated apoptosis in replicating immune progenitors, affecting lymphoid as well as myeloid development when hematopoietic progenitors are in competition in mixed bone marrow chimeras. However, Zbtb1-deficient mice do not have an apparent myeloid deficiency. We report here that Zbtb1-deficient lymphoid-primed multipotent progenitors (LMPPs) are biased to develop towards the myeloid fate in detriment of lymphoid development, contributing to the apparent unaffected myeloid development. Zbtb1 expression was maintained during lymphoid development of LMPP cells but downregulated during myeloid development. Deficiency of Zbtb1 in LMPP cells was sufficient to direct a myeloid fate in lymphoid-inducing conditions and in the absence of myeloid cytokines as shown by upregulation of a myeloid gene signature and the generation of myeloid cells in vitro. Finally, biased myeloid differentiation of Zbtb1-deficient LMPP cells was not due to increased p53-dependent apoptosis as it was not reverted by transgenic Bcl2 expression or p53 deficiency. Altogether, our results show that Zbtb1 expression prevents activation of a default myeloid program in LMPP cells, ensuring the generation of lymphoid cells.

MEF2C and EBF1 Co-regulate B Cell-Specific Transcription.

Hematopoietic stem cells are capable of self-renewal or differentiation along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for blood progenitor cells is whether to adopt the lymphoid or myeloid fate. Previous work had shown that myocyte enhancer factor 2C (MEF2C) is indispensable for the lymphoid fate decision, yet the specific mechanism of action remained unclear. Here, we have identified early B cell factor-1 (EBF1) as a co-regulator of gene expression with MEF2C. A genome-wide survey of MEF2C and EBF1 binding sites identified a subset of B cell-specific genes that they target. We also determined that the p38 MAPK pathway activates MEF2C to drive B cell differentiation. Mef2c knockout mice showed reduced B lymphoid-specific gene expression as well as increased myeloid gene expression, consistent with MEF2C’s role as a lineage fate regulator. This is further supported by interaction between MEF2C and the histone deacetylase, HDAC7, revealing a likely mechanism to repress the myeloid transcription program. This study thus elucidates both activation and repression mechanisms, identifies regulatory partners, and downstream targets by which MEF2C regulates lymphoid-specific differentiation.

Interleukin-1 Drives Precocious Myeloid Differentiation of Hematopoietic Stem Cells at the Expense of Self-Renewal

Hematopoietic stem cells (HSCs) maintain lifelong blood homeostasis. While many of the cell-intrinsic mechanisms regulating HSC function at steady state have been well characterized, the role of inflammatory cytokines and other environmental factors in tailoring blood production following physiological insults has become a topic of emerging interest. The cytokine interleukin-1 (IL-1) is a prototypical pro-inflammatory cytokine that plays a key role in host inflammatory responses to injury and infection, and is associated with elevated myeloid cell production. Importantly, IL-1 also drives a wide range of chronic inflammatory conditions such as diabetes, obesity, and arthritis that are often characterized by deregulated blood homeostasis. Here, we show at single-cell resolution using continuous tracking technology that IL-1 drives accelerated HSC cell division kinetics and myeloid differentiation via the rapid activation of a precocious PU.1-dependent myeloid gene program. Activation of this program requires direct IL-1R signaling and subsequent activation of IKK kinases, and instructively primes HSCs to adopt a myeloid fate. Strikingly, we demonstrate that IL-1 produced by myeloid cells and endothelial cells of the bone marrow (BM) niche exerts similar effects in vivo, and is required for efficient myeloid recovery following acute challenges such as transplantation or myeloablation. On the other hand, we find that chronic IL-1 exposure substantially remodels HSC blood output, resulting in myeloid overproduction and expansion of myeloid-biased multipotent progenitor (MPP) compartments at the expense of lymphoid and erythroid lineages. Critically, chronic IL-1 erodes HSC self-renewal, significantly impairing their regenerative capacity following transplantation. On the other hand, chronically exposed HSCs recover their function upon IL-1 withdrawal. Collectively, these findings identify IL-1 as a critical regulator of HSC fate and lineage specification via activation of a PU.1 circuit. They also demonstrate a role for IL-1 as a double-edged sword in HSC biology, promoting HSC regeneration in response to acute insults while severely disrupting HSC self-renewal and lineage output during chronic exposure, hence identifying IL-1 as an important and therapeutically targetable factor underwriting myeloid overproduction and other deregulations that contribute to the pathogenesis of a variety of chronic inflammatory diseases and blood disorders. DisclosuresNo relevant conflicts of interest to declare.

Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.

In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34(+) cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult "two-tier" hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.

Irf4 Regulates the Choice between T Lymphoid-Primed Progenitor and Myeloid Lineage Fates during Embryogenesis

T lymphoid-primed progenitors are hematopoietic progenitors destined to enter the thymus. The in vivo characterization of these embryonic progenitors is challenging, however, due to the intrauterine development of mouse embryos. Thus, how the fate of these cells is determined has not been fully defined in mammals. Here we use zebrafish embryos to show that the homing of T lymphoid-primed progenitors to the thymus is impaired, concomitant with a decrease in ccr9a expression, in the absence of irf4a. Strikingly, fate mapping assays at the single-cell level showed a fate change of irf4a-deficient T lymphoid-primed progenitors to myeloid cells, accompanied by an increase in Pu.1 expression. These data indicate that in addition to regulating ccr9a expression, Irf4a is essential in T lymphoid-primed progenitors for repressing Pu.1 expression to prevent an alternate fate. Our findings provide insight into the fate determination mechanism of T lymphoid-primed progenitors.

Mechanisms of MEF2C-Dependent Transcription in Lymphoid Differentiation

Hematopoiesis is a multi-step developmental process that requires an intricate coordination of signal relays and transcriptional regulation to give rise to all blood lineages in the organism. Hematopoietic stem/progenitor cells (HSPCs) can be driven to differentiate along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for HSPCs is whether to adopt the lymphoid or myeloid fate. Despite the discovery of several transcription factors required for different lineages of hematopoietic differentiation, the understanding of how gene expression allows HSPCs to adopt the lymphoid fate still remains incomplete. A study using an inducible hematopoietic-specific (Mx1-Cre) KO mouse line found that Myocyte Enhancer Factor 2C (MEF2C) is required for multi-potent HSPCs to differentiate into the lymphoid lineage (Stehling-Sun et al, 2009). However, the mechanisms of how MEF2C is activated and in turn, drives lymphoid fate specification are not known.Through a candidates approach with co-expression and co-immunoprecipitation, we have identified Early B Cell Factor 1 (EBF1) to be a specific interacting partner of MEF2C, and not other B cell specific transcription factors such as E12, E47, or PAX5. Genome-wide survey of MEF2C and EBF1 binding sites via chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in a proB cell line revealed that these two sequence-specific transcription factors co-occupy the promoters and intragenic regions of many B cell specific genes such as Il7ra, Myb, Foxo1, Ets1, Ebf1 itself, and Pou2af1. Regulatory regions of Il7ra and Foxo1 derived from the ChIP-seq data, as well as an artificial enhancer containing trimerized MEF2C and EBF1 binding sites, were examined in luciferase reporter assays and found to be sufficient to drive transcription from a minimal reporter in 293T cells. Further, this activation was co-dependent on MEF2C and EBF1 expression. The functional relevance of MEF2C binding was further supported by gene expression analyses of MEF2C-regulated B lineage genes in Mx1-Cre Mef2c KO mice compared to WT mice. Consistent with ChIP-seq and luciferase reporter assays, Myb, Ebf1, Il7ra, and Foxo1 all had significantly decreased expression levels in MEF2C-null HSPCs as well as B lineage progenitor cells, compared to sex-matched littermate control mice.Interestingly, myeloid gene expression in Mef2c-KO mice was increased compared to WT control. MEF2C ChIP-seq in a murine HSPC line revealed that it binds myeloid lineage gene targets that are not regulated by MEF2C in proB cells. These results suggest that MEF2C can repress myeloid gene expression in HSPCs. To elucidate the mechanism of this functional switch, we tested the requirement for MAPK pathways to phosphorylate and activate MEF2C at three previously identified residues in order to drive B cell differentiation. Inhibition of p38 MAPK (p38i), but not ERK1/2/5, decreased the potential of HSPCs to differentiate into B220+CD19+ B cells cultured with cytokines that drive this particular lineage fate. Instead, p38i-treated progenitor cells gave rise to more myeloid cells. 65% of this phenotype was rescued by over-expressing a phosphomimetic mutant of MEF2C that can bypass p38 inhibition. Furthermore, MEF2C is known to bind class II HDAC proteins to repress gene expression, providing a possible mechanism for its repression of myeloid transcription program. Supporting this mechanism, phosphomimetic and HDAC-binding double mutant of MEF2C can rescue p38 inhibition phenotype almost 100%.Taken together, this study elucidated the molecular mechanisms of a key lymphoid-specific lineage fate determinant, MEF2C. We discovered that p38 MAPK converts MEF2C from a transcriptional repressor to an activator by phosphorylation in B cell specification, which can be applied to understanding other cell differentiation processes regulated by this important stress-induced signaling pathway. Furthermore, we identified MEF2C’s binding and co-activating partner EBF1, several novel B cell specific targets that it activates in proB cells, and a novel myeloid transcription program that it represses in hematopoietic progenitors. Therefore, these results expanded our understanding of the intricate transcription network that regulates B cell differentiation. DisclosuresNo relevant conflicts of interest to declare.

Twist-1 Increases Hematopoietic Stem Cell Self-Renewal and Causes Myeloid Skewing

Twist-1 protein belongs to the large family of basic Helix-Loop-Helix transcription factors with diverse physiological functions in mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, there is little information available regarding its expression pattern and functional role in the hematopoietic system. We analyzed Twist-1 expression patterns in different hematopoietic cell populations from normal mouse bone marrow, and found that Twist-1 was most highly expressed in long-term hematopoietic stem cells (LT-HSCs) but showed a low abundance in more differentiated descendants. To investigate Twist-1 gene function, retroviral-mediated overexpression or removal experiments were performed. Competitive repopulation studies demonstrated that enforced expression of Twist-1 in HSC-enriched Lin−c-Kit+Sca-1+ (LKS) cells resulted in an increase in the size of the G0 population, and in their reconstitution ability after the first and a second transplantation. Conversely, removal of Twist-1 in LKS cells impaired their ability to repopulate. In addition, increased Twist-1 expression caused a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activated myeloid lineage-determining factors PU.1 and GATA-1 and down-regulated lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms revealed that Twist-1 overexpression lead to altered expression of Runx1/c-Mpl/Tie2 regulatory pathway, and quiescence-associated N-cadherin and Hes1 in LKS cells, which may contribute to the phenotypes observed in Twist-1-overexpressing mice. These studies shed additional light on the mechanisms involved in the maintenance of normal HSC and myeloid lineage differentiation, and may also provide clues to the mechanisms controlling pathogenesis and preservation of leukemic stem cells. DisclosuresNo relevant conflicts of interest to declare.

Genome-Scale Analysis of the Pu.1 Driven Transcriptional Programme That Overcomes the Differentiation Block in a Novel Pu.1-Inducible AML Cell Line

Transcription factors (TFs) play a key role in determining normal haematopoiesis; haematological cancers often being characterised by TF dysregulation. Pu.1 is a TF that plays a major role in myeloid differentiation, with mutations and down regulation of Pu.1 activity described in human acute myeloid leukemia (AML). To investigate the role of Pu.1 in normal and leukemic myeloid fate, we developed a model of inducible Pu.1 rescue in a murine AML cell line, and utilised it to assess genome-wide Pu.1-DNA-binding profiles and the subsequent effects on histone remodelling, gene expression and binding of the cooperative TF CEBPA.We used a previously described radiation-induced murine AML cell line known to be deficient in functional Pu.1 (Cook et al, Blood 2004; 104:3437-3444) and transduced cells with 4-hydroxy-tamoxifen (OHT)-inducible Pu.1 (PuER) or empty vector (EV). Clonal populations were produced by limiting dilution, and genotype and protein phenotype confirmed by PCR and Western blotting respectively. Incubation in 100nM OHT caused cessation of proliferation, and increased expression of the maturation markers CD11b and Gr-1.To assess the effects of Pu.1 binding, cells were incubated for 2 hours in 100nM OHT and cross-linked chromatin harvested for ChIP with antibodies against Pu.1, CEBPA and H3K27Ac. ChIP samples were amplified and sequenced, reads were mapped to the mouse reference genome, binding peaks identified, and binding levels normalised for peak length and total read count. RNA from was extracted from the same samples and analysed by microarray expression profiling.Binding peak analysis revealed an increase in Pu.1 binding sites from 5283 to 15675 following Pu.1 induction, showing that the mutant Pu.1 is able to perform limited DNA-binding, but not in a manner sufficient to induce transcription of genes necessary for normal differentiation. To investigate the effect of Pu.1 binding on histone remodelling, we selected a set of 613 peaks with ≥4-fold increase in Pu.1 read count, ≥4fold increase in H3K27Ac read count, and post-induction minimal H3K27Ac read count of ≥50 reads/region. Interestingly, these highly “dynamic” Pu.1 binding peaks were restricted to non-promoter regions, suggesting increased binding at distal enhancer sites.These 613 peaks mapped to 553 genes, which showed a small but significant overexpression post-Pu.1 induction, compared to genes adjacent to peaks with no change in Pu.1 (p = 5.357e-16). These genes were intersected with the 284 genes overexpressed in the gene expression array post Pu.1-induction, producing a list of 46 genes of relevant Pu.1 targets. Using Gene Ontology and Functional Term Enrichment analysis, we discovered a significant enrichment for genes overexpressed in bone marrow macrophages post-lipopolysaccharide stimulation (z score = -2.19). Investigating the effect of Pu.1 induction on CEBPA binding, we found that increased Pu.1 binding was enriched in CEBPA peaks which were ≥4fold increased post-Pu.1 induction (p< 0.001), suggesting recruitment of CEBPA in a significant proportion of regions. Further evidence of the relationship between Pu.1 and CEBPA comes from motif analysis of the ≥4fold Pu.1 peaks which showed CEBP motifs present in 30.0% of targets. Similarly, analysis of ≥4fold CEBPA peaks showed Ets motifs in 34.5% of these peaks with ≥ 4fold Pu.1 binding, but only 7.5% in those without.In conclusion, we present a novel model system where we have examined at genome-scale the effects of Pu.1-DNA binding to overcome the differentiation block in a Pu.1-deficient AML model. Functional Pu.1 binding rapidly induces changes in H3K27Ac profile at key enhancer sites, which subsequently drives the cell into a maturation pathway. The interaction between Pu.1 and CEBPA is also confirmed, with evidence of Pu.1 recruiting CEBPA to DNA-binding sites. DisclosuresNo relevant conflicts of interest to declare.

Twist-1, a novel regulator of hematopoietic stem cell self-renewal and myeloid lineage development.

Transcription factor Twist-1 plays essential roles in specification and differentiation of mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, the role of Twist-1 in hematopoietic stem cell (HSC) remains largely uncharacterized. We report that Twist-1 is more highly expressed in murine HSC and its expression declines with differentiation. To investigate Twist-1 gene function, retroviral-mediated overexpression or removal experiments are performed. Competitive repopulation studies demonstrate that enforced expression of Twist-1 in HSC-enriched Lin(-) c-Kit(+) Sca-1(+) (LKS) cells results in an increase in the size of the G(0) population, and in their reconstitution ability after the first and a second transplantation. Conversely, removal of Twist-1 in LKS cells impairs their ability to repopulate. In addition, increased Twist-1 expression causes a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activates myeloid lineage-determining factors PU.1 and GATA-1 and downregulates lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms reveals that Runx1/c-Mpl/Tie2 regulatory pathway could possibly account for the observed effects caused by Twist-1 overexpression. Our study provides the first evidence supporting a role for Twist-1 in hematopoiesis.