Myeloid leukaemias share the common characteristics of being stem cell-derived clonal diseases, characterised by excessive proliferation of one or more myeloid lineage. Chronic myeloid leukaemia (CML) arises from a genetic alteration in a normal haemopoietic stem cell (HSC) giving rise to a leukaemic stem cell (LSC) within the bone marrow (BM) ‘niche’. CML is characterised by the presence of the oncogenic tyrosine kinase fusion protein breakpoint cluster region-abelson murine leukaemia viral oncogene homolog 1 (BCR-ABL), which is responsible for driving the disease through activation of downstream signal transduction pathways. Recent evidence from our group and others indicates that important regulatory networks involved in establishing primitive and definitive haemopoiesis during development are reactivated in myeloid leukaemia, giving rise to an LSC population with altered self-renewal and differentiation properties. In this review, we explore the role the bone morphogenic protein (BMP) signalling plays in stem cell pluripotency, developmental haemopoiesis, HSC maintenance and the implication of altered BMP signalling on LSC persistence in the BM niche. Overall, we emphasise how the BMP and Wnt pathways converge to alter the Cdx–Hox axis and the implications of this in the pathogenesis of myeloid malignancies.
The bone morphogenic protein pathway
The bone morphogenic protein (BMP) ligands are part of the transforming growth factor-β (TGFβ) superfamily, which includes TGFβs, Activins/Inhibins, Nodal, Myostatin, and Anti-Mullerian Hormone. BMP ligands possess different affinities for the BMP receptors, which determine their specificity of action [1,2]. Activation of the BMP pathway occurs either in a canonical SMAD-dependant or SMAD-independent manner. The former is dependent on SMAD proteins for regulation, whereas the latter involves extracellular signal-regulated kinases, Janus N-terminal kinase, and p38 mitogen-activated protein kinase (MAPK) pathways. The canonical BMP pathway relies on a series of signal transduction steps, which is initiated when BMP ligands bind to type II receptors. Ligand binding then leads to the heterodimerization of the type I and type II receptors. This, in turn, phosphorylates the corresponding receptor-regulated SMADs (R-SMADs) and drives the formation of an R-SMAD–Co-SMAD complex. This complex translocates to the nucleus where it binds to relevant transcription factors/initiators, resulting in the transcription of BMP target genes involved in the cell cycle, proliferation, and self-renewal [1,2].
The role of BMP pathway in the maintenance of pluripotency
Human and mouse embryonic stem cells (ESCs) maintain their pluripotency through the expression of a complex transcription factor network with the key transcription factors being Oct4, Nanog, Sox2, Klf4, Rex1, and Cited2 . Several signalling pathways co-ordinate this transcriptional network, with the TGFβ superfamily signalling pathway contributing to the maintenance of pluripotency and early differentiation decisions . Transcriptome analyses of human ESCs (hESCs) cultured on feeder cells revealed that these cells require signals mediated by fibroblast growth factor 2 (FGF2), activin, TGFβ, insulin, and insulin-like growth factor (IGF) to maintain their undifferentiated state . In hESCs, TGFβ and FGF2 co-operate to inhibit BMP signalling and sustain the expression of pluripotency markers, especially via activated SMAD3 binding to the NANOG promoter. Activation of SMAD2/3 signals is required as a downstream event to maintain the undifferentiated state of hESCs [4,6]. On the other hand, the proximal NANOG promoter contains the TGFβ/activin responsive element, which is negatively regulated by signals from the BMP pathway. Xu et al. demonstrated that culturing hESCs in high amounts of FGF2 in combination with inhibiting the BMP pathway using the BMP antagonist noggin (NOG) maintains pluripotency and long-term, undifferentiated proliferation of hESCs [4,7]. FGF2 maintains hESCs by activating the MAPK pathways, whereas the signals mediated by Activin/Nodal and IGF maintain pluripotency via activation of Ras and phosphatidylinositol-3 kinase (PI3K) pathways [4,7] (Figure 1).
Maintenance of pluripotency in mouse and human stem cells.
In murine ES cells (mESCs), self-renewal capacity can be maintained by the cytokine leukaemia inhibitory factor (LIF) . However, the presence of LIF is not totally sufficient as the cells also require the presence of foetal calf serum to maintain pluripotency. It was shown that the BMPs present in the serum are the key additional signalling molecules that act in conjunction with LIF, enhancing the self-renewal and pluripotency of mESCs [4,9]. Further work revealed that activation of the BMP pathway through BMP4 stimulation leads to the expression of inhibitor of differentiation gene 1 (Id1), critical for suppressing ESC differentiation and sustaining pluripotency . This is supported by other studies showing that overexpression of Id1 encourages mESCs to self-renew in the absence of BMP4, with suppression of Id1 leading to decreased Nanog expression, which results in the failure of mESCs to maintain self-renewal .
BMP pathway and the initiation and maintenance of embryonic haemopoiesis
BMP signalling plays a key role during early embryogenesis and subsequent organogenesis. The BMP and Wnt pathways are essential for mesodermal patterning and subsequent specification of haemopoiesis in the developing embryo . Haemopoietic and endothelial cells are among the earliest cells to form in the embryo, with BMP pathway involvement in the initiation of this process conserved throughout evolution [11,12]. These cells arise during gastrulation from mesodermal progenitors in the epiblast . Studies using targeted gene disruption in murine models revealed that BMP2 , BMP4 , anaplastic lymphoma kinase 3 (ALK3)  and BMP type II receptor (BMPRII)  play important roles in early haemopoiesis as all these defects cause early embryonic lethality and reduced formation of mesoderm. In particular, BMP4 plays a key role in mesoderm induction and haemopoietic commitment, and is fundamental for inducing haemopoietic differentiation in murine  and human  ESCs in vitro. In mESCs, BMP4 induces the formation of mesoderm and early haemopoietic precursors, whereas inhibition of BMP signalling impaired haemopoietic development [17,19]. In hESCs, BMP4 in combination with cytokines also enhanced haemopoietic differentiation . Developmental studies indicate that the BMP and Wnt pathways converge to regulate the Cdx family of homeobox transcription factors, master regulators of Hox gene expression [20,21]. Activation of BMP pathway induces Wnt3a and thereby the canonical Wnt pathway, which in turn activates the Cdx–Hox axis . Furthermore, in mESCs, overexpression of Cdx1 and Cdx4 induces Wnt3a, suggesting that Wnt signalling also acts downstream of Cdx proteins . Studies from zebra fish and mESCs indicate that Cdx-driven patterning is necessary for the development of mesoderm derivatives such as the haemopoietic system [20,21]. During early development, Cdx genes show a similar expression pattern to Hox genes. In mice, this expression is detected in the posterior epiblast and the overlaying mesoderm at the posterior end of the primitive streak. As reviewed by Lengerke and Daley  during the development in the posterior trunk zone, anterior trunk tissues are exposed to Cdx genes but as cells move anteriorly, Cdx transcripts decrease. Cdx genes expression persists in the posterior region of the embryo. This is then followed by the expression of more posterior Hox genes, which enables the development of posterior trunk mesoderm and tail. Cdx4-deficient mice have been shown to produce reduced numbers of yolk sac-derived erythroid colonies .
Primitive haemopoiesis during murine development takes place in the yolk sac between E7 and E11, whereas in the human system primitive erythropoiesis is initiated in the yolk sac approximately week 3 of gestation and remains active until week 6. During mammalian development, BMP4 is involved in the induction of the yolk sac haemopoietic programme. It has been shown that signals from the visceral endoderm, such as Indian hedgehog (Hh), are critical for the development of the yolk sac haemopoietic programme and the regulation of BMP4 expression . Mouse studies illustrate that BMP4 knockout mice die between E7.5 and E9.5 exhibiting severe defects in mesoderm formation, and the embryos that survive up to E9.5 show defective blood islands . Studies on other components of the BMP pathway further highlight the role of this family in haemopoietic development. Disruption of the Smad5 gene in mice leads to severe defects in yolk sac circulation and subsequent death around mid-gestation . Yolk sacs from these embryos (Smad5−/− E9.0–9.5 yolk sac) have increased numbers of high-proliferative potential colony forming cells with enhanced re-plating potential. These data suggest that Smad5 transduces signals that inhibit the early specification events of haemopoietic precursors as well as their subsequent expansion .
Primitive yolk sac haemopoiesis is then replaced by the definitive, haemopoietic stem cells (HSCs) derived multilineage blood system that is sustained throughout life . The area of the embryo known as the para-aortic splanchnopleura (P-Sp), E8.5–9.5 in the mouse, is the first site to produce haemopoietic progenitors with myeloid and lymphoid potential ; however, these cells are unable to sustain long-term reconstitution of irradiated recipients . The P-Sp gives rise to the developing aorta, gonads, and mesonephros (AGM) region by E10–11.5 . The AGM is the site where the first definitive HSCs arise, these emerge as clusters of cells from the ventral wall of the dorsal aorta from E10.5, and these cells are multipotent and display long-term reconstitution potential [27–29]. In mouse and human embryos, BMP4 is expressed in the mesenchyme underlying the aorta and is required for HSC maintenance [30–32]. It has been shown using BMP responsive element green fluorescent protein (GFP) transgenic mouse embryos that the first HSCs emerging in vivo in the AGM region are GFP+, i.e. BMP activated, and have long-term reconstituting potential. However, the proportion of BMP-activated HSCs decreases through ontogeny, with approximately three quarters of the HSCs in the foetal liver (FL) being GFP+ BMP activated, whereas the majority of HSCs found in the bone marrow (BM) are GFP−, i.e. non-BMP activated. The two types of HSCs found in the FL and BM display distinct differences in their lineage output and their intrinsic molecular programmes, with BMP-activated HSCs favouring a more myeloid–lymphoid balanced differentiation programme . Explant studies using E11 AGM have revealed that over the 3-day culture period GFP− non-BMP-activated HSCs emerge and these cells are controlled by the Hh/vascular endothelial growth factor (VEGF) pathway. Elegant transplantation experiments and transcriptome data indicate that the GFP+ BMP-activated HSCs can give rise to the GFP− Hh/VEGF responsive HSCs, with the GFP+ expressing a more haemogenic endothelial molecular programme, whereas the GFP− express a haemopoietic programme . As in the mouse, the AGM region in humans contains definitive haemopoietic cells prior to their detection in the FL . Between E12 and E13 in the mouse, the HSCs migrate and populate the developing FL, which becomes the principal site of haemopoiesis throughout the remainder of foetal life. Whereas in humans the FL continues as a major site of haemopoiesis until week 22, by which time the foetal bones, which start to become colonised by HSCs in weeks 8–12, then takes over as the major site of haemopoiesis . By birth, the BM is established as the major site of haemopoiesis and continues as the major source of haemopoietic cells throughout life. In adults, the embryonic morphogens, including the TGFβ superfamily, play an important role in maintaining haemopoietic homeostasis. Osteoblasts, stromal cells, megakaryocytes, and platelets all secrete BMPs, which have been implicated in the self-renewal and maintenance of HSCs, as well as regulating the expansion of progenitor cells and their differentiation into the mature lineages of the haemopoietic system  (Figure 2).
Haemopoietic development timeline in mouse and human.
Reactivation of embryonic pathways in leukaemia
HSCs have three key properties of long life, multipotency, and self-renewal. Within the BM, control of steady-state haemopoiesis is regulated by extrinsic and intrinsic factors that control the balance between HSC self-renewal and differentiation. Cell extrinsic cues are governed by the stem cell niche and include growth factors, cytokines, chemokines, oxygen tension, nutrients, and cell-to-cell interactions with stroma, endothelium, osteoblasts, and the extracellular matrix. These signals merge into a network of intrinsic regulators, resulting in the activation of signalling pathways, induction of transcription factors, and changes to epigenetic marks [37,38]. All of which govern the balance between self-renewal and differentiation of HSCs. Recent evidence indicates that important regulatory networks involved in establishing primitive and definitive haemopoiesis during development are reactivated in leukaemias, giving rise to a leukaemic stem cell (LSC) population with altered self-renewal and differentiation properties. In particular, altered activation of the conserved embryonic morphogenic pathways, including Wnt, Hh, BMP, TGFβ, and Notch as well as the Cdx–Hox transcription factors and the BMI1/polycomb transcriptional regulators, is becoming an emerging theme in myeloid leukaemia . In mixed lineage leukaemia (MLL), it has been shown that the LSCs are maintained in a self-renewing state through expression of a transcription programme more akin to ESCs than adult stem cells, and that the LSCs occupy the apex of a leukaemia cell hierarchy, supporting the cancer stem cell model of disease .
Role of BMP pathway in leukaemia and LSCs
BMP ligands are naturally produced and secreted by cells in the BM microenvironment . There are more than 500 genes controlled by BMP and TGFβ pathways, which are involved in HSC maintenance and self-renewal . TGFβ signalling is important for HSC quiescence through its antiproliferative properties, as well as immune cell homeostasis and its ability to decrease meylopoiesis. BMPs are known to regulate HSCs self-renewal and differentiation through intrinsic and extrinsic mechanisms . BMP2, BMP4, BMP7 and Smad proteins have been implicated in the self-renewal and maintenance of HSCs, expansion of progenitor cells, and their differentiation into the mature lineages of the haemopoietic system [42,43]. In normal haemopoiesis, BMP2, BMP4, and BMP7 regulate proliferation, maintenance, clonogenicity, and repopulating activities of immature blood progenitors .
Given the important roles, BMPs play in stem cell biology and haemopoiesis, it is surprising that deregulation of this pathway has only been shown to occur in two types of leukaemia to date, namely acute promyelocytic leukaemia (APL)  and chronic myeloid leukaemia (CML) [36,46]. However, recent research has started to focus on the leukaemic BM niche, especially the leukaemic endosteal niche. In many myeloid leukaemias, there is enhanced osteoblastic proliferation and a marked increase in LSCs and progenitor expansion [37,46]. LSCs strongly rely on the BM niche for their self-renewal and proliferation and may also modify it to their advantage. Recent research on the role of the microenvironment in CML confirms elevated levels of BMPs in the BM of chronic phase (CP) patients . The present study illustrates that deregulation of the BMP pathway may lead to HSC expansion due to exposure of CD34+ cells, which overexpress BMPR1B (ALK6) to the increased amount of soluble BMP2/ BMP4 in the BM . Moreover, transcriptome studies of CML LSCs and progenitors indicate that the TGFβ and BMP pathways are down-regulated in CP CML, which suggests an extrinsic mechanism for TGFβ involvement in this disease . Deregulation of this pathway has also been shown in murine models where Bmpr1a/Alk3 conditional knockout mouse have impaired BMP signalling, which leads to increased niche size and thereby enhanced numbers of HSCs .
Although the BMP pathway has not been directly linked to many myeloid malignancies, its downstream target genes, the Cdx–Hox axis are an emerging theme in leukaemia (Figure 3). It is known that Wnt and BMP signalling converge to regulate the Cdx family of homeobox transcription factors, master regulators of Hox gene expression [21,48]. Wnt/glycogen synthase kinase 3 (GSK3)β/-βcatenin signalling, which plays an important role in HSC homeostasis, is altered in blast crisis CML [37,49]. Increased levels of active β-catenin and components of the Wnt pathway have also been shown to occur in acute myeloid leukaemia (AML) patients, with the enhanced re-plating capacity of these LSCs abrogated by inhibiting Wnt signalling [37,50]. Consensus binding sites for the three Cdx homologues Cdx1, Cdx2, and Cdx4 are present in the promoters of multiple HOX genes . Cdx2 is aberrantly expressed in AML and promotes leukemogenesis via deregulation of Hox genes . The study by Scholl et al.  indicated that CDX2 is overexpressed in 90% of patients with AML. In the present study, overexpression of Cdx2 in primary murine haemopoietic progenitors resulted in transplantable AML in vivo, coinciding with a three-fold up-regulation in Hoxb6 expression. Interestingly, overexpression of HOXB6 has been documented in approximately 40% of human AMLs that do not have chromosomal translocations . Furthermore, other family members of CDX2, such as CDX1 and CDX4, are direct targets of the Wnt pathway and are up-regulated in AML .
Main morphogenic signalling pathways activating Hox genes.
Substantial evidence has now linked aberrant expression of HOX genes to the pathogenesis of myeloid malignancies, especially AML and CML [54,55]. HOX genes have been reported to be deregulated in AML through several different mechanisms. Fusions of the HOXA9 or HOXD13 genes with NUP98, a gene that encodes a component of the nuclear pore complex, have been described in AML and recapitulates AML in murine models of disease [55–57]. Furthermore, overexpression of HOXA6, HOXA7, HOXA9, and the HOX cofactor myeloid ecotropic viral integration site 1 (MEIS1) has also been correlated with chromosome 11q23 abnormalities involving the MLL protein, which regulates the expression of HOX genes . The t(8;16)(p11;p13) translocation in AML results in the expression of the MYST3–CREBBP fusion protein and the up-regulation of HOXA9, HOXB9, HOXA10, and MEIS1 . In addition, the expression of HOXC4 was shown to be up-regulated in the NB4 promyelocytic leukaemia (PML)-RARα cell line following all-trans retinoic acid-induced (ATRA-induced) differentiation as well as in BM from acute PML patients during ATRA treatment . Overexpression of individual Hox family members, including HoxB3 , HoxB8 , or HoxA10 , by retroviral expression or retroviral insertion mutagenesis also generates AML in murine models. In addition, up-regulation of specific Hox genes, such as HoxB4 or HoxA9, is associated with expansion of the HSC compartment in vitro and in vivo and results in enhanced competitive repopulating activity in murine transplantation experiments [63,64]. Changes in Hox gene expression patterns have also been linked to clinical variables in myeloid leukaemias, most notably in CML, where hypermethylation of HOXA5 and HOXA4 strongly correlates with progression to blast crisis . Considering both the BMP and Wnt signalling pathways are known to be deregulated in CML, it is feasible to speculate that the elevated BMP ligands observed in the BM of the CML patients  may alter the Cdx–Hox gene axis resulting in an altered balance in LSCs self-renewal and myelopoiesis.
Overall, these data demonstrate that the BMP signalling pathway plays a fundamental role during embryogenesis and haemopoietic ontogeny. This family of morphogens acts at multiple levels during development, starting at the earliest stage by influencing pluripotent stem cell self-renewal, followed by mesodermal specification, then later on during the establishment of primitive and definitive haemopoiesis in the embryo, and finally playing a role in haemopoietic equilibrium in the adult. Evidence from the literature indicates that BMP activation of HSCs during AGM emergence in the murine setting may sustain their myeloid–lymphoid differentiation potential as they migrate and expand within different anatomical sites during development. Interestingly by the time, the HSCs populate the BM, the majority are no longer BMP activated, which could potentially reflect cell autologous changes or the influence of the different extrinsic signals within the FL and BM microenvironments. The influence of BMP signalling on lineage specification has important implications for leukaemia and may enhance our understanding in the future as to why paediatric leukaemia is predominantly lymphoid, whereas adult leukaemia is predominantly myeloid. Future studies will unravel the complexities of how altered BMP signalling within the malignant haemopoietic niche perturbs the equilibrium to aid aberrant haemopoiesis and the development of disease.
AGM, aorta–gonad–mesonephros; ALK, anaplastic lymphoma kinase; AML, acute myeloid leukaemia; APC, adenomatous polyposis coli; APL, acute promyelocytic leukaemia; ATRA, all-trans retinoic acid; BCR-ABL, breakpoint cluster region-abelson murine leukaemia viral oncogene homolog 1; BM, bone marrow; BMP, bone morphogenic protein; BMPRII, bone morphogenic protein type II receptor; CML, chronic myeloid leukaemia; DVL, dishevelled; FGF, fibroblast growth factor; FZD, frizzled; GFP, green fluorescent protein; GSK3, glycogen synthase kinase 3; hESCs, human embryonic stem cells; Hh, hedgehog; HSCs, haemopoietic stem cells; Id, inhibitor of differentiation; IGF, insulin-like growth factor; LEF, lymphoid enhancing factor; LIF, leukaemia inhibitory factor; LRP, low-density lipoprotein receptor-related protein; LSCs, leukaemic stem cells; MAPK, mitogen-activated protein kinase; MEIS1, myeloid ecotropic viral integration site 1; mESCs, murine embryonic stem cells; MLL, mixed lineage leukaemia; PI3K, phosphatidylinositol-3 kinase; PML, promyelocytic leukaemia; P-Sp, para-aortic splanchnopleure; TGFβ, transforming growth factor-β; VEGF, vascular endothelial growth factor; Wnt, wingless-type MMTV integration site family member.
This work was supported by a PhD studentship from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs).
The Authors declare that there are no competing interests associated with the manuscript.