Normal Hematopoiesis Is a Balancing Act of Self-Renewal and Regeneration

Abstract

The hematopoietic system is highly organized to maintain its functional integrity and to meet lifelong organismal demands. Hematopoietic stem cells (HSCs) must balance self-renewal with differentiation and the regeneration of the blood system. It is a complex balancing act between these competing HSC functions. Although highly quiescent at steady state, HSCs become activated in response to inflammatory cytokines and regenerative challenges. This activation phase leads to many intrinsic stresses such as replicative, metabolic, and oxidative stress, which can cause functional decline, impaired self-renewal, and exhaustion of HSCs. To cope with these insults, HSCs use both built-in and emergency-triggered stress-response mechanisms to maintain homeostasis and to defend against disease development. In this review, we discuss how the hematopoietic system operates in steady state and stress conditions, what strategies are used to maintain functional integrity, and how deregulation in the balance between self-renewal and regeneration can drive malignant transformation.

The hematopoietic system is organized in a hierarchical manner, with hematopoietic stem cells (HSCs) sitting at the apex of its differentiation hierarchy. HSCs give rise to progeny transiting through multiple differentiation states, progressing from lineage-biased but still multipotent progenitors to lineage-specific committed progenitors and precursors, ultimately producing all populations of mature blood cells. As an organ, the hematopoietic system is highly plastic and serves a diverse set of biological processes: generating the myeloid and lymphoid cells of the innate and adaptive immune systems to defend the organisms against a host of attacks, making the red blood cells that transport oxygen throughout the body to oxygenate all tissues, and producing the platelets that stop bleeding and orchestrate tissue repair. All these functions are critical for the survival of the organism throughout its life span, and the hematopoietic system has evolved significant levels of regulation and protection to maintain its functional integrity and meet blood production demands.

The hematopoietic system is designed to regenerate continuously, both to maintain homeostatic replacement of blood cells at steady state and to rapidly increase output to adequately compensate for acute blood loss in the context of physical trauma, infection, and metabolic or toxic stress. This adaptive response requires the hematopoietic system to switch from its steady state, slow production mode to an activated state known as “emergency hematopoiesis.” Although emergency hematopoiesis involves adaptive regulation in progenitors, as will be discussed later in the review, this is fundamentally a process that starts at the level of HSCs. HSCs must proliferate and balance two opposing cell-fate decisions, self-renewal and differentiation, to maintain the stem cell compartment and produce all the needed downstream progenitors and mature blood cells. Under homeostatic conditions, HSCs cycle infrequently and are largely in a dormant state known as quiescence (Wilson et al. 2008; Foudi et al. 2009). HSC quiescence serves as a protective mechanism by limiting replicative stress, which can cause the functional decline of HSCs especially on aging (Flach et al. 2014). In response to inflammatory signals that coordinate regeneration, HSCs quickly become activated, proliferate, and expand the needed progenitor compartments to replenish the blood system and produce the required effector cells (Wilson et al. 2008; Sato et al. 2009). When regenerative signaling becomes hyperactivated, differentiation can be prioritized over self-renewal, leading to the loss of the immature stem and progenitor cell (HSPC) compartment. Conversely, in conditions in which differentiation is inhibited and self-renewal is prioritized, as the result of somatic mutation or environmental cues, the HSC compartment becomes hyperplastic at the expense of effective maintenance of blood production. In both cases, the hematopoietic system becomes exhausted, leading to cytopenias and ultimately bone marrow (BM) failures. In the context of malignancies like myeloproliferative neoplasms (MPNs) or acute myeloid leukemia (AML), this regulatory axis is also disrupted but with increased self-renewal and regenerative signaling pathways working in concert to drive leukemic progression. In this review, we discuss how normal hematopoiesis balances regeneration and self-renewal, and how deregulation of these regulatory mechanisms leads to malignant hematopoiesis.

HSC IDENTIFICATION: SELF-RENEWAL AND MULTIPOTENCY

HSCs are defined by multipotency, the ability to differentiate into all lineages of mature blood cells, and the capacity to self-renew for the duration of an organism’s life. It is the investigation of these two features that has driven research in the field over the last century and led to the discovery of bona fide HSCs. The earliest conceptual understanding of the BM as a reservoir of self-renewing and multipotent HSCs originates from the first transplant to cure a patient with aplastic anemia (Osgood et al. 1939). Although anemia was previously treated with transfusions, the infusion of BM cells identified the source of blood-producing progenitor cells. Subsequent experimentation revealed that infusion of mouse BM cells could rescue mice from radiation-induced lethality (Jacobson et al. 1951) by restoring both myeloid and lymphoid lineage production (Ford et al. 1956), hence reinforcing the idea of multipotent blood-producing cells. In the 1960s, spleen colony-forming unit (CFU-S) assays provided further evidence for the concept of stem cells. Mice transplanted with syngeneic BM cells formed cellular colonies in the spleen, with differential compositions of myeloid and erythroid cells and self-renewal capacity (Till and McCulloch 1961; Becker et al. 1963). It was also noted at this time that self-renewal and multipotency were related to quiescence (Becker et al. 1965; Bruce et al. 1966).

In the 1980s, with the advance of technologies such as multicolor fluorescence-activated cell sorting (FACS) and monoclonal antibodies, HSCs were prospectively isolated from mouse BM as Lin⁻/Thy-1^low/Sca-1⁺ cells and shown to be capable of long-term, multilineage reconstitution in irradiated mice (Muller-Sieburg et al. 1986; Spangrude et al. 1988). Later in the 1990s, single-cell transplantation experiments were performed to truly show the capacity of a single blood-forming HSC to regenerate all hematopoietic lineages (Osawa et al. 1996). The phenotypic characterization of HSCs has considerably progressed with a current consensus on identifying long-term engrafting murine HSCs as Lin⁻/Sca-1⁺/c-Kit⁺/Flk2⁻/CD48⁻/CD150⁺/CD34⁻ BM cells (Cabezas-Wallscheid et al. 2014) and human HSCs as Lin⁻/CD34⁺/CD38⁻/CD90⁺/CD45RA⁻ cord blood and BM cells (Gentles et al. 2010). This significant body of research, conducted during the last century, has defined our understanding of HSC self-renewal and differentiation properties through transplantation assays and has set the stage for further mechanistic investigations of HSC biology.

The immediate downstream progeny of HSCs, the multipotent progenitors (MPPs), produce all hematopoietic lineages but have lost the capacity for extensive self-renewal in transplantation assays, although they retain the ability to maintain long-term blood production in native condition (Busch et al. 2015). The mouse MPP compartment is composed of MPP2 (Lin⁻/Sca-1⁺/c-Kit⁺/Flk2⁻/CD48⁺/CD150⁺/CD34⁺), MPP3 (Lin⁻/Sca-1⁺/c-Kit⁺/Flk2⁻/CD48⁺/CD150⁻/CD34⁺) and MPP4 (Lin⁻/Sca-1⁺/c-Kit⁺/Flk2⁺/CD48⁺/CD150⁻/CD34⁺) populations, which have megakaryocyte, granulocyte-macrophage, and lymphoid lineage-differentiation biases, respectively (Cabezas-Wallscheid et al. 2014; Pietras et al. 2015). These lineage-biased MPPs give rise to more lineage-restricted progenitors such as common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs). CMPs further differentiate into granulocyte-macrophage progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs), which eventually produce all mature myeloid cells (Fig. 1A). In response to stress, GMPs form patches in the bone marrow, which function as an amplification compartment, allowing an acute increase of myeloid output (Hérault et al. 2017). This hematopoietic tree-like model of hematopoiesis suggests that HSCs pass through a series of differentiation states with increasingly restricted self-renewal and lineage potentiality (Kondo et al. 1997; Akashi et al. 2000). This model is based on discrete bifurcating cell-fate decisions that restrict potentiality at each relatively homogenous progenitor stage and has guided research and provided the framework for the prospective identification of different multi- and bipotent progenitors by cell surface markers. However, it has become well appreciated that each stem and progenitor population is in fact heterogeneous, and that the majority of HSCs produce a lineage-biased rather than balanced output (Müller-Sieburg et al. 2002, 2004; Dykstra et al. 2007; Morita et al. 2010; Yamamoto et al. 2013). Lineage tracing studies using either polylox (Pei et al. 2017), viral (Naik et al. 2013), or transposon (Sun et al. 2014; Rodriguez-Fraticelli et al. 2018) barcoding strategies to assess the clonal output of single HSCs have gone further to show how heterogenous the HSCs are in terms of lineage bias and output. These approaches have also shown that the megakaryocyte lineage can arise directly from HSCs (Rodriguez-Fraticelli et al. 2018). Collectively, these studies indicate that cell-fate decisions are influenced by cell-intrinsic regulators and are likely occurring at early stages of hematopoietic differentiation. The advent of single-cell technology has clarified these findings by revealing that hematopoietic cells acquire lineage-biased expression in a rather continuous manner (Fig. 1B), and that the classical tree-like model of hematopoiesis represents a semi-artificial segmentation of the data (Pina et al. 2012; Macaulay et al. 2016; Nestorowa et al. 2016; Velten et al. 2017; Karamitros et al. 2018; Tusi et al. 2018). These new differentiation–continuum models of hematopoiesis suggest that differentiation trajectories can be established early on during development, and although these early biases are not immutable to external pressure, they explain, in large part, the bias in lineage output observed in the hematopoietic system (Laurenti and Göttgens 2018).

Figure 1.

Models of hematopoietic stem cell (HSC) lineage commitment. (A) In the classical tree-like model of hematopoietic differentiation, HSCs give rise to multipotent progenitors, which in turn produce lineage-restricted progenitors that ultimately produce all mature hematopoietic cells. This model assumes a relatively uniform developmental and differentiation status at each progenitor stage, and that bifurcating lineage choices occur in either a stochastic manner or in response to instructive cytokines. (B) The continuous differentiation model represents the gradual acquisition of lineage restricted features and the presence of transcriptional lineage bias at earlier stages than lineage commitment occurs. Boxes represent progenitor states based on phenotypic markers and illustrate the heterogeneity within each of these populations. MPP, multipotent progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte-erythroid progenitor; GMP, granulocyte-macrophage progenitor.

HSC DEFENSE MECHANISMS: NICHE REGULATION, GENOME MAINTENANCE, AND METABOLIC DORMANCY

The Hematopoietic Stem Cell Niche

To maintain their lifelong functional integrity, HSCs are tightly regulated both in a cell-intrinsic manner and by extrinsic cues from the BM microenvironment known as the BM niche. Much of the regulation of HSC quiescence is controlled by this hypoxic BM microenvironment, in which quiescence-enforcing and self-renewal promoting cytokines are provided by stromal and endothelial niche cells (Schepers et al. 2015; Crane et al. 2017; Wei and Frenette 2018). The c-x-c motif chemokine ligand 12 (CXCL12) and the c-Kit receptor ligand stem cell factor (SCF) have appeared as the most important factors for HSC retention in the niche (Ding et al. 2012). Stromal-derived CXCL12 is also required for HSCs to colonize the BM niche during development (Ara et al. 2003), and following DNA damaging injury from chemotherapy or radiation, CXCL12 expression is increased to promote the migration and repopulation of the BM niche by HSCs (Ponomaryov et al. 2000). Niche cells can also contribute to HSC activation, especially by secreting pro-inflammatory cytokines, and endothelial cells have been shown to sense pathogens and activate HSCs to drive emergency hematopoiesis (Boettcher et al. 2014). In this manner, niche cells work to support HSC maintenance and function, both by maintaining HSC quiescence and self-renewal and by coordinating regeneration in response to physiologic insult. Indeed, hematopoietic function and the niche are so inextricably linked that mutation in the niche compartment is sufficient to drive leukemogenesis (Kode et al. 2014). Although we will include discussion of the BM niche where relevant, as it pertains to HSC function in normal and malignant hematopoiesis, this topic is discussed in depth in a number of recent reviews (see Schepers et al. 2015; Crane et al. 2017; Wei and Frenette 2018).

DNA Damage and HSC Functional Decline

HSCs reside in the quiescent G₀ state, which serves as the first line of defense against genomic instability, metabolic stress, and functional decline (Wilson et al. 2008; Bakker and Passegué 2013; Chandel et al. 2016). DNA damage and genomic instability can arise from multiple intrinsic and extrinsic sources such as reactive oxygen species (ROS) produced during mitochondrial respiration (Ito and Suda 2014), replication errors and telomere attrition generated during cell division (Rossi et al. 2007), as well as irradiation, ultraviolet (UV), or exposure to genotoxic agents (Biechonski et al. 2017). HSCs use various mechanisms to cope with these intrinsic and environmental stresses to prevent DNA damage accrual. Indeed, an evolutionarily conserved feature of the BM niche is the protection of HSCs from UV (Kapp et al. 2018). HSCs also have high levels of ATP-binding cassette (ABC) transporters to efflux genotoxins and protect from environmental stress (Goodell et al. 1996; Zhou et al. 2002). Replication, however, is the main driver of somatic mutation over the life span (Tomasetti and Vogelstein 2015) and of functional decline in HSCs (Flach et al. 2014). Quiescence protects HSCs from replicative stress, with many studies showing an inverse correlation between proliferative status and engraftment of HSCs, in which dormant cells have the greatest long-term reconstitution potential (Orford and Scadden 2008). Cell cycle regulators such as the retinoblastoma (Rb) family (Viatour et al. 2008), the cyclin D-Cdk4/6 complex (Kozar et al. 2004; Malumbres et al. 2004), and the CIP/KIP family—in particular, p57 (Matsumoto et al. 2011; Zou et al. 2011)—control HSC dormancy in a redundant manner. Interestingly, p53 also regulates HSC quiescence in part by inducing the CIP family member p21 (Asai et al. 2011). Niche factors such as transforming growth factor β1 (TGF-β1) (Scandura et al. 2004; Yamazaki et al. 2009), angiopoietin-1 (Ang-1) (Arai et al. 2004), CXCL12 (Nagasawa et al. 1996), SCF (Thorén et al. 2008), or thrombopoietin (TPO) (Qian et al. 2007) enforce HSC quiescence, either by modulating cell cycle regulators or by promoting DNA damage repair. Additionally, the evolutionarily conserved developmental signaling pathways Wnt, Notch, and Hedgehog (HH) control HSC self-renewal through cell cycle regulation and inhibition of differentiation (Duncan et al. 2005; Trowbridge et al. 2006; Niehrs and Acebron 2012). Ironically, quiescence restricts HSCs to the use of the error-prone nonhomologous end-joining (NHEJ) pathway to repair nonreplicative DNA damage, such as radiation-induced DNA double-strand breaks, which can contribute to mutation and chromosomal instability (Mohrin et al. 2010). Of note, the protective quiescent status of HSCs is unique to adulthood, as fetal and postnatal HSCs are largely cycling (Orkin and Zon 2008; Pietras et al. 2011), which suggest that distinct mechanisms may operate to protect fetal or postnatal HSCs from DNA damage, functional decline, and malignant transformation.

In addition to the tight control of proliferation in HSCs, regulation of ROS, a primary oxidative stress molecule causing nucleotide oxidation and DNA breaks (Sedelnikova et al. 2010), is critical to suppressing the accumulation of DNA damage and HSC functional decline (Yahata et al. 2011). ROS production is regulated in HSCs through a coordinated antioxidant response and the suppression of aerobic metabolism (Ito and Suda 2014). HSCs deficient for either the forkhead box protein O (FOXO) transcription factors or nuclear factor erythroid 2-related factor 2 (NRF2), all of which are master regulators of the anti-oxidant response, lose quiescence and have reduced self-renewal capacity (Tothova et al. 2007; Tsai et al. 2013). ROS drives HSCs out of quiescence and impairs self-renewal by activating p38 mitogen-activated protein kinase (MAPK) (Ito et al. 2006). Indeed, not only is ROS itself a mutagen but its accumulation in response to replicative stress and unresolved DNA damage plays an important role in triggering differentiation and the elimination of damaged HSCs (Ito et al. 2004; Yahata et al. 2011). Collectively, these findings show how tightly activation and proliferation are controlled to suppress DNA damage and maintain HSC genome integrity and illustrate how ROS signaling integrates stress signaling and determines cell fate.

Metabolism and HSC Activation

Despite the high vascularity of the BM, direct measurement of oxygen tension indicates a hypoxic microenvironment (Spencer et al. 2014). Hypoxia-inducible factor 1α (HIF1α) is stabilized in HSCs by low oxygen tension, and with myeloid ecotropic viral integration site 1 (MEIS1) regulates quiescent HSC metabolism through the utilization of anaerobic glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) (Simsek et al. 2010). Indeed, quiescent HSCs have an abundance of glycolytic metabolites, and a relative absence of tricarboxylic acid cycle (TCA) metabolites (Simsek et al. 2010; Takubo et al. 2013). Maintaining the glycolytic metabolic state is critical to HSC quiescence and mice lacking HIF1α (Takubo et al. 2010) or the glycolytic enzymes pyruvate dehydrogenase kinase 2 (PDK2) and PDK4 (Takubo et al. 2013), which prevent the flux of pyruvate through the TCA cycle, display increased mitochondrial respiration and ROS generation with a concomitant loss of HSC quiescence and reduced self-renewal capacity. Similarly, loss of lactase dehydrogenase (LDH), the terminal enzyme in anaerobic glycolysis, results in a loss of HSCs and long-term BM reconstitution capacity (Wang et al. 2014). Collectively, these studies show the importance of anaerobic glycolysis in maintaining quiescent LT-HSCs.

HSCs have relatively high levels of mitochondria compared with some downstream progenitors, but they are usually small with low activity (Simsek et al. 2010; Norddahl et al. 2011; Ho et al. 2017). The activity of HSC mitochondria is inhibited in part through Sirtuin 7 (SIRT7) and nuclear respiratory factor 1 (NRF1), which suppress the mitochondrial unfolded protein response and metabolic activation (Mohrin et al. 2015). The relatively high, but dormant, mitochondrial content in HSCs likely ensures rapid engagement of OXPHOS to meet the increased energy demands when HSCs activate and proliferate. Indeed, although a metabolic shift to OXPHOS is sufficient to activate HSCs, ongoing oxidative metabolism is required for differentiation, and HSCs deficient for protein tyrosine phosphatase mitochondrial 1 (PTPMT1), a main OXPHOS machinery component, fail to differentiate, leading to rapid hematopoietic failure (Yu et al. 2013). Autophagy is critical for suppressing levels of activated mitochondria and the metabolic switch to OXPHOS, and HSCs with impaired autophagy display increased mitochondria number and ROS with increased proliferation, myeloid bias, and impaired regenerative capacity in transplantation assays (Mortensen et al. 2011; Ho et al. 2017). Phenotypically, this impaired autophagy and increased mitochondrial OXPHOS resemble the main features of aged HSCs (Ho et al. 2017). In Tie2⁺ HSCs, peroxisome proliferator–activated receptor (PPAR)δ and fatty acid oxidation induce mitophagy and the clearance of mitochondria is critical to HSC self-renewal and expansion (Ito et al. 2016). Thus, a switch from the quiescent glycolytic state to a pro-proliferative/pro-differentiation OXPHOS state is a critical for HSC cell-fate decisions and for balancing HSC self-renewal with regeneration of the hematopoietic system.

Recent studies have uncovered mechanisms by which HSC metabolic status regulates proliferation, differentiation, and self-renewal capacity. Intracellular ROS, a by-product of mitochondrial OXPHOS, appears to function as a rheostat to control HSC response to changing metabolic states through p38 MAPK (Ito et al. 2006; Jang and Sharkis 2007) and p53 (Asai et al. 2011). FOXO3 is also critical in maintaining HSC self-renewal by triggering autophagy in response to nutrient stress (Warr et al. 2013), in addition to its role in suppressing ROS (Miyamoto et al. 2007; Warr et al. 2013). FOXO transcription factors are inhibited by activation of the PI3K–AKT–mTOR signaling pathway, which coordinates HSC proliferation and promotes differentiation in part through regulating mitochondria activity and ROS production (Miyamoto et al. 2007; Tothova et al. 2007). Indeed, the PI3K-negative regulator phosphatase and tensin homolog (PTEN) is required for the maintenance of HSCs, and its loss leads to myeloproliferative disorder and exhaustion of normal HSCs (Yilmaz et al. 2006; Zhang et al. 2006). Similarly, liver kinase B (LKB1), which activates AMPK and negatively regulates the mTOR signaling pathway, also regulates HSC quiescence via a mitochondria-dependent but ROS-independent mechanism (Gan et al. 2010; Gurumurthy et al. 2010; Nakada et al. 2010). Loss of LKB1 leads to the down-regulation of PPARγ coactivators (Gan et al. 2010) and elevates levels of fatty acid metabolites (Gurumurthy et al. 2010). PPAR family members are important regulators of fatty acid oxidation (FAO), which is highly activated in HSCs, and among them PPARδ is highly expressed in HSCs (Ito et al. 2012). PPARδ deletion or pharmacological inhibition impairs FAO and results in HSC exhaustion (Ito et al. 2012). It is proposed that FAO might be a critical source of NADPH and thus serve as a negative regulator of ROS in HSCs, indicating another regulation of HSC self-renewal by the PPAR–FAO metabolic axis (Carracedo et al. 2013). Taken together, HSCs maintain their quiescent metabolic status to ensure self-renewal capacity by suppressing mitochondrial activity and utilizing FAO and deregulation of these pathways exhausts HSCs and leads to BM failure.

Of note, the metabolic switch of HSC activation is not merely a signal driving HSC proliferation and differentiation, it also plays a critical role in the metabolically intensive process of epigenetic remodeling required for differentiation. Upon HSC differentiation, global changes in the DNA methylome occur and these changes are critical to the demethylation and activation of lineage-specific transcriptional programs (Bock et al. 2012; Cabezas-Wallscheid et al. 2014; Farlik et al. 2016). Demethylation of DNA is an energy intensive process requiring ATP, oxygen, and the TCA metabolite α-ketoglutarate (α-KG) and is significantly impaired in a hypoxic and glycolytic metabolic state (Ryall et al. 2015). The DNA demethylase tet methylcytosine dioxygenase 2 (TET2), which requires an active TCA and robust levels of α-KG, is critical to HSC lineage commitment, and its loss leads to impaired differentiation and HSC expansion (Figueroa et al. 2010). The histone demethylase lysine-specific demethylase 1 (LSD1), which uses another OXPHOS metabolite, flavin adenine dinucleotide (FAD), as a cofactor, is also important for normal HSC differentiation and its loss leads to impaired repression of HSPC transcriptional programs during blood cell maturation (Sprüssel et al. 2012; Kerenyi et al. 2013). Therefore, the shift from glycolysis to OXPHOS is crucial to providing the energy and metabolites necessary not only for cellular proliferation but also for the epigenetic remodeling of lineage commitment and differentiation. In this manner, we can understand this stem cell state to be a metabolic ON/OFF switch, which transitions the HSC from its normally protected and self-renewing compartment into a metabolically active cell capable of regenerating the entire blood system.

DEMAND-ADAPTED HEMATOPOIESIS

Lineage Bias and Reprogramming during Emergency Hematopoiesis

Hematopoiesis is a demand-adapted system, which allows the organism to respond to physiological stress and pathogenic challenge in an informed and specific manner. Under steady state conditions, roughly one trillion cells are made in the BM of an adult human on a daily basis (Dancey et al. 1976; Doulatov et al. 2012). The continuous production of immune cells, red blood cells, and platelets is a metabolically intensive process. Therefore, the hematopoietic system tailors its output in a manner that conserves resources and maximizes the production of critical effector cells. Understanding how lineage bias arises from HSC heterogeneity and how it is affected by extrinsic signals is essential to our understanding of hematopoietic regulation by physiological stress. Inflammation can drive demand-adapted hematopoietic response by either activating specific subsets of lineage-biased HSCs or by reprogramming lineage bias in HSPCs more broadly. Indeed, there is evidence that both forms of regulation occur in response to different physiological stressors. TGF-β1 promotes the activation and differentiation of myeloid-biased HSCs (Challen et al. 2010). Conversely, myeloid-biased HSCs have diminished response to the lymphopoietic cytokine interleukin (IL)-7 (Muller-Sieburg et al. 2004). von Willebrand factor (VWF)-expressing HSCs contain quiescent stem cell–like megakaryocyte progenitors, which are activated by acute inflammation to produce only megakaryocytes and restore platelet levels in response to infection or tissue damage (Sanjuan-Pla et al. 2013; Haas et al. 2015). These VWF⁺ HSCs express megakaryocyte-specific transcripts, demonstrating intrinsic lineage commitment as early as the stem cell state (Sanjuan-Pla et al. 2013; Haas et al. 2015). In response to platelet depletion alone, these same HSC-like megakaryocyte progenitors become specifically activated in a thrombopoietin-dependent manner, rather than in response to generalized inflammation (Sanjuan-Pla et al. 2013). These studies show that activation of megakaryopoiesis can occur in a targeted manner or as part of a larger emergency hematopoietic response.

Although activation of lineage-biased HSC subpopulations does occur, inflammation generally reprograms the whole HSPC compartment and instructs production of specific lineages. Both IL-1 and M-CSF signaling lead to precocious activation of the myeloid lineage transcription factor PU.1 in HSCs (Mossadegh-Keller et al. 2013; Pietras et al. 2016). In concert with precocious lineage instruction in HSCs, the MPP compartment responds to inflammatory signals in a dynamic manner to alter blood production (Fig. 2). Under steady state conditions, lymphoid-biased MPP4 represents the dominant population relative to myeloid-biased MPP2 and MPP3 (Pietras et al. 2015). However, upon chronic IL-1 exposure, myeloid-biased MPP2 and MPP3 are expanded, and MPP4 output is redirected toward the myeloid lineage (Pietras et al. 2016). This remodeling of the MPP compartment is also evident during regenerative conditions to meet the intense myeloid demand following transplantation (Pietras et al. 2015), suggesting this is a common regenerative mechanism. IL-6 has also been shown to promote myeloid production by reprogramming lymphoid-biased MPP4 to myeloid-biased production (Reynaud et al. 2011). Downstream from the HSPC compartment, rapidly differentiating GMP clusters in the BM fuel mature myeloid cell production during emergency hematopoiesis (Hérault et al. 2017). Although stress-induced changes in lineage bias occur in HSCs and continue at every progenitor stage, the extent to which such bias at the HSC level reprograms downstream progenitors remains an open question.

Figure 2.

Dynamic alterations to the hematopoietic stem and progenitor cell (HSPC) compartment during regenerative stress. Quiescent hematopoietic stem cells (HSCs) depend on glycolysis and fatty acid oxidation to meet their metabolic needs. Upon activation, HSCs switch to mitochondrial oxidative phosphorylation (OXPHOS). Under steady state conditions, HSCs predominantly produce the lymphoid-biased MPP4. Under regenerative conditions, HSCs overproduce the granulocyte/macrophage (G/M)-biased MPP3 and megakaryocyte-biased MPP2 at the expense of MPP4. Furthermore, MPP4 is reprogrammed toward an almost exclusively myeloid output.

Inflammatory Signals Drive Hematopoietic Regeneration

The activation of the HSC compartment to trigger emergency hematopoiesis is largely driven by inflammatory cytokines. HSCs rapidly exit quiescence and proliferate in response to a number of inflammatory signals including type I and type II interferons (IFNs) (Essers et al. 2009; Sato et al. 2009; Baldridge et al. 2010), G-CSF (Wilson et al. 2008; Schuettpelz et al. 2014), TPO (Sanjuan-Pla et al. 2013), and IL-1 (Ueda et al. 2009; Pietras et al. 2016; Weisser et al. 2016) to increase regeneration of the blood system. Additionally, HSCs can directly sense pathogen-associated molecular-patterns (PAMPs), such as the toll-like receptor (TLR) ligands LPS and Pam3CSK4 (Nagai et al. 2006; Liu et al. 2015; Takizawa et al. 2017). Much of the functional effects of these inflammatory cytokines are mediated through activation of JAK/STAT, MyD88/NF-κB signaling, and MAPK signaling (Baldridge et al. 2011). Indeed, loss of JAK1 is sufficient to impair regeneration following BM transplant and 5-fluorouracil (5-FU) mediated myeloablation (Kleppe et al. 2017). This is associated with increased HSC quiescence and a loss of responsiveness to type I IFNs and IL-3 signaling (Kleppe et al. 2017). Downstream from the progenitor level, activation of STAT3 is crucial for emergency hematopoiesis, where it controls the proliferative expansion of GMPs in response to G-CSF (McLemore et al. 2001; Panopoulos et al. 2006; Zhang et al. 2010). Although there is significant functional overlap between many of these cytokines in driving emergency hematopoiesis, each of these inflammatory mediators activate HSCs and instruct lineage bias and reprogram differentiation in distinct ways (Schultze et al. 2019). This allows the hematopoietic system to expertly integrate extrinsic signals and efficiently tailor blood production to organismal demand.

Activation of Emergency Hematopoiesis in Response to Pathogens

Emergency hematopoiesis can be further distinguished based on whether the regenerative response is driven by pathogen sensing or sterile inflammation (Manz and Boettcher 2014). This distinction is supported by mechanistic evidence of overlapping but divergent signaling. IL-1 receptor signaling is dispensable for granulopoiesis in response to TLR ligands (Boettcher et al. 2012), whereas it is critical in the context of sterile inflammation such as 5-FU-induced myeloablation (Pietras et al. 2016). In the context of systemic infection, hematopoietic growth factors and cytokines including G-CSF, M-CSF, GM-CSF, IL-3, IL-6, and FLT3 ligand are significantly up-regulated in the circulation (Cheers et al. 1988; Watari et al. 1989; Kawakami et al. 1990; Cebon et al. 1994; Selig and Nothdurft 1995; Tanaka et al. 1996; Presneill et al. 2000). Stromal TLR4/MyD88 signaling is critical for LPS-induced G-CSF secretion and activation of emergency hematopoiesis (Boettcher et al. 2014). However, HSPCs can also directly sense LPS, proliferate, and secrete cytokines through NF-κB signaling, thereby directly initiating emergency hematopoiesis (Zhao et al. 2014a; Takizawa et al. 2017). Nonhematopoietic pathogen sensing is also necessary for mobilizing immune cells from the BM. In response to circulating TLR ligands, BM niche cells secrete CCL2 to drive mobilization of Ly6C^hi/CCR2⁺ inflammatory monocytes into the circulation (Shi et al. 2011). It is believed that homeostatic density-dependent mechanisms sense the number of monocytes and neutrophils available in the BM, either through cell–cell contacts with myeloid cells or through myeloid secreted cytokines, and therefore mobilization of myeloid cells and emptying of these reservoirs stimulates regenerative pathways. Indeed, depletion of neutrophils is sufficient to induce HSC activation and emergency hematopoiesis (Scumpia et al. 2010; Cain et al. 2011; Hérault et al. 2017). Furthermore, clearance of aged and apoptotic neutrophils by BM resident macrophages leads to increased production of G-CSF to promote granulopoiesis and restore homeostasis (Furze and Rankin 2008). Together, emergency programs driven by PAMPs regulate both the production of innate immune cell as well as their release from the BM and, through cross talk with physiological systems throughout the body, coordinate hematopoietic regeneration and prevent the development of neutropenia.

Resolution of Emergency Hematopoiesis

Resolution of emergency hematopoiesis and the return of HSCs to quiescence is critical to hematopoietic system function. TGF-β1 in particular has been shown to be a critical inducer of HSC quiescence (Sitnicka et al. 1996; Batard et al. 2000), and within the BM niche megakaryocytes are the dominant source of TGF-β1 and play an important role in maintaining quiescence under steady state conditions (Zhao et al. 2014b). In response to myeloablative chemotherapy, TGF-β1 secreted by megakaryocytes is critical to resolving hematopoietic regeneration and restoring HSC quiescence (Hérault et al. 2017). In the context of type 1 IFN signaling, the response to acute stimulation causes HSCs to become desensitized and no longer competent to respond to further IFN signaling (Pietras et al. 2014). Similarly, activation of STAT3 during emergency hematopoiesis leads to the up-regulation of its own negative regulator, suppressor of cytokine signaling 3 (SOCS3), which desensitizes the cells to further stimulation and terminates the response (Kimura et al. 2004). The restoration of interferon regulatory factor 8 (IRF8) activity in GMPs is also critical to reestablishing normal function and resolving emergency hematopoiesis (Hu et al. 2016). In addition, it appears that repopulation of mature hematopoietic cells in the BM is required to restore homeostatic conditions as loss of BM neutrophils on its own is sufficient to drive emergency hematopoiesis (Cain et al. 2011; Hérault et al. 2017). Although dispensable for steady state hematopoiesis, Fanconi anemia (FA) pathway proteins are also critical for emergency hematopoiesis responses and the rapid proliferation of GMPs (Hu et al. 2013). In the absence of these proteins, DNA damage leads to TP53-dependent apoptosis, and HSCs become exhausted because of their inability to effectively mount an emergency hematopoiesis response leading to aplastic anemia (Hu et al. 2018). Restoration of cell viability, however, increases the population of mature granulocytes within the BM niche and resolves the regenerative response (Hu et al. 2018). In response to chronic inflammation, the normal regulatory processes that control HSC activation and their return to quiescence can fail. Although HSCs are normally protected from chronic IFN signaling by returning to quiescence (Pietras et al. 2014), when negative regulators of IFN signaling are abrogated, HSCs experience chronic IFN exposure, remain cycling, and eventually exhaust (Hartner et al. 2009; Sato et al. 2009). Similarly, chronic low-level activation of TLR signaling leads to a myeloid bias and loss of reconstitution potential of HSCs in a manner reminiscent of aging (Esplin et al. 2011). Indeed, the phenotypes associated with aging of the hematopoietic system are driven by chronic low-grade inflammation (Kovtonyuk et al. 2016). Collectively, the negative regulators that terminate emergency hematopoiesis are as critical to maintaining HSC function, as the initiators of the regenerative response.

HEMATOPOIETIC MALIGNANCY

The architecture of the hematopoietic system is inherently tumor-suppressive, with self-renewal capacity restricted to tightly regulate the size of the HSC pool, and unidirectional differentiation leading to the elimination of acquired oncogenic mutation in non-self-renewing cells. Furthermore, the small size of the HSC population and their largely quiescent cell state makes oncogenic mutation events unlikely. However, malignant transformation does occur, with transformed disease-initiating leukemic stem cells (LSCs) acquiring both dysregulated self-renewal and unbalanced or impaired differentiation and often an activated, highly proliferative, regenerative state (Fig. 3). This occurs through the expansion of the HSC population with accumulated somatic mutations that drive proliferation and block differentiation and the acquisition of self-renewal capacity in downstream progenitors (Passegue et al. 2003; Attolini et al. 2010; Altrock et al. 2015). Although the cell of origin remains an active area of research in the hematological malignancy field, the study of steady state and emergency hematopoiesis in native conditions has significant relevance for understanding the events of transformation and their consequences. The mechanisms that regulate HSC quiescence and limit self-renewal are inherently tumor-suppressive and, conversely, those pathways that drive HSC and progenitor expansion in response to stress are oncogenic in the context of malignant transformation. Furthermore, given that emergency hematopoiesis can dynamically alter self-renewal and differentiation phenotypes, reviewing how these processes promote disease initiation and progression during leukemogenesis is of significant value to the study of hematological malignancies.

Figure 3.

Dysregulation of hematopoietic stem and progenitor cell (HSPC) self-renewal and activation of regenerative pathways during malignant progression. Hematopoietic malignancy arises through the accumulation of mutations that dysregulate self-renewal, inhibit differentiation, and activate proliferation. In age-related clonal hematopoiesis (ARCH), somatic mutations cause the relative expansion of single hematopoietic stem cell (HSC) clones without obvious changes to the hematopoietic system. The accrual of mutations that activate emergency hematopoiesis pathways leads to excessive production of mature cells in myeloproliferative neoplasms (MPNs). In parallel, increased self-renewal and inhibition of differentiation cause dysplasia of the HSPC compartment and mature cell cytopenias in myelodysplastic syndromes (MDSs). In acute myeloid leukemia (AML), both activation of emergency hematopoiesis pathways and inhibition of differentiation combine to drive aggressive proliferation and expansion of leukemic blasts. Although there is evidence that malignant disease can progress in a stepwise manner, MPN, MDS, and AML can also arise de novo from normal hematopoiesis, and ARCH can likely progress directly to AML.

Dysregulated Self-Renewal in Clonal Hematopoiesis and Malignancy

HSC self-renewal is normally tightly regulated, intrinsically and extrinsically, for the lifelong fitness of the blood system, and mutations causing dysregulation of self-renewal, alone or in conjunction with additional driver mutations, often lead to the development of malignancy. DNA damage associated with replication and mitosis is normally suppressed by HSCs staying largely in a quiescent state and only cycling roughly every 40 weeks in humans (Ito et al. 2004; Rossi et al. 2007; Mohrin et al. 2010; Catlin et al. 2011; Flach et al. 2014). However, upon aging, the toll of replicative stress and the lifelong usage of the error-prone NHEJ DNA repair pathway causes HSCs to accumulate somatic mutations (Mohrin et al. 2010; Flach et al. 2014). When these mutated HSCs acquire increased fitness, they expand in a phenomenon termed age-related clonal hematopoiesis (ARCH) (Young et al. 2016). ARCH is defined as the disproportionate expansion of single HSC clones relative to other clones, without the significant dysplasia or cytopenia associated with myelodysplastic syndromes (MDSs) (Bowman et al. 2018). In this manner, ARCH represents both somatic mutations in HSCs and the clear dysregulation of HSC homeostasis independent of clinically significant effects on hematopoietic output. Studies from either healthy cohorts or patients with cancer, type 2 diabetes, or cardiovascular disease show that ARCH has a clear correlation with aging and development of hematological malignancy, indicating that antecedent ARCH can progress to hematological malignancy. However, the frequency of malignant transformation is very low (∼4%) and the relevance of ARCH in malignant transformation is still not fully understood despite the fact that ARCH incidence is higher in cancer patients (Busque et al. 2012; Genovese et al. 2014; Jaiswal et al. 2014, 2017; Xie et al. 2014; Buscarlet et al. 2017; Coombs et al. 2017; Takahashi et al. 2017). An unbiased sequencing study using 2700 germline control blood samples from 11 cancer types identified ARCH-associated recurrent mutations in DNA methyltransferase 3A (DNMT3A), TET2, Janus kinase 2 (JAK2), additional sex-comb like-1 (ASXL1), splicing factor 3B subunit 1 (SF3B1), protein phosphatase, Mg²⁺/Mn²⁺-dependent 1D (PPM1D), and tumor protein p53 (TP53), which are all known to be mutated in hematological malignancies such as MPN, MDS, and AML (Rampal et al. 2014; Xie et al. 2014; Lindsley et al. 2017). In the case of mutations in epigenetic modifiers such as DNMT3A, TET2, and ASXL1, these mutations can provide selective advantages over nonmutated clones by enhancing self-renewal and blocking differentiation (Tadokoro et al. 2007; Moran-Crusio et al. 2011; Quivoron et al. 2011; Abdel-Wahab et al. 2013; Challen et al. 2014). Alternatively, mutations in DNA damage regulators such as TP53 and PPM1D can provide fitness advantage under conditions of genotoxic stress and drive clonal enrichment (Marusyk et al. 2010; Hsu et al. 2018). In this manner, accumulated mutations which favor competitive fitness, either through increased self-renewal or resistance to genotoxic stress, lead to clonal expansion and predisposition to malignant transformation. Indeed, ARCH progression to a lethal MPN and AML can be modeled in the mouse by sequential activation of DNMT3A and NPM1 mutations (Loberg et al. 2019).

Epigenetic modifiers, in particular, are known to regulate HSC self-renewal, and their mutation promotes malignant transformation. Knocking out DNMT3A/B in mice results in decreased HSC self-renewal (Tadokoro et al. 2007), and loss of DNMT3A alone can drive HSC expansion and both lymphoid and myeloid leukemia upon aging (Challen et al. 2014). Similarly, conditional TET2 loss-of-function (LOF) mutation also leads to HSPC expansion (Moran-Crusio et al. 2011; Quivoron et al. 2011). HSC metabolism, integral to epigenetic remodeling, is also dysregulated in malignancy. LSCs rely on OXPHOS, resembling activated HSCs and downstream progenitors more than quiescent HSCs (Lagadinou et al. 2013; Wang et al. 2014). Mutations in the TCA enzymes isocitrate dehydrogenase 1 and 2 (IDH1/2), lead to the production of the onco-metabolite 2-hydroxygluterate (2HG), which inhibits TET2 and thereby impairs differentiation and increases LSC self-renewal (Dang et al. 2009; Figueroa et al. 2010; Lu et al. 2012). Of note, IDH1/2 and TET2 mutations are mutually exclusive, indicating their redundant role in regulating differentiation and self-renewal in malignancy (Figueroa et al. 2010).

The BM niche also has important roles in the development of malignancy through its regulation of LSC self-renewal. Extrinsic signaling pathways controlling HSC self-renewal and differentiation such as Wnt, Notch, and Hh lead to the development of hematological malignancies when their activity is dysregulated. This occurs either through the constitutive activation of the pathways by mutation or epigenetic alteration (Irvine and Copland 2012; Lobry et al. 2014; Staal et al. 2016) or by remodeling the BM niche into a tumor-promoting state (Schepers et al. 2015). Mildly increased Wnt activity enhances HSC repopulating activity (Luis et al. 2011) and the canonical Wnt signaling transducer β-catenin is critical for LSC activity in both chronic myelogenous leukemia (CML) and AML mouse models (Zhao et al. 2007; Wang et al. 2010). Loss of β-catenin can prevent disease onset in a retroviral HOXA9 and MEIS1 overexpression model of AML, indicating dysregulated Wnt activity is required for malignancy (Wang et al. 2010). Additionally, a high level of nuclear β-catenin is found in GMPs from blast crisis and an imatinib-resistant CML patient and is shown to engage a self-renewal circuit in GMPs and drive GMP cluster formation in both regenerative and leukemic contexts (Jamieson et al. 2004; Wang et al. 2010; Hérault et al. 2017). Hypermethylation of several Wnt antagonist genes is also observed in the BM of AML patient samples, explaining in part the high Wnt activity in leukemic conditions (Valencia et al. 2009). Notch signaling can be either an oncogene or a tumor suppressor depending on cellular contexts. In a lymphoid malignancy such as T-cell acute lymphoblastic leukemia (T-ALL), B-chronic lymphocytic leukemia (B-CLL), or splenic marginal zone lymphoma, Notch signaling functions as an oncogene, whereas in a myeloid malignancy such as chronic myelomonocytic leukemia (CMML) or AML, it functions as a tumor suppressor (Lobry et al. 2014). LOF mutations in Notch pathway genes are found in CMML patient samples (Klinakis et al. 2011), and reduced Notch activity is observed in AML blast cells (Kannan et al. 2013). Hh signaling is also involved in both myeloid and lymphoid malignancy (Irvine and Copland 2012). Loss of Hh signaling impairs HSC self-renewal and reduces the onset of CML driven by BCR-ABL, with sustained activation leading to HSC exhaustion (Zhao et al. 2009). Mutations in Hh pathway genes are also found in T-ALL patients (Burns et al. 2018). Taken together, impaired self-renewal by intrinsically accumulated mutations and/or dysregulated external signaling pathways leads to malignant transformation in a context-dependent manner.

Activation of Regenerative Pathways in Malignancy

In hematological malignancies, mutations conferring clonal fitness advantages, such as DNMT3A and TET2, often synergize with oncogenic mutations that drive proliferation, thereby driving clonal expansion of transformed malignant LSCs in a manner that disrupts the balancing act between self-renewal and regeneration. The fact that dysregulated HSC self-renewal can result in ARCH without leukemia development further shows the importance of additional regenerative signaling in driving malignant transformation. In fact, TET2 mutations require microbial-driven inflammatory signals to activate an emergency hematopoietic response and induce preleukemic myeloproliferation, or additional FLT3, JAK2, or NRAS mutations to develop myeloid leukemia (Ortmann et al. 2015; Shih et al. 2015; Kunimoto et al. 2018; Meisel et al. 2018). In adult AML patients, TET2 mutation co-occurs with FLT3 mutation along with hypermutation in many loci or with loss of Notch signaling (Lobry et al. 2013; Shih et al. 2015). Similarly, FLT3 and RAS signaling pathways are comutated with DNMT3A (Cancer Genome Atlas Research et al. 2013; Papaemmanuil et al. 2016). Although in normal hematopoiesis HSC self-renewal and continuous proliferation are mutually exclusive, in malignancy LSCs are able to achieve both by dysregulating differentiation.

Oncogenic mutations that drive leukemia often activate pathways involved in hematopoietic regeneration. The BCR-ABL fusion oncogene, a constitutively active tyrosine kinase, interacts with multiple downstream signaling pathways such as JAK/STAT and Ras/MAPK/ERK (Cilloni and Saglio 2012), resulting in the transformation of HSCs in a manner analogous to permanent activation of emergency hematopoiesis. Indeed, GMP clusters, a hallmark of emergency myelopoiesis, can be found in the BM of a CML mouse model (Hérault et al. 2017). JAK2 mutations are also commonly found in hematologic malignancies and lead to the constitutive activation of regenerative signaling pathways in a ligand-independent manner (Baxter et al. 2005; James et al. 2005; Kralovics et al. 2005; Levine et al. 2005). As discussed in the context of normal hematopoiesis, the chronic activation of regenerative pathways and the increased proliferation of HSCs is detrimental to their genomic stability, and the same is true for LSCs. BCR-ABL-mediated signaling is associated with defective DNA repair and inhibition of apoptosis, leading to additional mutations and further genomic instability, which may in part explain the aggressiveness of advanced blast crisis CML (Calabretta and Perrotti 2004). Similarly, receptor tyrosine kinase FLT3 internal tandem duplications, found in one-third of AML patients, induce aberrant STAT5 signaling and confer factor-independent proliferation and radio resistance and enhance leukemogenesis in vivo (Hayakawa et al. 2000; Mizuki et al. 2000). Mutations in the key lymphoid transcription factor IKZF1 found in blast crisis CML, ALL, AML, and MDS also reduce apoptosis, enhance survival, and impair lymphoid differentiation, thereby contributing to leukemogenesis (Mullighan et al. 2008; John and Ward 2011; Gowda et al. 2017). Nras^G12D promotes HSC survival under ER stress by activating the IRE1α-XBP1 axis through the MAPK pathway and inhibiting IRE1α-XBP1 activation decreases the competitive advantage of NRAS^G12D HSCs in transplantation (Liu et al. 2019) confirming that mutations altering regenerative capacity can lead to leukemic transformation. Collectively, mutations that dysregulate HSC self-renewal synergize with mutations that drive a regenerative and proliferative response and together transition the hematopoietic system into a state of malignancy.

CONCLUSION

The hematopoietic system continuously regenerates to meet blood production needs of the organism. For this reason, the protection of HSCs is critical to the lifelong function of the hematopoietic system and this is achieved, in part, through their location in a hypoxic and quiescence-enforcing BM niche microenvironment. The suppression of mitochondrial aerobic respiration in quiescent HSCs, in particular, is an important part of this privileged state. Quiescent HSCs, however, are not invulnerable and the rapid repair of DNA damage through error-prone DNA repair mechanisms makes these cells susceptible to mutation accumulation and functional decline. The activation of HSCs is driven by the metabolic switch to OXPHOS, which controls both the proliferation as well as the epigenetic remodeling and differentiation of HSCs. Indeed, the intersection of metabolism and epigenetics in stem cell biology is an area of active research with the potential to greatly enhance our understanding of hematopoiesis. HSC activation is largely driven by inflammatory cytokines, which coordinate the regenerative response and reprogram the hematopoietic system to produce the required number of mature effector cells for homeostasis and response to physiologic insults. In contrast, much less is known about the return of HSCs to quiescence and the development of quiescence-enforcing therapeutic strategies will be critical to improving hematopoietic function and longevity.

The hematopoietic system normally balance regeneration with HSC self-renewal and, when these two functions are no longer kept at cross purposes, the result is malignancy. New single-cell technologies are set to drive a period of rapid discovery and increase our resolution in the study of HSCs, their multiple cellular states, and the transition between them. This biology is critically relevant for understanding malignancy, and although it was long thought that highly quiescent LSCs drive therapeutic resistance and relapse, it has recently been shown that in fact LSCs become activated in response to the inflammatory environment of therapeutic treatment and initiate a regenerative response (Boyd et al. 2018). This response of LSCs to chemotherapeutic treatment is very similar to that of normal HSCs and shows how our understanding of native hematopoiesis can inform the study and treatment of cancer. With a better understanding of the processes by which normal HSCs return to quiescence, we may in fact be able to develop strategies that enforce quiescence in LSCs and normalize disease. Conversely, our understanding of the mechanisms of HSC activation can be leveraged to develop therapeutic strategies that activate latent reservoirs of quiescent LSCs and enforce their differentiation and exhaustion. Indeed, the success of current therapies likely result from these principles. Redundant and multifactorial signaling pathways that stimulate regeneration allow the body to tailor hematopoietic output with precision. However, in the context of malignancy this redundancy of signaling helps explain why therapeutic development has been so difficult in the context acute leukemias. Further study of normal hematopoiesis will hopefully identify core biology and provide new insight into specific dependencies of hematopoietic regeneration that can serve as therapeutic targets to normalize or eliminate malignant disease.