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Myeloid cells are known to suppress antitumour immunity1. However, the molecular drivers of immunosuppressive myeloid cell states are not well defined. Here we used single-cell RNA sequencing of human and mouse non-small cell lung cancer (NSCLC) lesions, and found that in both species the type 2 cytokine interleukin-4 (IL-4) was predicted to be the primary driver of the tumour-infiltrating monocyte-derived macrophage phenotype. Using a panel of conditional knockout mice, we found that only deletion of the IL-4 receptor IL-4Rα in early myeloid progenitors in bone marrow reduced tumour burden, whereas deletion of IL-4Rα in downstream mature myeloid cells had no effect. Mechanistically, IL-4 derived from bone marrow basophils and eosinophils acted on granulocyte-monocyte progenitors to transcriptionally programme the development of immunosuppressive tumour-promoting myeloid cells. Consequentially, depletion of basophils profoundly reduced tumour burden and normalized myelopoiesis. We subsequently initiated a clinical trial of the IL-4Rα blocking antibody dupilumab2–5given in conjunction with PD-1/PD-L1 checkpoint blockade in patients with relapsed or refractory NSCLC who had progressed on PD-1/PD-L1 blockade alone (ClinicalTrials.gov identifier NCT05013450). Dupilumab supplementation reduced circulating monocytes, expanded tumour-infiltrating CD8 T cells, and in one out of six patients, drove a near-complete clinical response two months after treatment. Our study defines a central role for IL-4 in controlling immunosuppressive myelopoiesis in cancer, identifies a novel combination therapy for immune checkpoint blockade in humans, and highlights cancer as a systemic malady that requires therapeutic strategies beyond the primary disease site.

Definitive haematopoiesis is the process by which haematopoietic stem cells, located in the bone marrow, generate all haematopoietic cell lineages in healthy adults. Although highly regulated to maintain a stable output of blood cells in health, the haematopoietic system is capable of extensive remodelling in response to external challenges, prioritizing the production of certain cell types at the expense of others. In this Review, we consider how acute insults, such as infections and cytotoxic drug-induced myeloablation, cause molecular, cellular and metabolic changes in haematopoietic stem and progenitor cells at multiple levels of the haematopoietic hierarchy to drive accelerated production of the mature myeloid cells needed to resolve the initiating insult. Moreover, we discuss how dysregulation or subversion of these emergency myelopoiesis mechanisms contributes to the progression of chronic inflammatory diseases and cancer.

Hematopoietic stem cells (HSCs), which govern the production of all blood lineages, transition through a series of functional states characterized by expansion during fetal development, functional quiescence in adulthood, and decline upon aging. We describe central features of HSC regulation during ontogeny to contextualize how adaptive responses over the life of the organism ultimately form the basis for HSC functional degradation with age. We particularly focus on the role of cell cycle regulation, inflammatory response pathways, epigenetic changes, and metabolic regulation. We then explore how the knowledge of age-related changes in HSC regulation can inform strategies for the rejuvenation of old HSCs.

Haematopoietic ageing is marked by a loss of regenerative capacity and skewed differentiation from haematopoietic stem cells (HSCs), leading to impaired blood production. Signals from the bone marrow niche tailor blood production, but the contribution of the old niche to haematopoietic ageing remains unclear. Here we characterize the inflammatory milieu that drives both niche and haematopoietic remodelling. We find decreased numbers and functionality of osteoprogenitors at the endosteum and expansion of central marrow LepR+mesenchymal stromal cells associated with deterioration of the sinusoidal vasculature. Together, they create a degraded and inflamed old bone marrow niche. Niche inflammation in turn drives the chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors, which promotes myeloid differentiation and hinders haematopoietic regeneration. Moreover, we show how production of interleukin-1β (IL-1β) by the damaged endosteum acts in transto drive the proinflammatory nature of the central marrow, with damaging consequences for the old blood system. Notably, niche deterioration, HSC dysfunction and defective regeneration can all be ameliorated by blocking IL-1 signalling. Our results demonstrate that targeting IL-1 as a key mediator of niche inflammation is a tractable strategy to improve blood production during ageing.

Autophagy is central to the benefits of longevity signaling programs and to hematopoietic stem cell (HSC) response to nutrient stress. With age, a subset of HSCs increases autophagy flux and preserves regenerative capacity, but the signals triggering autophagy and maintaining the functionality of autophagy-activated old HSCs (oHSCs) remain unknown. Here, we demonstrate that autophagy is an adaptive cytoprotective response to chronic inflammation in the aging murine bone marrow (BM) niche. We find that inflammation impairs glucose uptake and suppresses glycolysis in oHSCs through Socs3-mediated inhibition of AKT/FoxO-dependent signaling, with inflammation-mediated autophagy engagement preserving functional quiescence by enabling metabolic adaptation to glycolytic impairment. Moreover, we show that transient autophagy induction via a short-term fasting/refeeding paradigm normalizes glycolytic flux and significantly boosts oHSC regenerative potential. Our results identify inflammation-driven glucose hypometabolism as a key driver of HSC dysfunction with age and establish autophagy as a targetable node to reset oHSC regenerative capacity.

Haematopoiesis is governed by haematopoietic stem cells (HSCs) that produce all lineages of blood and immune cells. The maintenance of blood homeostasis requires a dynamic response of HSCs to stress, and dysregulation of these adaptive–response mechanisms underlies the development of myeloid leukaemia. Leukaemogenesis often occurs in a stepwise manner, with genetic and epigenetic changes accumulating in pre-leukaemic HSCs prior to the emergence of leukaemic stem cells (LSCs) and the development of acute myeloid leukaemia. Clinical data have revealed the existence of age-related clonal haematopoiesis, or the asymptomatic clonal expansion of mutated blood cells in the elderly, and this phenomenon is connected to susceptibility to leukaemic transformation. Here we describe how selection for specific mutations that increase HSC competitive fitness, in conjunction with additional endogenous and environmental changes, drives leukaemic transformation. We review the ways in which LSCs take advantage of normal HSC properties to promote survival and expansion, thus underlying disease recurrence and resistance to conventional therapies, and we detail our current understanding of leukaemic ‘stemness’ regulation. Overall, we link the cellular and molecular mechanisms regulating HSC behaviour with the functional dysregulation of these mechanisms in myeloid leukaemia and discuss opportunities for targeting LSC-specific mechanisms for the prevention or cure of malignant diseases.

Although the role of promyelocytic leukemia/retinoic acid receptor α (PML/RARA) fusion protein is well recognized in acute promyelocytic leukemia (APL), its contribution to initiation and maintenance of leukemogenesis is not completely understood. Transcriptome analysis in the murine MRP8-PML/RARAAPL model has demonstrated modest alterations in gene expression accompanied by expansion of the promyelocyte compartment. Of particular interest, mice expressing PML/RARA showed downregulation of the transcription factor Irf8mRNA. Interferon regulatory factor 8 (IRF8) is a known regulator of hematopoiesis. Previous research had implicated IRF8 as a tumor suppressor for myeloid neoplasia, and mice lacking IRF8 develop a well-differentiated myeloproliferative neoplasm characterized by expansion of neutrophilic lineage cells. We hypothesized that PML/RARA-mediated downregulation of Irf8transcript levels contributes to the initiation of APL. We observed significant downregulation of IRF8 protein levels in highly purified promyelocyte populations of PML/RARA transgenic mice. We also found that loss of IRF8 results in expansion of promyelocytes in vivo, partially phenocopying the impact of PML/RARA expression. Moreover, survival experiments showed that complete loss of IRF8 leads to acceleration of APL onset in our PML/RARA mice. Collectively, these data identify IRF8 downregulation as an important factor in APL initiation and highlight a tumor-suppressor role for IRF8 in this acute leukemia.

Hematopoietic stem cells (HSC) and downstream lineage-biased multipotent progenitors (MPP) tailor blood production and control myelopoiesis on demand. Recent lineage tracing analyses revealed MPPs to be major functional contributors to steady-state hematopoiesis. However, we still lack a precise resolution of myeloid differentiation trajectories and cellular heterogeneity in the MPP compartment. Here, we found that myeloid-biased MPP3 are functionally and molecularly heterogeneous, with a distinct subset of myeloid-primed secretory cells with high endoplasmic reticulum (ER) volume and FcγR expression. We show that FcγR+/ERhigh MPP3 are a transitional population serving as a reservoir for rapid production of granulocyte/macrophage progenitors (GMP), which directly amplify myelopoiesis through inflammation-triggered secretion of cytokines in the local bone marrow (BM) microenvironment. Our results identify a novel regulatory function for a secretory MPP3 subset that controls myeloid differentiation through lineage-priming and cytokine production and acts as a self-reinforcing amplification compartment in inflammatory stress and disease conditions.

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1β, G-CSF, TGFβ and CXCL4) and activation of an inducible Irf8 and β-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

With age, haematopoietic stem cells lose their ability to regenerate the blood system, and promote disease development. Autophagy is associated with health and longevity, and is critical for protecting haematopoietic stem cells from metabolic stress. Here we show that loss of autophagy in haematopoietic stem cells causes accumulation of mitochondria and an activated metabolic state, which drives accelerated myeloid differentiation mainly through epigenetic deregulations, and impairs haematopoietic stem-cell self-renewal activity and regenerative potential. Strikingly, most haematopoietic stem cells in aged mice share these altered metabolic and functional features. However, approximately one-third of aged haematopoietic stem cells exhibit high autophagy levels and maintain a low metabolic state with robust long-term regeneration potential similar to healthy young haematopoietic stem cells. Our results demonstrate that autophagy actively suppresses haematopoietic stem-cell metabolism by clearing active, healthy mitochondria to maintain quiescence and stemness, and becomes increasingly necessary with age to preserve the regenerative capacity of old haematopoietic stem cells.

Many tissues and organ systems in metazoans have the intrinsic capacity to regenerate, which is driven and maintained largely by tissue-resident somatic stem cell populations. Ageing is accompanied by a deregulation of stem cell function and a decline in regenerative capacity, often resulting in degenerative diseases. The identification of strategies to maintain stem cell function and regulation is therefore a promising avenue to allay a wide range of age-related diseases. Studies in various organisms have revealed a central role for metabolic pathways in the regulation of stem cell function. Ageing is associated with extensive metabolic changes, and interventions that influence cellular metabolism have long been recognized as robust lifespan-extending measures. In this Review, we discuss recent advances in our understanding of the metabolic control of stem cell function, and how stem cell metabolism relates to homeostasis and ageing.

Haematopoietic stem cells (HSCs) maintain lifelong blood production and increase blood cell numbers in response to chronic and acute injury. However, the mechanism(s) by which inflammatory insults are communicated to HSCs and their consequences for HSC activity remain largely unknown. Here, we demonstrate that interleukin-1 (IL-1), which functions as a key pro-inflammatory ‘emergency’ signal, directly accelerates cell division and myeloid differentiation of HSCs through precocious activation of a PU.1-dependent gene program. Although this effect is essential for rapid myeloid recovery following acute injury to the bone marrow, chronic IL-1 exposure restricts HSC lineage output, severely erodes HSC self-renewal capacity, and primes IL-1-exposed HSCs to fail massive replicative challenges such as transplantation. Importantly, these damaging effects are transient and fully reversible on IL-1 withdrawal. Our results identify a critical regulatory circuit that tailors HSC responses to acute needs, and is likely to underlie deregulated blood homeostasis in chronic inflammation conditions.

Despite great advances in understanding the mechanisms underlying blood production, lineage specification at the level of multipotent progenitors (MPPs) remains poorly understood. Here, we show that MPP2 and MPP3 are distinct myeloid-biased MPP subsets that work together with lymphoid-primed MPP4 cells to control blood production. We find that all MPPs are produced in parallel by hematopoietic stem cells (HSCs), but with different kinetics and at variable levels depending on hematopoietic demands. We also show that the normally rare myeloid-biased MPPs are transiently overproduced by HSCs in regenerating conditions, hence supporting myeloid amplification to rebuild the hematopoietic system. This shift is accompanied by a reduction in self-renewal activity in regenerating HSCs and reprogramming of MPP4 fate toward the myeloid lineage. Our results support a dynamic model of blood development in which HSCs convey lineage specification through independent production of distinct lineage-biased MPP subsets that, in turn, support lineage expansion and differentiation.

Hematopoietic stem cells (HSCs) rely on instructive cues from the bone marrow (BM) niche to maintain their quiescence and adapt blood production to the organism’s needs. Alterations in the BM niche are commonly observed in blood malignancies and directly contribute to the aberrant function of disease-initiating leukemic stem cells (LSCs). Here, we review recent insights into the cellular and molecular determinants of the normal HSC niche and describe how genetic changes in stromal cells and leukemia-induced BM niche remodeling contribute to blood malignancies. Moreover, we discuss how these findings can be applied to non-cell-autonomous therapies targeting the LSC niche.

Type I interferons (IFN-1s) are antiviral cytokines that suppress blood production while paradoxically inducing hematopoietic stem cell (HSC) proliferation. Here, we clarify the relationship between the proliferative and suppressive effects of IFN-1s on HSC function during acute and chronic IFN-1 exposure. We show that IFN-1–driven HSC proliferation is a transient event resulting from a brief relaxation of quiescence-enforcing mechanisms in response to acute IFN-1 exposure, which occurs exclusively in vivo. We find that this proliferative burst fails to exhaust the HSC pool, which rapidly returns to quiescence in response to chronic IFN-1 exposure. Moreover, we demonstrate that IFN-1–exposed HSCs with reestablished quiescence are largely protected from the killing effects of IFNs unless forced back into the cell cycle due to culture, transplantation, or myeloablative treatment, at which point they activate a p53-dependent proapoptotic gene program. Collectively, our results demonstrate that quiescence acts as a safeguard mechanism to ensure survival of the HSC pool during chronic IFN-1 exposure. We show that IFN-1s can poise HSCs for apoptosis but induce direct cell killing only upon active proliferation, thereby establishing a mechanism for the suppressive effects of IFN-1s on HSC function.

Multipotent stromal cells (MSCs) and their osteoblastic lineage cell (OBC) derivatives are part of the bone marrow (BM) niche and contribute to hematopoietic stem cell (HSC) maintenance. Here, we show that myeloproliferative neoplasia (MPN) progressively remodels the endosteal BM niche into a self-reinforcing leukemic niche that impairs normal hematopoiesis, favors leukemic stem cell (LSC) function, and contributes to BM fibrosis. We show that leukemic myeloid cells stimulate MSCs to overproduce functionally altered OBCs, which accumulate in the BM cavity as inflammatory myelofibrotic cells. We identify roles for thrombopoietin, CCL3, and direct cell-cell interactions in driving OBC expansion, and for changes in TGF-β, Notch, and inflammatory signaling in OBC remodeling. MPN-expanded OBCs, in turn, exhibit decreased expression of many HSC retention factors and severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of MPN-affected patients and prevention of myelofibrosis.

Adult hematopoiesis occurs primarily in the BM space where hematopoietic cells interact with stromal niche cells. Despite this close association, little is known about the specific roles of osteoblastic lineage cells (OBCs) in maintaining hematopoietic stem cells (HSCs), and how conditions affecting bone formation influence HSC function. Here we use a transgenic mouse model with the ColI(2.3) promoter driving a ligand-independent, constitutively active 5HT4 serotonin receptor (Rs1) to address how the massive increase in trabecular bone formation resulting from increased Gs signaling in OBCs impacts HSC function and blood production. Rs1 mice display fibrous dysplasia, BM aplasia, progressive loss of HSC numbers, and impaired megakaryocyte/erythrocyte development with defective recovery after hematopoietic injury. These hematopoietic defects develop without compensatory extramedullary hematopoiesis, and the loss of HSCs occurs despite a paradoxical expansion of stromal niche cells with putative HSC-supportive activity (ie, endothelial, mesenchymal, and osteoblastic cells). However, Rs1-expressing OBCs show decreased expression of key HSC-supportive factors and impaired ability to maintain HSCs. Our findings indicate that long-term activation of Gs signaling in OBCs leads to contextual changes in the BM niche that adversely affect HSC maintenance and blood homeostasis.

Adult hematopoiesis occurs primarily in the BM space where hematopoietic cells interact with stromal niche cells. Despite this close association, little is known about the specific roles of osteoblastic lineage cells (OBCs) in maintaining hematopoietic stem cells (HSCs), and how conditions affecting bone formation influence HSC function. Here we use a transgenic mouse model with the ColI(2.3) promoter driving a ligand-independent, constitutively active 5HT4 serotonin receptor (Rs1) to address how the massive increase in trabecular bone formation resulting from increased Gssignaling in OBCs impacts HSC function and blood production. Rs1 mice display fibrous dysplasia, BM aplasia, progressive loss of HSC numbers, and impaired megakaryocyte/erythrocyte development with defective recovery after hematopoietic injury. These hematopoietic defects develop without compensatory extramedullary hematopoiesis, and the loss of HSCs occurs despite a paradoxical expansion of stromal niche cells with putative HSC-supportive activity (ie, endothelial, mesenchymal, and osteoblastic cells). However, Rs1-expressing OBCs show decreased expression of key HSC-supportive factors and impaired ability to maintain HSCs. Our findings indicate that long-term activation of Gssignaling in OBCs leads to contextual changes in the BM niche that adversely affect HSC maintenance and blood homeostasis.

The bone marrow (BM) niche is essential for lifelong hematopoietic stem cell (HSC) maintenance, proliferation and differentiation. Several BM cell types, including osteoblast lineage cells (OBC), mesenchymal stem cells (MSC) and endothelial cells (EC) have been implicated in supporting HSC location and function, but the relative importance of these cell types and their secreted ligands remain controversial. We recently found that the cell surface receptors Robo4 and CXCR4 cooperate to localize HSC to BM niches. We hypothesized that Slit2, a putative ligand for Robo4, cooperates with the CXCR4 ligand SDF1 to direct HSC to specific BM niche sites. Here, we have isolated OBC, MSC and EC by flow cytometry and determined their frequency within the bone marrow and the relative mRNA levels of Slit2, SDF1 and Robo4. We found that expression of Slit2 and SDF1 were dynamically regulated in MSC and OBC-like populations following radiation, while Robo4 expression was restricted to EC. Radiation also significantly affected the cellularity and frequency of both the non-adherent and adherent cells within the BM stroma. These data support a physiological role for Slit2 in regulating the dynamic function of Robo-expressing cells within BM niches at steady state and following radiation.

Hematopoiesis, the process by which all mature blood cells are generated from multipotent hematopoietic stem cells (HSCs), is a finely tuned balancing act in which HSCs must constantly decide between different cell fates: to proliferate, to self‐renew or differentiate, to stay quiescent in the bone marrow niche or migrate to the periphery, to live or die. These fates are regulated by a complex interplay between cell‐extrinsic cues and cell‐intrinsic regulatory pathways whose function is to maintain a homeostatic balance between HSC self‐renewal and life‐long replenishment of lost blood cells. Improper regulation of these competing cellular programs can transform HSCs and progenitor cells into disease‐initiating leukemic stem cells (LSCs). Strikingly, many of the mechanisms required for maintenance of normal HSC fate decisions are equally critical for the aberrant functions of LSCs. Because of the inherent complexities of these molecular mechanisms, a systematic approach to understanding the regulatory networks underlying HSC self‐renewal is critical for uncovering the similarities and differences between HSCs and LSCs. In this review, we focus on recent developments in elucidating the regulatory networks governing normal HSC self‐renewal programs and their implications for leukemic transformation. We describe the current technical and methodological limitations in isolating and characterizing HSCs and LSCs, and the emerging approaches that may afford a better understanding of the regulation of normal and leukemic hematopoiesis. Finally, we discuss how such basic mechanistic information may be of use for the design of novel therapies that will selectively reprogram and/or eliminate LSCs. WIREs Syst Biol Med2011 3 681–701 DOI: 10.1002/wsbm.145

Adhesion properties of hematopoietic stem cells (HSCs) in the bone marrow (BM) niches control their migration and affect their cell-cycle dynamics. The serum response factor (Srf) regulates growth factor–inducible genes and genes controlling cytoskeleton structures involved in cell spreading, adhesion, and migration. We identified a role for Srf in HSC adhesion and steady-state hematopoiesis. Conditional deletion of Srf in BM cells resulted in a 3-fold expansion of the long- and short-term HSCs and multipotent progenitors (MPPs), which occurs without long-term modification of cell-cycle dynamics. Early differentiation steps to myeloid and lymphoid lineages were normal, but Srf loss results in alterations in mature-cell production and severe thrombocytopenia. Srf-null BM cells also displayed compromised engraftment properties in transplantation assays. Gene expression analysis identified Srf target genes expressed in HSCs, including a network of genes associated with cell migration and adhesion. Srf-null stem cells and MPPs displayed impair expression of the integrin network and decreased adherence in vitro. In addition, Srf-null mice showed increase numbers of circulating stem and progenitor cells, which likely reflect their reduced retention in the BM. Altogether, our results demonstrate that Srf is an essential regulator of stem cells and MPP adhesion, and suggest that Srf acts mainly through cell-matrix interactions and integrin signaling.

The HOXA9 homeoprotein exerts dramatic effects in hematopoiesis. Enforced expression of HOXA9 enhances proliferation of primitive blood cells, expands hematopoietic stem cells (HSCs), and leads to myeloid leukemia. Conversely, loss of HOXA9 inhibits proliferation and impairs HSC function. The pathways by which HOXA9 acts are largely unknown, and although HOXA9 is a transcription factor, few direct target genes have been identified. Our previous study suggested that HOXA9 positively regulates Pim1, an oncogenic kinase. The hematologic phenotypes of Hoxa9- and Pim1-deficient animals are strikingly similar. Here we show that HOXA9 protein binds to the Pim1promoter and induces Pim1mRNA and protein in hematopoietic cells. Pim1 protein is diminished in Hoxa9−/−cells, and Hoxa9and Pim1mRNA levels track together in early hematopoietic compartments. Induction of Pim1 protein by HOXA9 increases the phosphorylation and inactivation of the proapoptotic BAD protein, a target of Pim1. Hoxa9−/−cells show increased apoptosis and decreased proliferation, defects that are ameliorated by reintroduction of Pim1. Thus Pim1appears to be a direct transcriptional target of HOXA9 and a mediator of its antiapoptotic and proproliferative effects in early cells. Since HOXA9 is frequently up-regulated in acute myeloid leukemia, Pim1 may be a therapeutic target in human disease.

The HOXA9 homeoprotein exerts dramatic effects in hematopoiesis. Enforced expression of HOXA9 enhances proliferation of primitive blood cells, expands hematopoietic stem cells (HSCs), and leads to myeloid leukemia. Conversely, loss of HOXA9 inhibits proliferation and impairs HSC function. The pathways by which HOXA9 acts are largely unknown, and although HOXA9 is a transcription factor, few direct target genes have been identified. Our previous study suggested that HOXA9 positively regulates Pim1, an oncogenic kinase. The hematologic phenotypes of Hoxa9- and Pim1-deficient animals are strikingly similar. Here we show that HOXA9 protein binds to the Pim1 promoter and induces Pim1 mRNA and protein in hematopoietic cells. Pim1 protein is diminished in Hoxa9−/− cells, and Hoxa9 and Pim1 mRNA levels track together in early hematopoietic compartments. Induction of Pim1 protein by HOXA9 increases the phosphorylation and inactivation of the proapoptotic BAD protein, a target of Pim1. Hoxa9−/− cells show increased apoptosis and decreased proliferation, defects that are ameliorated by reintroduction of Pim1. Thus Pim1 appears to be a direct transcriptional target of HOXA9 and a mediator of its antiapoptotic and proproliferative effects in early cells. Since HOXA9 is frequently up-regulated in acute myeloid leukemia, Pim1 may be a therapeutic target in human disease.

Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers, including myeloid malignancies. We investigated the effects of an oncogenic KrasG12D allele on phosphorylated signaling molecules in primary c-kit+ lin−/low hematopoietic stem/progenitor cells. Comparison of wild-type and KrasG12D c-kit+ lin−/low cells shows that K-RasG12D expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5, ERK, and S6 under basal and stimulated conditions. Whereas KrasG12D cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor, we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell–targeted interventions.

Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RASgenes contribute to many cancers, including myeloid malignancies. We investigated the effects of an oncogenic KrasG12Dallele on phosphorylated signaling molecules in primary c-kit+lin−/lowhematopoietic stem/progenitor cells. Comparison of wild-type and KrasG12Dc-kit+lin−/lowcells shows that K-RasG12Dexpression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5, ERK, and S6 under basal and stimulated conditions. Whereas KrasG12Dcells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor, we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell–targeted interventions.

Abstract: Leukemias can now be viewed as aberrant hematopoietic processes initiated by rare cancer stem cells, or leukemic stem cells (LSCs) that have maintained or reacquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of LSCs and the mechanisms responsible for their emergence in the course of the disease. Mouse models of human leukemias have provided a unique system to study the mechanisms influencing LSC generation and function, and were recently used to demonstrate that LSCs can arise from both self-renewing hematopoietic stem cells (HSCs) and committed progenitor populations. This striking finding indicates that LSC identity is largely dictated by the nature of the oncogenic events and by how these events perturb essential processes such as self-renewal, proliferation, differentiation, and survival. Such approaches in the mouse are essential for the basic understanding of leukemogenesis and for the conceptual design of novel therapeutic strategies that could lead to improved treatments for human leukemias.

The activator protein 1 (AP-1) member JunB has recently been implicated in leukemogenesis. Here we surveyed human lymphoma samples for expression of JunB and other AP-1 members (c-Jun, c-Fos, Fra1, JunD). JunB was strongly expressed in T-cell lymphomas, but non-Hodgkin B-cell lymphomas do not or only weakly express JunB. We therefore asked whether JunB acted as a negative regulator of B-cell development, proliferation, and transformation. We used transgenic mice that expressed JunB under the control of the ubiquitin C promoter; these displayed increased JunB levels in both B- and T-lymphoid cells. JunB transgenic cells of B-lymphoid, but not of T-lymphoid, origin responded poorly to mitogenic stimuli. Furthermore, JunB transgenic cells were found to be less susceptible to the transforming potential of the Abelson oncogene in vitro. In addition, overexpression of JunB partially protected transgenic mice against the oncogenic challenge in vivo. However, transformed B cells eventually escaped from the inhibitory effect of JunB: the proliferative response was similar in explanted tumor-derived cells from transgenic animals and those from wild-type controls. Our results identify JunB as a novel regulator of B-cell proliferation and transformation. (Blood. 2003;102:4159-4165)

Inflammation exerts multiple effects on the early hematopoietic compartment. Best studied is the role of proinflammatory cytokines in activating adult hematopoietic stem and progenitor cells to dynamically replenish myeloid lineage cells in a process known as emergency myelopoiesis. However, it is increasingly appreciated that the same proinflammatory signaling pathways are used in diverse hematopoietic scenarios. This review focuses on inflammatory signaling in the emergence of the definitive hematopoietic compartment during embryonic life, and tonic inflammatory signals derived from commensal microbiota in shaping the adult hematopoietic compartment in the absence of pathogenic insults. Insights into the unique and shared aspects of inflammatory signaling that regulate hematopoietic stem and progenitor cell function across the lifespan and health span of an individual will enable better diagnostic and therapeutic approaches to hematopoietic dysregulation and malignancies.

While young blood can restore many aged tissues, its effects on the aged blood system itself and old hematopoietic stem cells (HSCs) have not been determined. Here, we used transplantation, parabiosis, plasma transfer, exercise, calorie restriction, and aging mutant mice to understand the effects of age-regulated systemic factors on HSCs and their bone marrow (BM) niche. We found that neither exposure to young blood, nor long-term residence in young niches after parabiont separation, nor direct heterochronic transplantation had any observable rejuvenating effects on old HSCs. Likewise, exercise and calorie restriction did not improve old HSC function, nor old BM niches. Conversely, young HSCs were not affected by systemic pro-aging conditions, and HSC function was not impacted by mutations influencing organismal aging in established long-lived or progeroid genetic models. Therefore, the blood system that carries factors with either rejuvenating or pro-aging properties for many other tissues is itself refractory to those factors.

Hematopoietic stem cells (HSCs) must at times exit quiescence to divide and produce differentiated blood cells. In this issue of Cell Stem Cell, Takubo et al. (2013) and Yu et al. (2013) show that these opposing actions require distinct metabolic programs to meet the changing energy demands of self-renewing HSCs.

Before one can think of the challenges that face women in science and the hurdles that impair their development into leadership positions, it is worth considering the diversity within the collective of women scientists at the level of culture and past experience and life events.

Targeting commonly altered mechanisms in leukemia can provide additional treatment options. Here, we show that an inducible pathway of myeloid regeneration involving the remodeling of the multipotent progenitor (MPP) compartment downstream of hematopoietic stem cells (HSCs) is commonly hijacked in myeloid malignancies. We establish that differential regulation of Notch and Wnt signaling transiently triggers myeloid regeneration from HSCs in response to stress, and that constitutive low Notch and high Wnt activity in leukemic stem cells (LSCs) maintains this pathway activated in malignancies. We also identify compensatory crosstalk mechanisms between Notch and Wnt signaling that prevent damaging HSC function, MPP production, and blood output in conditions of high Notch and low Wnt activity. Finally, we demonstrate that restoring Notch and Wnt deregulated activity in LSCs attenuates disease progression. Our results uncover a mechanism that controls myeloid regeneration and early lineage decisions in HSCs and could be targeted in LSCs to normalize leukemic myeloid cell production.

Blood-related cancers, or leukemias, have been shown to arise from a rare subset of cells that escape normal regulation and drive the formation and growth of the tumor. The finding that these so-called cancer stem cells, or leukemic stem cells (LSC), can be purified away from the other cells in the tumor allows their precise analysis to identify candidate molecules and regulatory pathways that play a role in progression, maintenance, and spreading of leukemias. The analyses of the other, numerically dominant, cells in the tumor, while also interesting, do not directly interrogate these key properties of malignancies. Mouse models of human myeloproliferative disorder and acute myelogenous leukemia have highlighted the remarkable conservation of disease mechanisms between both species. They can now be used to identify the LSC for each type of human leukemia and understand how they escape normal regulation and become malignant. Given the clinical importance of LSC identification, the insights gained through these approaches will quickly translate into clinical applications and lead to improved treatments for human leukemias.

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.

Chronic inflammation is associated with bone marrow (BM) failure, aging and hematological malignancies. TNFα is a major pro-inflammatory cytokine overproduced in many hematological diseases, which is also known as a prototypical death ligand that can trigger programmed cell death in effector cells. Hematopoietic stem cells (HSCs: Lin-cKit+Sca1+Flk2-CD48-CD150+) are highly responsive to an altered cytokine milieu in the BM, and show unique response to cell death stimuli compared to downstream progenitors. Yet, how TNFα regulates HSCs and downstream progenitors remains controversial.

Acute Promyelocytic Leukemia (APL) is characterized by the accumulation in the blood and bone marrow of abnormal promyelocytes, which have the ability to transfer the disease to secondary recipients in animal models. The PML-RARα fusion protein is thought to be the primary abnormality implicated in the pathology, and is believed to prevent transcription of genes necessary for normal myeloid development and differentiation. Identifying PML-RARα targets is critical for understanding the road to leukemic transformation. However, such targets have so far been identified using cell line assays in vitro, murine cells differentiated into promyelocytes in vitro, or fully transformed murine or human leukemic cells. Focusing on the cell population in which the transforming potential is acquired, we describe here a novel strategy to identify the transcriptomic dysregulation induced by PML-RARα expression in maturing myeloid populations in vivo.We utilize a murine model of human APL in which the human PML-RARα fusion gene is expressed under the control of the MRP8 promoter, driving its expression in maturing myeloid populations. Those animals can be described as pre-leukemic since they eventually develop leukemia when additional mutations occur. Fresh bone marrows from normal (Fvb/n) or pre-leukemic (PML-RARα) animals were harvested. Using an improved cell surface antigen staining strategy and fluorescence-activated cell sorting, three populations of increasingly differentiated myeloid populations have been sorted (Granulocyte Macrophage Progenitor, Early promyelocyte and Late promyelocyte). RNA was extracted and submitted for whole-genome microarray analysis. In addition, we are using a variety of bioinformatics approaches to decipher the network of novel interactions driven by PML-RARα expression.Markers used in our sorting strategy were validated in the dataset, including CD34 and Gr1. In the normal samples, markers of neutrophil maturation increased, largely as expected, and a number of early transcription factors decreased in an expected manner including Hoxa9 and Meis1. One remarkable finding was that despite the previously described ability of PML-RARα to regulate transcription from multiple sites in the genome, only a small number of genes were differentially impacted by the expression of this protein. Surprisingly, well-known regulators of myeloid differentiation that have been implicated in the retinoic acid responsiveness of APL including Sfpi1 (PU.1) and Cebpa were not differentially expressed. However, in pre-leukemic samples PML-RARα did cause decreased expression of multiple neutrophilic granule genes including Ltf, Mmp9 and Ngp. The gene most upregulated in the pre-leukemic samples was Spp1 which encodes the osteopontin phosphoprotein. Of interest, we identified the myeloid tumor suppressor Irf8 to be downregulated 5 fold in the presence of PML-RARα. To investigate the importance of IRF8 levels in APL initiation, we transplanted Irf8+/+ PML-RARα or Irf8+/− PML-RARα bone marrow into irradiated recipients. Despite the potential for decreased expression of IRF8 to contribute to APL, we observed no difference. This result does not confirm a role for IRF8 in APL pathogenesis, but further investigations are needed to exclude such a role.Bioinformatics studies highlighted enrichment in cell cycle-related genes upon PML-RARα expression, suggesting a possible difference in the proliferation capacity of the pre-leukemic cells, which is currently under investigation.We found that in vivo the transcriptome was only modestly dysregulated by the presence of PML-RARα. These observations open up new questions on the role of the fusion protein in pathogenesis: How does PML-RARα prime pre-leukemic cells for full transformation? How do secondary events allow an initiated cell to advance to a fully transformed state? Such questions are currently being investigated, with a special interest on looking at the cooperation between PML-RARα and activated cytokine signaling in leukemia initiation.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Hematopoietic development is organized hierarchically, starting with a rare population of hematopoietic stem cells (HSCs) that gives rise to a series of committed myeloid progenitors and mature cells with particular functional and immunophenotypic properties. HSCs are operationally defined by their ability to provide long-term multilineage reconstitution when transplanted into hematopoietically compromised recipient, and are the only cells within the myeloid series that self-renew throughout life. Under steady-state conditions, HSCs are largely quiescent, slowly cycling cells. However, in response to environmental stresses, HSCs can undergo dramatic expansion and contraction to ensure proper homeostatic replacement of blood cells. HSCs are found predominantly in the bone marrow (BM) associated with several recently described vascular and endosteal niches. A complex balance of cell extrinsic factors present in these niches maintains HSCs in relatively dormant states and regulate their trafficking to and from these BM microenvironments. Recent studies have highlighted the roles of specific signal transducer (Pten), polycomb group protein (Bmi1), transcription factors (JunB, Gfi-1, Hox genes), and extrinsic regulatory pathways (Notch, TGF-beta, Wnt) in controlling HSC selfrenewal, proliferation, and differentiation. Here, we will review the various experimental approaches used to identify and functionally assess HSC and myeloid progenitor activity in the mouse. We will discuss how HSCs are molecularly equipped to integrate signals from the microenvironment, and how such regulations control the balance between proliferation and early myeloid differentiation. The case of the JunB/AP-1 transcription factor will be used as an example of pathological situation. Inactivation of JunB leads to the development of a myloproliferative disease (MPD) arising from the HSC compartment. JunB normally controls HSC proliferation by regulating the expression levels of key cell cycle regulators, including Bmi1, and limits the rate at which HSCs produce early myeloid progenitors by maintaining appropriate responsiveness to both Notch and TGF-beta signaling pathways. The transforming effect of JunB loss is, therefore, due to a broad deregulation of the regulatory mechanisms controlling HSC differentiation, leading to overproduction of myeloid progenitors and MPD development in vivo. These results provide insights into fundamental issues in hematopoiesis and leukemogenesis.

Fetal haematopoietic stem cells (HSCs) self-renew extensively to build the blood system from scratch. The protein Lin28b negatively regulates the microRNA let-7 to keep levels of its target Hmga2 high, hence conferring high self-renewal potential to fetal HSCs. This regulatory circuit can be experimentally modulated to boost the lower self-renewal activity of quiescent adult HSCs.