The Role of Smad Signaling in Hematopoiesis and Translational Hematology

The role of Smad signaling in hematopoiesis and translational hematology

U Blank and S Karlsson

Division of Molecular Medicine and Gene Therapy, Laboratory Medicine, Lund Stem Cell Center, Lund University Hospital, Lund, Sweden

Hematopoietic stem cells (HSCs) reside in the bone marrow ligands are reflected in vitro, often leading to opposing findings (BM) of adult individuals and function to produce and between in vitro and in vivo systems. regenerate the entire blood and immune system over the In this review, we will discuss the role of TGF-b and Smad course of an individual’s lifetime. Historically, HSCs are among the most thoroughly characterized tissue-specific stem cells. signaling in normal hematopoiesis, featuring aspects of transla- Despite this, the regulation of fate options, such as self-renewal tional hematology, particularly the role of Smad signaling in the and differentiation, has remained elusive, partly because of the development of hematologic malignancies and how the Smad expansive plethora of factors and signaling cues that govern circuitry may be exploited for the purpose of stem cell HSC behavior in vivo. In the BM, HSCs are housed in expansion. It should be emphasized that although some of the specialized niches that dovetail the behavior of HSCs with the findings discussed here come from studies of human and need of the organism. The Smad-signaling pathway, which operates downstream of the transforming growth factor-b patients’ cells, the majority of the data derive from studies in (TGF-b) superfamily of ligands, regulates a diverse set of bio- well-defined mouse models. logical processes, including proliferation, differentiation and apoptosis, in many different organ systems. Much of the function of Smad signaling in hematopoiesis has remained Smad signaling nebulous due to early embryonic lethality of most knockout mouse models. However, recently new data have been The Smad-signaling circuitry embodies an evolutionary con- uncovered, suggesting that the Smad-signaling circuitry is served signaling module, which functions to convert biological intimately linked to HSC regulation. In this review, we bring the information from activated TGF-b receptor complexes at the cell Smad-signaling pathway into focus, chronicling key concepts and recent advances with respect to TGF-b-superfamily signal- surface to concrete transcriptional regulation in the nucleus. ing in normal and leukemic hematopoiesis. TGF-b ligands transmit signals through two types of serine/ Leukemia (2011) 25, 1379–1388; doi:10.1038/leu.2011.95; threonine kinase receptors known as type I and type II published online 13 May 2011 receptors.1 In vertebrates, seven different type I receptors Keywords: hematopoietic stem cell; smad signaling; TGF-b (ALK1–7) and five distinct type II receptors have been identified.1 Although some promiscuity occurs, each ligand generally signals through a specific combination of receptors (Figure 1). Following ligand binding, the type I receptor Introduction becomes activated through phosphorylation by the type II receptor. Activated type I receptors subsequently phosphorylate Transforming growth factor-b (TGF-b) is the founding member of the Smad proteins at residues in the C-terminus, leading to a large superfamily of secreted polypeptide growth factors, propagation of the signal intracellularly. The Smad family of which additionally includes activins, nodal, bone morpho- intracellular mediators is comprised of eight members in genetic proteins (BMPs), growth and differentiation factors and 1 mammals, Smad1–8, which can be further subdivided into others. From early development and continuously throughout three distinct classes based on structural properties and the adult life, TGF-b members carry out pivotal functions by specific functions they carry out.7 Receptor-regulated Smads (R- regulating biological events ranging from gastrulation and organ Smads), Smad1, 2, 3, 5 and 8, are the only Smads directly morphogenesis to homeostatic tissue turnover. Alterations in phosphorylated and activated by the kinase domain of type I components of the TGF-b-superfamily pathway lead to severe receptors. Phosphorylation of R-Smads results in a conforma- SPOTLIGHT developmental abnormalities and have been shown to underlie tional change, allowing complex formation with the common- a range of human diseases, including autoimmune and 2,3 Smad, Smad4. Activated complexes subsequently accumulate in cardiovascular disorders as well as cancer. A fundamental the nucleus, where they cooperate with other transcriptional co- feature of the TGF-b superfamily is its highly pleiotropic nature, regulators to modify target gene transcription. The third class of a phenomenon well illustrated within the hematopoietic system; Smads includes the inhibitory Smads, Smad6 and Smad7, which depending on the differentiation stage and environmental function in a negative feedback loop to inhibit TGF-b-super- context of the target cell, these factors can affect proliferation, 4–6 family signaling. TGF-b/activin/nodal and BMP/growth and differentiation and apoptosis either positively or negatively. differentiation factor employ different subsets of R-Smads. Part of the molecular basis for this is thought to stem from the R-Smad2 and 3 specifically relay signals from TGF-b and activin unique repertoire of transcriptional co-factors expressed by each receptors, whereas R-Smad1, 5 and 8 primarily operate specific cellular target. The context-dependent actions of TGF-b downstream of BMP receptors.1,8

Correspondence: Dr U Blank, Division of Molecular Medicine and Gene Therapy, Laboratory Medicine, Lund Stem Cell Center, Lund TGF-b in hematopoiesis University Hospital, BMC A12, Lund 221 84, Sweden. E-mail: [email protected] Received 13 January 2011; revised 25 March 2011; accepted 6 April TGF-b is categorized as one of the most potent inhibitors of 2011; published online 13 May 2011 hematopoietic stem cell (HSC) growth in vitro and a large body Smad signaling in hematopoiesis U Blank and S Karlsson 1380 Ligand Activin/Nodal TGF-β BMP

Receptor

ActR-IIA BMPR-II ActR-IIB ActR-IIA β TGF- RII ActR-IIB

ALK4 ALK1 ALK2 ALK7 ALK5 P ALK3 P ALK6 P P I-Smads Smad1 Smad6 Smad2 Smad5 R-Smads R-Smads Smad7 Smad3 Smad8 SPOTLIGHT

Smad4 Co-Smad Cytoplasm

Nucleus

DNA-binding partner P R-Smad P Smad4 R-Smad Smad4 Target gene

Figure 1 The TGF-b superfamily. Schematic representation of the various components of the TGF-b-superfamily pathway, including ligands, receptors and Smads. Following ligand binding to type I and type II receptors, R-Smads become phosphorylated by activated type I receptors. Phosphorylation of R-Smads results in a conformational change allowing for complex formation with Smad4. The R-Smad/Smad4 complex subsequently translocates to the nucleus, where target gene transcription is modiﬁed in cooperation with other DNA-binding factors. P indicates phosphorylation. R-Smad denotes receptor-activated Smad; Co-Smad, common-Smad and I-Smad indicates inhibitory Smad.

4,9–11 of work from a variety of culture systems supports this notion. Vasculature Owing to the naturally quiescent state of HSCs, TGF-b has been hypothesized to be a cardinal regulator of HSC quiescence, maintaining a slow-cycling state of HSCs in vivo (Figure 2). In keeping with this, neutralization of TGF-b in vitro was shown to release early hematopoietic progenitor cells from quiescence.12–14 Several molecular mechanisms have been proposed to account HSC

for TGF-b-mediated growth inhibition, including alterations in TGFβ cytokine receptor expression and up-regulation of cyclin- Stroma cell G0 dependent kinase inhibitors, such as p15, p21 and p27.13,15–21 Osteoblast BMP Smad4 However, it has been shown that TGF-b can exert growth Smad7 inhibitory actions independently of p21 and p27.22 Addition- ally, neutralization of TGF-b coupled with antisense knockdown ALK3 of p27 was shown to result in synergistically increased retroviral gene transfer efﬁciency in human CD34 þ bone marrow (BM) cells, implying that TGF-b and p27 work in separate pathways.16 þ b In human CD34 cells, TGF- -mediated cell-cycle arrest has Bone been suggested to occur through up-regulation of p57, another 23 member of the cyclin-dependent kinase inhibitor family. This Figure 2 The BM niche. Quiescent HSCs reside in the BM endosteal ﬁnding was further corroborated by the observation that p57 region in close proximity to osteoblastic cells and other cellular and was highly enriched in mouse CD34ÀKit þ lineageÀSca1 þ structural components with supportive and regulatory functions. BMPs (CD34ÀKLS) cells as opposed to the more mature CD34 þ KLS signal via ALK3 on osteoblastic cells, regulating the size of the fraction.24 Interestingly, a high level of p57 was shown to osteoblastic niche and consequently the size of the HSC pool. Autocrine and/or paracrine TGF-b is thought to induce quiescence of correlate with the activation status of Smad2 and Smad3, HSCs, contributing to maintenance of HSCs. Loss of Smad4 in HSCs which were reported to be uniquely phosphorylated in freshly results in impaired self-renewal capacity, whereas overexpression of isolated CD34ÀKLS cells but not in CD34 þ KLS progenitors.25 Smad7 leads to increased self-renewal of HSCs.

Leukemia Smad signaling in hematopoiesis U Blank and S Karlsson 1381 In addition, TGF-b was shown to up-regulate p57 in CD34ÀKLS of redundant mechanisms in vivo, its role as a critical regulator cells in vitro.25 These findings point to a mechanism, where of HSC quiescence in vivo remains to be fully proven, despite TGF-b functions to induce p57 within the most primitive HSC intense research and increased knowledge of TGF-b signaling. compartment, thus maintaining their quiescent state in vivo. Most work regarding TGF-b in hematopoiesis has been b b carried out using TGF- 1. However, TGF- exists in three TGF-b signaling diversified: a role for transcriptional b b isoforms: TGF- 1–3. Although all TGF- s share significant intermediary factor-1c in hematopoiesis sequence homology and signal through the same receptor 26,27 complex, differing responses have been reported. Most Smad4 has traditionally been viewed as the nexus of Smad notably, KLS cells exposed to TGF-b2 exhibited a biphasic signaling, as it functions as a core component of both TGF-b/ response, being growth inhibited at high doses and stimulated at activin and BMP-signaling branches. However, Smad2/3 have low concentrations.28 The findings are intriguing as the been shown to partner not only with Smad4 but also with b stimulatory component of TGF- 2 was shown to be dependent transcriptional intermediary factor-1g (TIF1g), suggesting that on serum factors, genetic background and age.29 However, this the Smad pathway is more diversified than previously thought b finding showed bearing in vivo as Tgf- 2 heterozygote knockout (Figure 3). In a model proposed by He et al.,40 TGF-b was shown cells exhibited a defective repopulative capacity upon trans- to mediate erythroid differentiation concomitantly with balan- plantation. This effect became more pronounced following cing growth inhibition in human hematopoietic stem/progenitor serial transplantation, suggesting that TGF-b2 functions cell cells. According to this model, TIF1g selectively binds Smad2 autonomously as a positive regulator of adult HSCs that have and Smad3 in competition with Smad4. In response to TGF-b, undergone replicative stress.28 the TIF1g/Smad2/3 complex stimulated erythroid differentiation, Recently, evidence has accumulated suggesting that the adult whereas Smad2/3 in association with Smad4 led to growth HSC compartment consists of a number of functionally distinct inhibition of human hematopoietic progenitors. Thus, the subsets with diverse self-renewal and differentiation poten- relative abundance of Smad4 and TIF1g appears crucially 30–32 33 b tials. Challen et al. identified the TGF- pathway as a important for determining the precise outcome of TGF-b potential mechanism for differential regulation among discrete stimulation. Interestingly, the zebrafish homolog of TIF1g, b HSC subtypes. Specifically, TGF- stimulated proliferation of encoded by moonshine, has been shown to be essential for myeloid-biased HSCs, whereas the opposite was true for 33 blood formation with mutants displaying severe red cell aplasia, lymphoid-biased HSCs. These bidirectional effects further indicating that the function of TIF1g may be preserved across substantiate the complexity of TGF-b signaling and it remains to 41 42 species. Furthermore, Bai et al. recently uncovered a role for be clarified whether or not the Smad pathway is differentially TIF1g in regulating transcription elongation of erythroid genes. regulated at low and high doses and between diverse HSC The model proposed suggests that TIF1g functions to release subtypes. Additionally, the roles of Smad2 and Smad3 in HSCs paused Pol II at erythroid genes by recruiting positive elongation have not yet been functionally gauged in conditional knockout factors to the blood-specific transcriptional complex, thus mouse models. This will be an important in vivo system to promoting transcription.42 TIF1g also functions in a broader characterize, in order to further unwind the intricate nature of context, as it was recently shown to have a role in erythroid/ TGF-b signaling in HSCs.

TGF-β TGF-b: lessons from in vivo models

TGF-b can affect most cell types throughout the hematopoietic hierarchy and depending on the context and differentiation stage of the target cell, different biological responses are elicited.4–6 In vivo, TGF-b has a principal role as regulator of β immune cell homeostasis and function, as unequivocally shown TGF- RII ALK5 by the development of a lethal inflammatory disorder in both ? Tgf-b1-ligand and receptor knockout mice.34–36 Furthermore, SPOTLIGHT Tgf-b1 null mice exhibited enhanced myelopoiesis, suggesting Smad2/3 Smad7 that TGF-b acts as a negative regulator of myelopoiesis in vivo.35 TAK1 When Tgf-b1 knockout mice were analyzed before the onset of multifocal inflammation, a range of HSC properties were shown p38 TIF1γ Smad4 to be altered.37 Most significantly, BM cells from Tgf-b1- deficient neonates exhibited impaired reconstitution ability JNK upon transplantation, a finding attributed to defective homing.37 In contrast, mice deficient in the TGF-b type I receptor (TbRI) Differentiation Growth inhibition displayed normal HSC self-renewal and regenerative capacity in vivo, even under extreme hematopoietic stress.38,39 Thus, there are both overlapping and non-overlapping phenotypes between Figure 3 Summary of TGF-b signaling. TGF-b signaling results in ligand and receptor knockout models and it appears to be growth inhibition of HSCs when Smad2/3 partners with Smad4. In significantly important at which level TGF-b signaling is human cells, binding of TIF1g to Smad2/3 stimulates erythroid disrupted. The apparent discrepancies related to in vitro and differentiation in response to TGF-b. Activation of non-canonical mitogen-activated protein kinase (MAPK)-signaling downstream of in vivo findings may reflect redundant functions of other type I TGF-b has been shown in other cell types, but is largely unexplored in receptors or alternatively other ligands, such as activin, which HSCs. However, TGF-b-activated kinase1 (TAK1) is expressed in the signals through the same R-Smad pathway. Due to the multi- KLS (Kit þ lineageÀSca1 þ ) compartment and is essential for HSC faceted nature of TGF-b coupled with a potentially complex set survival in vivo.

Leukemia Smad signaling in hematopoiesis U Blank and S Karlsson 1382 myeloid lineage bifurcation by modulating GATA1 and PU.1 lack of ventral mesoderm development and a complete absence expression.43 The role of the Smad pathway in these processes of blood cells.55 Additionally, to block BMP signaling after its has not been investigated and what signaling pathways function initial requirement during specification of mesoderm, Schmerer to control transcription elongation of blood genes remains to be and Evans56 devised a model where activity of the inhibitory determined. It is, however, interesting to note that the BMP Smad6 could be induced after gastrulation in Xenopus explants. pathway has been previously linked to stress erythropoiesis, as Using this approach, it was shown that Smad signals are will be discussed below.44,45 The mechanism by which TIF1g required for primitive erythropoiesis and maintenance of Gata1 cooperates with Smads continues to be a controversial issue. expression within specified mesoderm. Furthermore, when BMP Developmental studies from a variety of species have suggested signaling was disrupted in lateral mesoderm in the zebrafish that TIF1g, also known as Ectodermin, acts as a ubiquitin ligase model, by targeting a dominant-negative BMP receptor to for Smad4, thus functioning as a direct inhibitor of Smad4 Lmo2 þ cells, it was concluded that BMP signaling continues to downstream of TGF-b and BMP signaling.46,47 Mice deficient in function in the regulation of lineage specification after lateral TIF1g die during early somitogenesis, displaying phenotypes, mesoderm commitment.57 However, at this stage, BMP signal- which are consistent with excessive TGF-b/nodal signaling, ing functions to restrict hemato-vascular fate in favor of supporting a role for TIF1g as negative regulator of Smad4.48 pro-nephric development,57 indicating that the effect of BMP How these seemingly disparate molecular mechanisms can be signaling on hematopoiesis is affected by the exact stage of reconciled will require further investigation, but it is possible development. Moreover, in both murine and human embryonic that temporal aspects and differences in cellular context have stem cells, exposure to BMP4 has been reported to induce critical roles in determining the precise role of TIF1g. Regardless mesoderm formation, including hematopoietic commitment.58–61 SPOTLIGHT of the exact molecular mechanism, accumulated data suggest BMP4 has also been shown to enhance hematopoietic develop- that TIF1g functions to restrict Smad-signaling downstream of ment of rhesus monkey embryonic stem cells.62 Data obtained TGF-b/nodal. from the murine embryonic stem cell system further supports that BMP-signaling functions in a two-step manner, its role in mesoderm patterning being separable from its function in blood BMP signaling in hematopoietic development fate specification.63 Thus, in vertebrates, embryonic hematopoiesis depends on BMP signaling in a mechanism that appears to BMPs figure early during development as morphogens regula- be independent from its role in mesoderm patterning. ting mesoderm patterning. In mice, targeted deletions of a variety of BMP-signaling components, including Bmp2, Bmp4 and BmpRIa, resulted in severe mesoderm deficiency and Modeling BMP deficiency in vivo embryonic lethality.49–51 Because blood is derived from mesoderm, BMPs have been implicated as key regulators of The role of Smad-mediated BMP signaling in hematopoiesis has blood formation during embryonic development. However, recently been investigated in both mouse and zebrafish model since BMP signaling is required before the onset of hemato- systems, using mutants and targeted deletions of Smad1 and poietic development, the exact role of BMPs in hematopoietic Smad5 (Table 1). Intriguingly, data accumulated suggest that this induction has been challenging to study particularly in the pathway is subject to considerable species and temporal mouse. Therefore, much of the initial knowledge on the role of variation with a seemingly more pronounced role for Smad1 BMP signaling in hematopoietic development has been derived and Smad5 in zebrafish as compared with the mouse. Using from studies in lower vertebrates and from culture systems both loss-of-function approaches and hypomorphic mutants, in vitro.52 For example, studies performed in Xenopus, have the roles of Smad1 and Smad5 were studied in zebrafish.64 revealed an important role for BMP4 in induction of hema- Interestingly, distinct phenotypes were generated with respect to topoiesis.53,54 In zebrafish, BMP2 and BMP7 mutants displayed primitive erythropoiesis. While smad1 morphants exhibited

Table 1 Smad knockout/overexpression or mutant models and associated hematopoietic phenotypes

Smad signaling Organism Model system Hematopoietic phenotype Reference component

Smad1 Mouse Conditional knockout, Mx-Cre Normal HSC properties. 67 Smad1 Zebrafish Morpholino knockdown Enhanced primitive erythropoiesis, failure to 64 generate definitive hematopoietic progenitors. Smad5 Zebrafish Piggytail mutant, morpholino knockdown Defective primitive erythropoiesis, failure to 64 generate definitive hematopoietic progenitors. Smad5 Mouse Conditional knockout, Mx-Cre, Vav-Cre Normal adult HSC parameters. Unperturbed fetal 67,68 liver hematopoiesis. Smad5 Mouse Flexed-tail mutant Defective erythroid response during acute 44 anemia. Smad1/5 Mouse Conditional knockout, Mx-Cre, Vav-Cre Reduced myeloid component in PB upon 67 transplantation of fetal liver cells, but otherwise normal adult and fetal liver hematopoietic parameters. Smad4 Mouse Conditional deletion, Mx-Cre Impaired self-renewal of HSCs. 77 Smad7 Mouse Retroviral overexpression Enhanced self-renewal of HSCs in vivo. 75 Smad7 Human/ Retroviral overexpression in cord blood Altered cell fate, favoring myeloid commitment 76 xenograft SCID-repopulating cells over lymphoid. Abbreviations: HSC, hematopoietic stem cell; PB, peripheral blood.

Leukemia Smad signaling in hematopoiesis U Blank and S Karlsson 1383 enhanced erythropoiesis, knockdown of smad5 resulted in a has previously been shown to maintain and expand HSCs in failure to maintain the erythroid program and thus erythropoiesis long-term cultures.72 Interestingly, AFT024 cells were shown to failed.64 Both genes, however, were shown to be required for produce BMP4 and this contributed significantly to maintenance definitive hematopoiesis, as deficiency of either Smad1 or of co-cultured human hematopoietic progenitors from cord Smad5 caused a failure in the generation of definitive blood (CB).73 In the murine system, BMP4 does not appear to hematopoietic progenitors.64 To what extent this is a cell- affect proliferation of purified HSCs in vitro, although it is autonomous effect is not clear, as Hild et al.65 previously currently unclear whether BMP4 can extend the maintenance of showed that transplantation of somitabun (a Smad5 dominant- murine HSCs in culture, as this study did not assess in vivo negative mutation) mutant cells survived and formed blood reconstitution ability following BMP4 exposure.74 tissue in a wild-type environment. In the zebrafish model system, the initial requirement for BMP signaling can be overcome by injection of mRNA of the wild-type gene into Complete disruption of the Smad pathway fertilized eggs, thus allowing analysis of hematopoietic parameters in adult fish. Several allelic mutations of smad5 were To block the entire Smad-signaling network and to sidestep studied, revealing anemia with mutants exhibiting decreased potentially redundant mechanisms within this circuitry, two numbers of erythroid progenitors.66 Additionally, smad5 parallel approaches have been used: overexpression of the mutants had an altered response to hemolytic anemia, indicat- inhibitory Smad7 and deletion of Smad4. Smad7 was over- ing that Smad5 may be involved in regulating the kinetics of expressed in murine HSCs using a retroviral gene transfer recovery under conditions of acute anemia.66 This is particularly approach.75 Forced expression of Smad7 resulted in significantly interesting in the context of the flexed-tail mouse mutant, which increased self-renewal capacity of HSCs in vivo, indicating that carries a spontaneous mutation in the Smad5 gene. Neonatal the Smad pathway negatively regulates self-renewal in vivo. mutant mice are anemic, but recover in adulthood, except under Importantly, differentiation was unperturbed, suggesting that conditions of stressed hematopoiesis.44 Mice carrying the self-renewal is regulated independently of differentiation by flexed-tail mutation could not mount an effective response to Smad signaling. When a similar strategy was used in human acute anemia due to a specific defect in splenic erythropoietin- SRCs, overexpression of Smad7 resulted in altered differentia- responsive progenitors to respond to BMP4.44 Subsequently, it tion from lymphoid dominant engraftment toward increased was shown that stem cell factor and hypoxia synergize with myeloid contribution.76 Thus, in the xenograft model system, BMP4 to drive effective recovery upon stress anemia.45 forced expression of Smad7 modulates differentiation of Although acute anemia has never been studied in the context primitive multipotent human SRCs. Using a conditional knock- of complete deletion of Smad5 in the mouse, these studies stand out mouse model, disruption of the entire Smad pathway at the in sharp contrast to studies performed by Singbrant et al.Ina level of Smad4 was recently investigated. Intriguingly, Smad4- series of experiments, it was shown that Cre-mediated deletion deficient HSCs displayed a significantly reduced repopulative of Smad1 and/or Smad5 did not impair adult hematopoiesis in capacity of primary and secondary recipients, indicating that the mouse and mutant HSCs displayed normal self-renewal and Smad4 is critical for HSC self-renewal in vivo.77 Since differentiation capacities upon transplantation.67,68 Similarly, overexpression of Smad7 vs. deletion of Smad4 would be using a fetal liver-specific Cre-driver to induce deletion of anticipated to yield similar hematopoietic phenotypes, it is Smad5 or Smad1/Smad5 together, hematopoiesis was shown to conceivable that Smad4 functions as a positive regulator of self- occur normally upon transplantation into wild-type hosts.67 renewal independently of its role as a central mediator of the These differences are intriguing, but may suggest that canonical Smad pathway. The precise molecular mechanism for Smad-mediated BMP signaling is only required under very this is currently unknown, but it is possible that Smad4 specific stressed conditions. Taken together, the canonical BMP- participates in other signaling cascades, such as Wnt or signaling pathway does not seem to regulate critical aspects of Notch.78–80 HSC biology in the adult mouse, in spite of its pivotal function in earlier developmental events. However, BMPs are present in the BM, and a role for BmpRIa has been established in Smad signaling in hematopoietic malignancies the osteoblastic niche.69 BmpRIa-deficient mice exhibited an increase in the number of N-cadherin þ osteoblastic cells The role of TGF-b in hematologic malignancies has been resulting in an increase in the number of HSCs.69 Thus, BMPs reviewed in detail, including the role of TGF-b in leukemia, SPOTLIGHT are indirectly involved in regulating HSC frequency in adult lymphoma/lymphoproliferative disorders, multiple myeloma, mice. myeloproliferative diseases and myelofibrosis.81 Here, we will review briefly the role of TGF-b in hematologic malignancies and myelofibrosis. BMP signaling ex vivo Despite the pronounced anti-proliferative effect of TGF-b on HSCs in vitro and the fact that alterations in genes encoding In vitro, BMP4 has been shown to promote maintenance of components of the TGF-b pathway are frequently found in many human HSCs in culture, whereas lower concentrations of BMP4 epithelial neoplasms, such as pancreatic and colon cancer,82,83 induced proliferation and differentiation of human hematopoie- mutational inactivations involving the TGF-b-signaling pathway tic progenitors.70 Furthermore, Shh induced proliferation of are uncommon in leukemias and other hematological malig- primitive human hematopoietic progenitors in vitro, apparently nancies (reviewed in Kim and Letterio84). A potential role for through a BMP4-dependent mechanism.71 However, while TGF-b as a tumor suppressor has been demonstrated in vivo, BMP4 has been shown to maintain human nonobese diabetic/ where heterozygous knockout mice for Tgf-b1 developed severe combined immunodeficient repopulating cells (SRCs) increased numbers of lung and liver tumors upon exposure to in culture, it does not seem to cause an expansion, sugges- carcinogenic stimuli.85 In addition, Smad3 homozygous knock- ting that Shh may act through additional mechanisms. out mice live to adulthood, but spontaneously develop meta- The murine fetal liver stromal cell line AFT024 static colorectal cancer, clearly supporting a role for TGF-b

Leukemia Smad signaling in hematopoiesis U Blank and S Karlsson 1384 signaling in tumor suppression.86 The lack of leukemogenesis in Several cytokines have been reported to contribute toward our TGF-b-signaling-deficient mouse models implies that loss of accumulation of reticulin fibers in the BM of patients responsiveness to TGF-b is more important for progression rather with myelofibrosis. These include TGF-b, mainly TGF-b1, than initiation of leukemogenesis.38,67,75,77 These findings basic fibroblast growth factor and platelet-derived growth suggest that deficient TGF-b signaling alone is not sufficient to factor.105–108 Some of the strongest evidence for a prominent induce neoplastic transformation in hematopoietic cells. role of TGF-b in the generation of myelofibrosis involved the use Although loss-of-function mutations that disrupt the TGF-b of BM cells from Tgf-b1 null mice. To induce myelofibrosis, pathway are rare in hematological malignancies, a number of irradiated mice were transplanted with BM cells transduced with cases have been reported involving SMAD4 and TGFbRII in vectors containing the thrombopoietin gene. Importantly, patients with acute myelogenous leukemia (AML).87–90 Using prominent myelofibrosis only developed in mice receiving ultra-dense array comparative genomic hybridization on 86 transduced wild-type cells, but not Tgf-b1 null cells.107 These AML genomes, 18 copy number alterations regions were found data indicate that TGF-b1 produced by hematopoietic cells is a recurrently modified, one of which represented the deletion of vital component in the development of myelofibrosis. the SMAD4 gene, demonstrating that SMAD4 can be an AML- associated gene (Walter, PNAS, 2009). Furthermore, sporadic mutations in both TbRI and TbRII have been reported in Stem cell expansion toward advanced cell therapy lymphoid neoplasms.91,92 Another study reported that the SMAD3 protein could not be detected in fresh samples from One of the most important therapeutic modalities in hematology is patients with T-cell acute lymphocytic leukemia. The mechan- blood and marrow transplantation to cure leukemia and genetic SPOTLIGHT ism for the SMAD3 deficiency is not known, since the SMAD3 disorders. The process of finding donors that have compatible mRNA was present and no mutations could be detected in the histocompatibility antigens for patients that need blood and MADH3 gene, which encodes SMAD3.93 However, T-cell marrow transplantation is often a challenge, and a limiting factor leukemogenesis was promoted in mice with haploinsufficiency in current cell therapy is the shortage of available donors. As of of Smad3 and a complete deficiency of p27.93 These findings today, umbilical CB is being used increasingly as a source of HSCs are interesting because the p27 gene is frequently mutated in due to the common availability of CB cells and the diversity of pediatric acute lymphocytic leukemia, due to translocations and histocompatibility gene haplotypes that are available in banked deletions or germline mutations.94,95 Impaired TGF-b signaling CB samples.109 However, the number of HSCs in each CB sample in hematologic malignancies can also be caused by suppression is limited and it would, therefore, greatly increase the applicability of Smad-dependent transcriptional responses by oncoproteins of CB-derived HSCs if efficient expansion could be safely like TAX, EVI-1 and AML1-ETO.96–98 Similarly, down-regulation achieved ex vivo prior to transplantation. In order to achieve of the transcription factor ZEB1 and overexpression of Smad7 stem cell expansion, a detailed understanding of cell signaling is contribute to resistance to TGF-b1-mediated growth suppression required, including not only Smad signaling but also other major in adult T-cell leukemia/lymphoma without known mutations in signaling pathways. Therefore, we will discuss the possible role of TGF-b pathway genes.99 In addition, there have been a number Smad signaling and that of other pathways in future efforts to of reports on oncoproteins, which generate leukemias and expand stem cells in vitro. simultaneously neutralize the growth inhibitory signal of the Successful stem cell expansion involves symmetric self- Smad pathway by binding or interacting with Smads. Fusion renewal divisions of HSCs, where both daughter cells retain oncoproteins like TEL-AML1 and AML1-EVI1 have been shown HSC properties.110 More commonly, HSCs grown in vitro to bind to Smad3, impairing both TGF-b signaling and apoptosis undergo asymmetric divisions characterized by the production of transduced HSCs in vitro.97,100–102 Furthermore, Smad4 has of one HSC and a more differentiated progenitor, or alterna- been shown to physically associate with HoxA9, thus reducing tively, a symmetric division where both progeny cells have lost its ability to regulate transcriptional targets in hematopoietic their HSC potential. Although BMPs have been reported to cells in vitro.103 Recently, in vivo studies have shown that in contribute to HSC expansion ex vivo, manipulations of the wild-type mice overexpressing HoxA9 or Nup98-HoxA9, Smad-signaling pathway alone are not likely to result in effective Smad4 binds to the oncoproteins and sequestrates them to the stem cell expansion.70,71 Rather, stimulation of several signaling cytoplasm, suggesting a protective role of Smad4 against further circuitries and suppression of other pathways will be essential to promotion and growth of leukemic cells.104 Therefore, Smad generate stem cell expansion in vitro prior to transplantation in signaling is often reduced or neutralized in hematopoietic clinical settings. Positive and negative regulators ultimately malignancies, but in a majority of cases, this is not due to balance the transition from quiescence to proliferation of HSCs. primary mutations in genes encoding proteins of the Smad Thus, the strategies for stem cell expansion should involve circuitry. Rather, malignant cells may exploit other mechanisms activation of regulators that encourage HSC self-renewal and/or to reduce Smad signaling indirectly, through altered expression inhibition of pathways that mediate quiescence, differentiation or function of co-factors and oncoproteins, or alternatively via or apoptosis of HSCs. Some approaches, such as overexpression loss of or disruption of TGF-b target genes. of HoxB4, require viral vector-mediated gene transfer to HSCs Although TGF-b has a major role as tumor suppressor, TGF-b for efficient expansion.111 However, this strategy is not likely to can also paradoxically facilitate tumor growth, particularly in be accepted for clinical therapy due to the risk of insertional the later stages of the disease. This is due to effects on the tumor mutagenesis.112,113 The safest approaches would involve microenvironment and the immunosuppressive function of soluble factors, for example cytokines, developmental cues or TGF-b, rendering the patient/animal with reduced possibilities components, such as angiopoietin-like proteins. to reject tumor cells by immunological means. Developmental cues that activate Notch and Wnt signaling in Myelofibrosis with myeloid metaplasia is a chronic myelo- HSCs have been shown to affect self-renewal positively ex vivo. proliferative disease characterized by clonal myeloproliferation Most notably, Wnt3A was shown to expand murine repopu- and reactive BM fibrosis.81 Myelofibrosis appears in the later lating HSCs and injection of Wnt5A into nonobese diabetic/ stages of chronic myeloid leukemia and polycytemia vera, while severe combined immunodeficient mice repopulated with it occurs early in myelofibrosis with myeloid metaplasia. human hematopoietic cells increased the reconstitution and

Leukemia Smad signaling in hematopoiesis U Blank and S Karlsson 1385 number of primitive hematopoietic cells.114,115 Similarly, a Conflict of interest soluble form of the Notch ligand, Jagged 1, was reported to stimulate growth of human HSCs and may, therefore, be used to The authors declare no conflict of interest. expand stem cells ex vivo.116 However, the angiopoietin-like proteins, angiopoietin-like 2 and 3, are by far the most promising soluble factors identified to date for expansion of Acknowledgements murine HSCs.117 Using cultures containing angiopoietin-like 5 together with stem cell factor, thrombopoietin, fibroblast growth This work was supported by the European Commission (Stemex- factor-1 and insulin growth factor-binding protein 2, the number pand); Swedish Medical Research Council; Swedish Cancer of SRCs could be expanded by a factor of 20.118 Clinical benefits Society; Swedish Children Cancer Foundation; Clinical Research can also be achieved by expanding hematopoietic progenitors Award from Lund University Hospital and a grant from The Tobias ex vivo to prevent delayed myeloid engraftment following HSC Foundation awarded by the Royal Academy of Sciences. (SK). The transplantation. Engineered Notch ligand attached to tissue Lund Stem Cell Center is supported by a Center of Excellence culture plates was used to significantly expand SRCs and was grant in life sciences from the Swedish Foundation for Strategic also used in a clinical trial to improve engraftment and prevent Research. delayed myeloid reconstitution following transplantation of CB CD34 þ cells.119 As more detailed knowledge is unearthed concerning the regulatory pathways that govern HSC self- References renewal, it may be possible to modulate these pathways with small molecule drugs. Recently, it was demonstrated that 1 Massague J. TGF-beta signal transduction. Annu Rev Biochem chemicals that increase prostaglandin E2 synthesis could expand 1998; 67: 753–791. 120 HSC numbers in both zebrafish and mice. Similarly, a 2 Gordon KJ, Blobe GC. Role of transforming growth factor-beta chemical screen identified a purine derivative, StemRegenin 1, superfamily signaling pathways in human disease. Biochim which was shown to promote ex vivo expansion of human Biophys Acta 2008; 1782: 197–228. CD34 þ cells and SRCs.121 In the future, more detailed 3 Massague J. TGFbeta in cancer. Cell 2008; 134: 215–230. information on single-signaling pathways that determine cell 4 Fortunel NO, Hatzfeld A, Hatzfeld JA. Transforming growth factor-beta: pleiotropic role in the regulation of hematopoiesis. fate options will be required, in order to increase our Blood 2000; 96: 2022–2036. understanding of how integration of major signaling modules 5 Larsson J, Karlsson S. The role of Smad signaling in hematopoi- may be exploited in vitro. Such increased knowledge will open esis. Oncogene 2005; 24: 5676–5692. up new avenues for maintaining and expanding HSCs in vitro. 6 Ruscetti FW, Akel S, Bartelmez SH. 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