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Acute Myelogenous Leukemia and its Microenvironment: A Molecular Conversation
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AML and its Microenvironment: A molecular Conversation Gabriel Ghiaur, Mark Wroblewski, Sonja Loges
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Cite this article as: Gabriel Ghiaur, Mark Wroblewski, Sonja Loges, AML and its Microenvironment: A molecular Conversation, Semin Hematol , http://dx.doi.org/10.1053/j.seminhematol.2015.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AML AND ITS MICROENVIRONMENT: A MOLECULAR CONVERSATION Gabriel Ghiaur1, Mark Wroblewski2,3 and Sonja Loges 2,3
(1) Division of Hematological Malignancies, Department of Oncology, Johns Hopkins University, Baltiumore, MD, USA, (2) Department of Hematology and Oncology with sections BMT and Pneumology, Hubertus Wald Tumorzentrum, University Comprehensive Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, (3) Institute of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Acknowledgement: We thank Dr. Judy Karp for critical review and discussions on this manuscript. G. Ghiaur was supported by the Passano Foundation, Clinician Scientist Award and the Leukemia Lymphoma Society, Translational Research Program. S. Loges was supported by the
Max-Eder
group
leader
program from Deutsche
Krebshilfe,
the
Deutsche
Forschungsgemeinschaft (Grant #LO1863/3-1), the Jose Carreras Stiftung (DJCLS R14/06), the Roggenbuck Stiftung, the Hamburger Krebsgesellschaft, the Medical Faculty of the University of Hamburg (FFM program) and the Hamburger Exzellenzinitiative (LEXI program). Editorial Correspondance: Gabriel Ghiaur M.D., Ph.D., 1650 Orleans St., CRB 1, 243, Baltimore, MD, 21287, Phone: 1-410-502-3183, Fax: 1-410-614-7279, e-mail: [email protected]
Sonja Loges M.D., Ph.D., University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany, Phone +49-40-7410-51538, Fax: +49-40-7410-56546, e-mail: [email protected] Conflicts of interests: G. Ghiaur received Advisory Board Honoraria from Spectrum Pharmaceuticals, Inc.. S. Loges received Advisory Board Honoraria, Travel Support and Research Support from BerGenBio.
ABSTRACT Survival of patients with acute myelogenous leukemia (AML) depends on our ability to prevent relapse in patients that achieved complete remission after intensive chemotherapy. While studies focusing on the malignant clone brought many advances in understanding AML biology and chemoresistance, little improvement has been made in eliminating the last bastion of malignant cells, the minimal residual disease (MRD). Inspired by Sir Paget’s “soil and seed” hypothesis, it is becoming more clear that there is constant feedback between the malignant clone and the leukemic microenvironment. This “molecular conversation” dictates AML behavior and holds the key to eliminating MRD. Here we review recent advances in our understanding of how leukemia cells modify their microenvironment and how these changes reinforce AML homeostasis. In addition, we outline current clinical and pre-clinical efforts to disrupt these interactions and to therapeutically target MRD.
1. INTRODUCTION Acute myeloid leukemia (AML) encompasses a heterogenous group of myeloid malignancies, characterized by clonal expansion of abnormal hematopoietic progenitor cells. Patients with AML achieve a high rate of complete remission (CR) with induction chemotherapy but the majority eventualy relapse and die[1]. Rare leukemia cells with the capacity to re-create the disease in xenograft models have been identified as leukemia stem cells (LSCs)[2, 3] and have been shown to be present in patients in CR and predict for disease relapse[2]. Thus, there is critical need for overcoming the mechanisms mediating drug resistance of AML to eradicate minimal residual disease (MRD) and improve outcome in AML[4]. The bulk AML cells and LSCs in particular reside in a complex surrounding where multiple cell populations, some hematopoietic, some non-hematopoietic (stromal) contribute to their homeostasis. While a variety of cell autonomous mechanisms could account for persistance of MRD in AML[4], there is increasing evidence that LSCs survival and chemotherapy resistance depend on intimate interactions with their microenvironment. Current therapies are predominantly leukemia cell centered but alternative approaches targeting the bone marrow microenvironment are emerging. In this review we provide an overview of advances in the field of AML-bone marrow stroma-crosstalk and vice versa. In addition, we outline present and upcoming therapeutic strategies targeting these interactions.
2. AML IN THE BONE MARROW MICROENVIRONMENT Originaly proposed by R. Schofield, the niche concept defines a physical space where hematopoietic and non hematopoietic elements converge to modulate stem cell function to match the physiological demands of the organism[5, 6]. Studies of normal hematopoietic
stem cells (HSCs) behavior at steady state and during stress hematopoiesis have so far suggested the existence of two functionally and perhaps anatomicaly distinct compartments in the bone marrow microenvironment: endosteal and vascular. Biochemical differences between these two spaces in terms of calcium concentration, oxygen tension and pH coupled with distinct cellular components (osteoblasts, osteocytes, osteoclasts in the endosteal niche vs. endothelial cells and megakaryocytes in the vascular niche) translate into functionally different HSC populations. To this end, the endosteal space harbors a population of slowly or noncycling, radiation resistant HSCs while the vascular space can be home for either quiescent (arterioles niche) or actively dividing HSCs (sinusoidal niche). In addition to these two stem cell niches, a variety of mesenchymal and hematopoietic cells contribute to HSCs homeostasis and may have major implications in LSCs function.
2.1 AML in the endosteal niche Similar to HSCs, LSCs have been shown to localize to the surface of the osteoblasts in immonodeficient mice[7] and osteoblasts (OBs) protect AML cells from SDF1a induced apoptosis in vitro[8]. Yet, the number of OB is significantly reduced in preclinical AML and blast crisis CML models (refs 10, 11). Importantly, OB are also decreased in the bone marrow of patients with AML or MDS[9] posing an interesting question as to the role of a deregulated endosteal niche in the pathogenesis and homeostasis of AML. To this end, it appears that malignant myeloid cells can stimulate mesenchymal stromal cells (MSCs) to produce OB that have impaired capacity to retain normal HSCs and favor malignant HSCs[10] (Figure 1). Consequently, although the MSC are directed by the AML cells towards an osteoblast fate their maturation is interrupted resulting in accumulation of immature, osteoblast primed MSC and lack of fully differentiated OB[11]. Interestingly, genetic ablation of mature osteoblasts[9] or impaired osteoblastogenesis via inhibition of
β2adrenergic receptors[11] results in an increased AML burden and decreased survival in animal models. On the other hand, increasing OB number and function via tryptophan hydroxylase 1 inhibitors[9] or overexpression of PTH related peptide[12] leads to decreased AML burden and improved survival. In addition to changes in OB numbers, functional deregulation of these cells can result in altered HSCs behavior and predisposition to myeloproliferative conditions and even AML. It was recently demonstrated that activating β-catenin signaling via overexpression of Jagged-1 in OBs induces the transformation from MDS to AML as early as after 2 weeks, suggesting that there may be no need for cooperating mutations in AML cells in this cell extrinsic model of myeloid malignancy[13]. Also, knockout of Dicer1 (the RNAase III endonuclease essential for RNA and miRNA processing) in osteoprogenitor cells induces an MDS like disease, including progression to AML in mice[14]. This phenotype appears to be secondary to decreased Shwachman-Bodian-Diamond Syndrome (Sbds) gene function as it can be reproduced in mice with Sbds-/- microenvironment[14]. Thus, immature and/or dysfunctional OB elicited by leukemia cells promote while mature OB inhibit AML progression. In sum, there is a cyclical interaction between LSC and OBs: LSCs causes defective maturation of OBs which in turn facilitate LSCs expansion and survival.
2.2 AML in the vascular niche As with solid
tumors,
leukemic cells
have higher
oxygen demand requiring
neovascularization. To this end, patients with AML have increased marrow vascular density (MVD) at diagnosis and their microenvironment normalizes as patients enter complete remission[15-17]. The expansion of vascular structures in the BM of patients with AML predict for poor prognosis[18, 19]. Similar to the endosteal niche discussed above, the
vascular space has a unique repertoire of soluble factors and cellular elements that may contribute to leukemia homeostasis as well as response to treatment (Figure 1). While a great deal of data are available about how angiogenic factors such as VEGF, Angiopoetins and bFGF influence leukemia biology, it is difficult to discsriminate the cell intrinsic effects of these factors from their activity on the vascular space, since many AML cells express receptors that can be directly activated by these factors. For instance, VEGF, a prototypical angiogenic factor that promotes endothelial homeostasis, has been associated with poor prognosis when overexpressed by the AML blasts[20]. The clinical inhibition of VEGF with the monoclonal antibody Bevacizumab in combination with cytarabine and mitoxantrone in a non-randomized Phase II trial demonstrated significant efficacy in a cohort of relapsed and refractory AML patients[21]. Since both AML blasts and endothelial cells express VEGF receptors, it remains unclear if VEGF works in a paracrine or autocrine fashion or, more likely, both. Definitive genetic studies to dissect its role are currently lacking. Nevertheless, there is clear evidence that AML cells localize to and are functionally integrated into the vascular endothelial cell niche both in patients with AML as well as in xenograft models[22]. Elegant in vitro studies also show that AML cells induce proliferation of microvascular endothelial cells, at least in part via CXCL8[23]. Interestingly, in this setting, activation of the Tie2-Ang axis via Ang1 induced AML proliferation. Nonetheless, in vivo studies have shown that AML cells localized to the vascular endothelium appear rather quiescent[22], which is consistent with relative resistance to chemotherapy. However, Ang2 may also be able to activate the BM vascular endothelium and, by mechanisms that are as yet undefined, counteract resistance to chemotherapy because high levels of Ang2 is associated with better survival in AML patients[24]. Unfortunately, while the cellular composition of the vascular niche is better defined in murine models, there are still gaps in our understanding of how these apply to humans and
more so to AML. To complicate things further, given the potential common origins of hematopoietic and endothelial systems during ontogeny and the relatively promiscuous origins of leukemia cells, it is not surprising that the phenotype of endothelial cells and leukemia cells is at times difficult to discriminate. Most recent studies have suggested that endothelial cells and AML cells have either a common origin or that some AML cells can adopt phenotypic and functional characteristics of endothelial cells. For instance, patients with Polycythemia Vera (PV) who presented with Budd Chiari syndrome were found to have intrahepatic endothelial cells that express the Jak2V617F, the hallmark mutation of PV[25]. In addition, AML cells isolated from patients at diagnosis can be transdifferentiated into functional endothelial cell progenitors in vitro[22]. However, the role of endothelial progenitor cells in pathogenesis of myeloprolierative neoplasms and the clinical implications of transdifferentiation of AML cells into endothelial cells remain to be determined.
2.3 Interaction with mesenchymal and non-mesenchymal bone marrow compartment
2.3.1 Mesenchymal cells and extracellular matrix In the BM, a complex network of fibronectins, laminins, collagens and proteoglycans (extracellular matrix - ECM) forms the scaffold for hematopoiesis. These diverse types of stroma cells are mesenchymal in origin (MSCs) and not only produce but also balance the turnover of ECM components and maintain the structural and functional integrity of the BM microenvironment. Recent work indicates that AML cells closely interact with bone marrow stroma cells in a paracrine manner, which increases AML cell survival and resistance to chemotherapy. In vitro data show that, by producing IL-10 and GM-CSF, AML cells augment secretion of growth arrest-specific gene 6 (Gas6) by MSCs. Gas6 in turn promotes AML cell survival and
chemoresistance. Interestingly, approximately 50% of AML patients express the Gas6 receptor Axl, and high Axl expression was independently associated with worse prognosis in a cohort of AML patients[26]. In another cohort of AML patients, high Gas6 rather than Axl expression predicted for worse prognosis[27]. While patient outcomes may be due to many factors including heterogeneity in the populations studied at both clinical and molecular level, these findings suggest that AML cells may depend on Gas6-Axl signaling. Gain- and loss-of-function experiments confirmed that Axl promotes AML cell survival and proliferation. Blockade of Axl by the small molecule inhibitor BGB324 or by genetic means using shRNA approaches inhibited growth of AML xenografts and prolonged survival of mice in a HoxA9-Meis1 model[26, 28]. In addition, the alternative Gas6 receptor Mer has also been identified as potential novel target in AML[29]. The therapeutic potential of Gas6targeting approaches including novel high affinity extracellular domains of the Axl receptor acting as Gas6 traps still needs to be determined[30] but these results propose Gas6-Axl as a novel therapeutic target in AML. AML cells bind to BM mesenchymal elements and extracellular matrix via β1- and β2integrins[31] which increases their proliferation and decreases apoptosis. In addition, leukemia cells induce fibroblast-dependent production of IL8 to stimulate angiogenesis[32] and release of matrix metalloproteinases to degrade extracellular structures thereby stimulating AML progression[33]. This bidirectional crosstalk not only reinforces the malignant hematopoietic clone but also induces genetic instability in MSCs[34]. For instance, leukemia cells activate Hedgehog signaling in the surrounding MSCs[35] to induce their own proliferation. Furthermore, direct contact via VLA-4/VCAM mediated adhesion induces NFkB activation in MSC[36] which in turn up regulate anti-apoptotic pathways and resistance to chemotherapy in AML cells[37]. More so, direct contact of focal adhesion
kinase (FAK) expressing LSCs with MSCs induces the production of AML growth promoting cytokines[38]. At a cellular level, dysregulation of the entire bone marrow microenvironment foster leukemogenesis. For instance, a retinoic receptor (RAR) γ deficient microenvironment results in a myeloproliferative (MPN) like disease[39]. While these mice have elevated TNFα levels, it is tempting to hypothesize that the altered HSC behavior may be due to a dysequilibrium in retinoid function in the stem cell niche since cell extrinsic expression of CYP26, a RAR target gene, controls the balance between HSC proliferation and differentiation[40]. Similarly, deregulated balance between proliferation and differentiation of HSC with resultant MPN can be induced by concommittent loss of Retinoblastoma gene in myeloidderived and osteoblastic microenvironment[41].
2.3.2 Normal myeloid cells interact with AML cells A variety of mononuclear hematopoietic cells, including macrophages and mature neutrophils have been described as regulators of normal BM microenvironment. For instance, macrophages regulate nestin positive MSCs to retain normal HSCs in the BM[42] which may explain why depletion of phagocytic cells is associated with reduction of CXCL12 levels and HSC egress from the BM[43]. Leukemia cells alter macrophage function and promote disease progression either by releasing arginase II in the microenvironment and promoting an immunosuppressive M2 phenotype[44] or by directly inhibiting their phacytosis via expression of CD47[45]. Defective innate immunity might persist in AML in remission because immature multi-lobed granulocyte progenitor cells can be identified[46].
Thus, absence of normal hematopoiesis in patients with AML may alter BM microenvironment and promote niches that favor leukemia homeostasis.
3. CLINICAL RELEVANCE OF LEUKEMIA-BONE MARROW CROSSTALK
One of the most succesful stories in AML remains the use of ATRA in acute promyelocytic leukemia (APL). In addition to being the first targeted therapy in oncology, this approach also unvailed minimal residual disease as the culprit for relapse as single agent ATRA induces remission without cure in APL and PML-RARα fussion transcripts can still be detactable in remission[47]. This work transformed the clinical management of APL such that cure can now be achieved without traditional chemotherapy[48]. Unfortunately, outside APL, there are only sparse reports of benefit from ATRA in AML, particularly NPM1 mutated AML[49]. Preclinical work suggest that in APL, as well as non-APL AML, the BM microenvironment is able to metabolize ATRA and thus protect AML cells from systemic retinoids[50]. Targeting such mechanisms may expand the use of ATRA in non-APL AML. Similar to the inactivation of retinoids, mesenchymal stroma was found to express a variety of drug metabolizing enzymes[51] bringing into quention if systemic drug levels correlate with levels in the stem cell niche (Table 1).
It is increasingly clear that both leukemia and normal HSCs depend on constant interaction with their microenvironment. Given the fact that the malignant clone dominates the BM landscape at diagnosis, there is a common assumption that LSCs “hijack” the stem cell niche and create a self-enforcing microenvironment. In xeno-transplantation models of primary human AML, normal cord blood HSCs appear able to outcompete LSCs when these are displaced from their niche[52]. Thus, targeting such disruptive niches may open a therapeutic
window to eliminate LSCs while preserving HSCs. To this end, inhibition of CXCR4-SDF1α axis in xenograft models of AML results in decreased tumor burden while similar treatments did not affect normal HSCs[53]. Similarly combination of CXCR4 inhibitor with chemotherapy was shown to be safe in patients with relapse refractory AML and had a remission rate of 46% in a phase 1/2 single institution study[54]. This may be due to higher levels of CXCR4 required by the AML blasts to be anchored in the BM microenvironment[55]. While the reduction in disease burden and achievement of remission in these very difficult patients is welcomed, there is no evidence so far that this translates into elimination of LSCs and improved cure rate. A number of clinical trials combining targeting CXCR4-SDF1α axis with chemotherapy and either GCSF (NCT00906945) or targeted agents (NCT01236144) are currently underway and they provide further evidence into the clinical potential of this approach.
The promising results of combination VEGF inhibitor with chemotherapy[21] also generated awareness as to the therapeutic role of targeting the vascular niche in AML. While microvessel density and plasma VEGF levels in these patients were monitored, it remains unclear to what extent Bevacizumab therapeutic benefit came from directly targeting AML and how much from modulating the BM microenvironment. Nevertheless, antiangiogenic drugs are currently being tested in combination with chemotherapy in AML. Drugs such as Lenalidomide (NCT00885508) and Pomalidomide (NCT02029950), while used as immune mediators are also powerful antiangiogenic drugs. Correlative studies on these patients coupled with results from studies of novel angiogenic inhibitors (i.e. AZD2171, NCT00321581) should provide valuable input on vascular niche remodeling and effects on LSCs and MRD.
Targeting AML paracrine interactions with the stromal compartment are currently entering the clinics with the first-in-patient Phase 1a/b trial with the Axl blocking compound BGB324 (BGBC003). Previously, BGB324 was safely applied to healthy volunteers[56]. BGB324 will be applied as a monotherapy to refractory AML patients and as frontline therapy to elderly patients non-eligible for intensive chemotherapy alone and in combination with cytarabine.
Provocative studies on the role of Hedgehog in AML have led to initiation of a variety of clinical trials using Hedgehog inhibitors either as single agents or in combination with demethylating
agents
or
low
dose
Cytarabine
(NCT00953758,
NCT01841333,
NCT02038777, NCT01842646, NCT01546038). Most recent data shows that in addition to a cell-intrinsic role, paracrine Hedgehog signaling also contributes to leukemia homeostasis and may actualy represent a more „rational target“ in AML[57, 58].
Lastly, the lack of normal hematopoiesis rather than the disease burden dictates the vast majority of morbidity and mortality in AML. While in most cases of AML the malignant clone dominates the BM microenvironment at diagnosis, enough normal HSCs are present to reconstitute a quasi-normal hematopoiesis when the patient achieves complete remission. Until now, distinguishing normal HSCs from LSCs in patients with AML has been impossible. Recent work has shown that in some AML patients (i.e. in core binding factor mutated AMLs), aldehyde dehydrogenase activity can separate LSCs (CD34+CD38ALDHintermediate) from normal HSCs (CD34+CD38-ALDHbright) at diagnosis[2]. These results allow functional characterization of normal and malignant hematopoiesis in patients with AML at diagnosis and upon entering remission. The hope is that such studies may provide tools to boost normal hematopoiesis in patients with AML and, while not curing the disease alleviate morbidity and improve mortality.
4. CONCLUSION Drug resistance in AML has been a source of constant frustration for leukemia clinicians and despair for our patients. Studies of the malignant hematopoietic clone furthered our understanding of cellular and molecular biology of leukemia and made possible progress in management of this disease. Thanks to this approach, we are now using targeted therapies in APL (ATRA), BCR-ABL/FLT3 ITD leukemia (TKIs) and most recently, IDH1 inhibitors. The question remains, how come we can eliminate the bulk of the tumor but retain some minimal residual disease? Several potential explanations have been proposed: a) some leukemia cells are intrinsically resistant to chemotherapy (LSCs) and LSC directed therapies are currently under investigation (see anti-CD123 clinical trial NCT01632852) or b) some leukemia cells are in a microenvironment that provides signals such as Gas6 that makes them resistant to chemotherapy (see above). These two possibillities are not mutually exclusive. In fact, it is likely that they act in complementary and/or cooperative fashions. Targeting such microenvironments should eliminate MRD, decrease relapse rate and improve outcome in AML.
5. Acknowledgement We thank Dr. Judy Karp for critical review and discussions on this manuscript. G. Ghiaur was supported by the Passano Foundation, Clinician Scientist Award and the Leukemia Lymphoma Society, Translational Research Program. S. Loges was supported by the Max-Eder group leader program from Deutsche Krebshilfe, the Deutsche Forschungsgemeinschaft (Grant #LO1863/3-1), the Jose Carreras Stiftung (DJCLS R14/06), the Roggenbuck Stiftung, the Hamburger Krebsgesellschaft, the Medical
Faculty of the University of Hamburg (FFM program) and the Hamburger Exzellenzinitiative (LEXI program).
6. Conflicts of interests G. Ghiaur received Advisory Board Honoraria from Spectrum Pharmaceuticals, Inc.
S. Loges received Advisory Board Honoraria, Travel Support and Research Support from BerGenBio.
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FIGURE LEGENDS:
Figure 1: AML and its microenvironment. Interaction between leukemic cells and the bone marrow microenvironment occurs with in three major compartments. (A) In the vascular niche AML cells induce vessel proliferation via CXCL8 and possibly fuse with endothelial cells whereas endothelial cells induce quiescence in AML blasts that leads to increased chemoresistance. (B) Mesenchymal stroma cells: i) get educated to produce the mitogen Gas6 wich in turn induces proliferation and chemoresistance in AML cells; ii) release matrix metallo proteinases (MMP) that liberate matrix-bound growth factors (green dots) which fuel AML growth; iii) express retinoid-inactivating CYP26 and creat a retinoid low niche to protect AML cells. In addition, direct interactions of AML cells with MSC and/or ECM promote leukemia resistance to chemotherapy and apoptosis. (C) Within the endosteal niche, AML cells interfere with osteoblast differentiation and redirect this process towards immature osteoblasts. Instead of maintaining HSC-quiescence immature osteoblasts produce the mitogen TGFβ2 which leads to AML proliferation and egression of hematopoietic stem cells from the bone marrow.
TABLES
Table 1. Agents targeting the bone marrow microenvironment.
Target in the Niche
Agents
Study in AML
Drug metabolism
CYP26 inhibitors (R115866)
Preclinical
CYP3A4 inhibitors
Preclinical
(Ketoconazole) CXCR4-SDF1α
AMD3100
Preclinical, Phase I/II
VLA4-fibronectin
AS101
Preclinical, Phase II
Antiangiogenesis
Anti-VEGF antibodies
Phase II
(Bevacizumab)
Phase II
VEGFR inhibitor (AZD2171)
Phase II
IMiDs (Lenalidomide, Pomalidomide) Gas6-Axl
Hedgehog signaling
BGB324
Phase Ib
TP0903
Preclinical
PF-04449913
Phase Ib/II
Leukemic cells HSC Mature osteoblasts Immature osteoblasts
Vascular niche
EEndosteal d t l niche i h
Mesenchymal cells ECM
Inducon of quiescence MSC
V-AML CXCL8 CXCL L8
β1 - / β2 integrins
VCAM/VLA4
AML-ECfusion
IL10 L10 MCSF GMCSF
MSC
Gas 6 Ga
MMPs MM MMP MPs
CYP26
Vascular endothelium
A
BM stroma
B
C
AML and its Microenvironment: A molecular Conversation Gabriel Ghiaur, Mark Wroblewski, Sonja Loges
www.elsevier.com/locate/enganabound
PII: DOI: Reference:
S0037-1963(15)00027-X http://dx.doi.org/10.1053/j.seminhematol.2015.03.003 YSHEM50814
To appear in: Semin Hematol
Cite this article as: Gabriel Ghiaur, Mark Wroblewski, Sonja Loges, AML and its Microenvironment: A molecular Conversation, Semin Hematol , http://dx.doi.org/10.1053/j.seminhematol.2015.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AML AND ITS MICROENVIRONMENT: A MOLECULAR CONVERSATION Gabriel Ghiaur1, Mark Wroblewski2,3 and Sonja Loges 2,3
(1) Division of Hematological Malignancies, Department of Oncology, Johns Hopkins University, Baltiumore, MD, USA, (2) Department of Hematology and Oncology with sections BMT and Pneumology, Hubertus Wald Tumorzentrum, University Comprehensive Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, (3) Institute of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Acknowledgement: We thank Dr. Judy Karp for critical review and discussions on this manuscript. G. Ghiaur was supported by the Passano Foundation, Clinician Scientist Award and the Leukemia Lymphoma Society, Translational Research Program. S. Loges was supported by the
Max-Eder
group
leader
program from Deutsche
Krebshilfe,
the
Deutsche
Forschungsgemeinschaft (Grant #LO1863/3-1), the Jose Carreras Stiftung (DJCLS R14/06), the Roggenbuck Stiftung, the Hamburger Krebsgesellschaft, the Medical Faculty of the University of Hamburg (FFM program) and the Hamburger Exzellenzinitiative (LEXI program). Editorial Correspondance: Gabriel Ghiaur M.D., Ph.D., 1650 Orleans St., CRB 1, 243, Baltimore, MD, 21287, Phone: 1-410-502-3183, Fax: 1-410-614-7279, e-mail: [email protected]
Sonja Loges M.D., Ph.D., University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany, Phone +49-40-7410-51538, Fax: +49-40-7410-56546, e-mail: [email protected] Conflicts of interests: G. Ghiaur received Advisory Board Honoraria from Spectrum Pharmaceuticals, Inc.. S. Loges received Advisory Board Honoraria, Travel Support and Research Support from BerGenBio.
ABSTRACT Survival of patients with acute myelogenous leukemia (AML) depends on our ability to prevent relapse in patients that achieved complete remission after intensive chemotherapy. While studies focusing on the malignant clone brought many advances in understanding AML biology and chemoresistance, little improvement has been made in eliminating the last bastion of malignant cells, the minimal residual disease (MRD). Inspired by Sir Paget’s “soil and seed” hypothesis, it is becoming more clear that there is constant feedback between the malignant clone and the leukemic microenvironment. This “molecular conversation” dictates AML behavior and holds the key to eliminating MRD. Here we review recent advances in our understanding of how leukemia cells modify their microenvironment and how these changes reinforce AML homeostasis. In addition, we outline current clinical and pre-clinical efforts to disrupt these interactions and to therapeutically target MRD.
1. INTRODUCTION Acute myeloid leukemia (AML) encompasses a heterogenous group of myeloid malignancies, characterized by clonal expansion of abnormal hematopoietic progenitor cells. Patients with AML achieve a high rate of complete remission (CR) with induction chemotherapy but the majority eventualy relapse and die[1]. Rare leukemia cells with the capacity to re-create the disease in xenograft models have been identified as leukemia stem cells (LSCs)[2, 3] and have been shown to be present in patients in CR and predict for disease relapse[2]. Thus, there is critical need for overcoming the mechanisms mediating drug resistance of AML to eradicate minimal residual disease (MRD) and improve outcome in AML[4]. The bulk AML cells and LSCs in particular reside in a complex surrounding where multiple cell populations, some hematopoietic, some non-hematopoietic (stromal) contribute to their homeostasis. While a variety of cell autonomous mechanisms could account for persistance of MRD in AML[4], there is increasing evidence that LSCs survival and chemotherapy resistance depend on intimate interactions with their microenvironment. Current therapies are predominantly leukemia cell centered but alternative approaches targeting the bone marrow microenvironment are emerging. In this review we provide an overview of advances in the field of AML-bone marrow stroma-crosstalk and vice versa. In addition, we outline present and upcoming therapeutic strategies targeting these interactions.
2. AML IN THE BONE MARROW MICROENVIRONMENT Originaly proposed by R. Schofield, the niche concept defines a physical space where hematopoietic and non hematopoietic elements converge to modulate stem cell function to match the physiological demands of the organism[5, 6]. Studies of normal hematopoietic
stem cells (HSCs) behavior at steady state and during stress hematopoiesis have so far suggested the existence of two functionally and perhaps anatomicaly distinct compartments in the bone marrow microenvironment: endosteal and vascular. Biochemical differences between these two spaces in terms of calcium concentration, oxygen tension and pH coupled with distinct cellular components (osteoblasts, osteocytes, osteoclasts in the endosteal niche vs. endothelial cells and megakaryocytes in the vascular niche) translate into functionally different HSC populations. To this end, the endosteal space harbors a population of slowly or noncycling, radiation resistant HSCs while the vascular space can be home for either quiescent (arterioles niche) or actively dividing HSCs (sinusoidal niche). In addition to these two stem cell niches, a variety of mesenchymal and hematopoietic cells contribute to HSCs homeostasis and may have major implications in LSCs function.
2.1 AML in the endosteal niche Similar to HSCs, LSCs have been shown to localize to the surface of the osteoblasts in immonodeficient mice[7] and osteoblasts (OBs) protect AML cells from SDF1a induced apoptosis in vitro[8]. Yet, the number of OB is significantly reduced in preclinical AML and blast crisis CML models (refs 10, 11). Importantly, OB are also decreased in the bone marrow of patients with AML or MDS[9] posing an interesting question as to the role of a deregulated endosteal niche in the pathogenesis and homeostasis of AML. To this end, it appears that malignant myeloid cells can stimulate mesenchymal stromal cells (MSCs) to produce OB that have impaired capacity to retain normal HSCs and favor malignant HSCs[10] (Figure 1). Consequently, although the MSC are directed by the AML cells towards an osteoblast fate their maturation is interrupted resulting in accumulation of immature, osteoblast primed MSC and lack of fully differentiated OB[11]. Interestingly, genetic ablation of mature osteoblasts[9] or impaired osteoblastogenesis via inhibition of
β2adrenergic receptors[11] results in an increased AML burden and decreased survival in animal models. On the other hand, increasing OB number and function via tryptophan hydroxylase 1 inhibitors[9] or overexpression of PTH related peptide[12] leads to decreased AML burden and improved survival. In addition to changes in OB numbers, functional deregulation of these cells can result in altered HSCs behavior and predisposition to myeloproliferative conditions and even AML. It was recently demonstrated that activating β-catenin signaling via overexpression of Jagged-1 in OBs induces the transformation from MDS to AML as early as after 2 weeks, suggesting that there may be no need for cooperating mutations in AML cells in this cell extrinsic model of myeloid malignancy[13]. Also, knockout of Dicer1 (the RNAase III endonuclease essential for RNA and miRNA processing) in osteoprogenitor cells induces an MDS like disease, including progression to AML in mice[14]. This phenotype appears to be secondary to decreased Shwachman-Bodian-Diamond Syndrome (Sbds) gene function as it can be reproduced in mice with Sbds-/- microenvironment[14]. Thus, immature and/or dysfunctional OB elicited by leukemia cells promote while mature OB inhibit AML progression. In sum, there is a cyclical interaction between LSC and OBs: LSCs causes defective maturation of OBs which in turn facilitate LSCs expansion and survival.
2.2 AML in the vascular niche As with solid
tumors,
leukemic cells
have higher
oxygen demand requiring
neovascularization. To this end, patients with AML have increased marrow vascular density (MVD) at diagnosis and their microenvironment normalizes as patients enter complete remission[15-17]. The expansion of vascular structures in the BM of patients with AML predict for poor prognosis[18, 19]. Similar to the endosteal niche discussed above, the
vascular space has a unique repertoire of soluble factors and cellular elements that may contribute to leukemia homeostasis as well as response to treatment (Figure 1). While a great deal of data are available about how angiogenic factors such as VEGF, Angiopoetins and bFGF influence leukemia biology, it is difficult to discsriminate the cell intrinsic effects of these factors from their activity on the vascular space, since many AML cells express receptors that can be directly activated by these factors. For instance, VEGF, a prototypical angiogenic factor that promotes endothelial homeostasis, has been associated with poor prognosis when overexpressed by the AML blasts[20]. The clinical inhibition of VEGF with the monoclonal antibody Bevacizumab in combination with cytarabine and mitoxantrone in a non-randomized Phase II trial demonstrated significant efficacy in a cohort of relapsed and refractory AML patients[21]. Since both AML blasts and endothelial cells express VEGF receptors, it remains unclear if VEGF works in a paracrine or autocrine fashion or, more likely, both. Definitive genetic studies to dissect its role are currently lacking. Nevertheless, there is clear evidence that AML cells localize to and are functionally integrated into the vascular endothelial cell niche both in patients with AML as well as in xenograft models[22]. Elegant in vitro studies also show that AML cells induce proliferation of microvascular endothelial cells, at least in part via CXCL8[23]. Interestingly, in this setting, activation of the Tie2-Ang axis via Ang1 induced AML proliferation. Nonetheless, in vivo studies have shown that AML cells localized to the vascular endothelium appear rather quiescent[22], which is consistent with relative resistance to chemotherapy. However, Ang2 may also be able to activate the BM vascular endothelium and, by mechanisms that are as yet undefined, counteract resistance to chemotherapy because high levels of Ang2 is associated with better survival in AML patients[24]. Unfortunately, while the cellular composition of the vascular niche is better defined in murine models, there are still gaps in our understanding of how these apply to humans and
more so to AML. To complicate things further, given the potential common origins of hematopoietic and endothelial systems during ontogeny and the relatively promiscuous origins of leukemia cells, it is not surprising that the phenotype of endothelial cells and leukemia cells is at times difficult to discriminate. Most recent studies have suggested that endothelial cells and AML cells have either a common origin or that some AML cells can adopt phenotypic and functional characteristics of endothelial cells. For instance, patients with Polycythemia Vera (PV) who presented with Budd Chiari syndrome were found to have intrahepatic endothelial cells that express the Jak2V617F, the hallmark mutation of PV[25]. In addition, AML cells isolated from patients at diagnosis can be transdifferentiated into functional endothelial cell progenitors in vitro[22]. However, the role of endothelial progenitor cells in pathogenesis of myeloprolierative neoplasms and the clinical implications of transdifferentiation of AML cells into endothelial cells remain to be determined.
2.3 Interaction with mesenchymal and non-mesenchymal bone marrow compartment
2.3.1 Mesenchymal cells and extracellular matrix In the BM, a complex network of fibronectins, laminins, collagens and proteoglycans (extracellular matrix - ECM) forms the scaffold for hematopoiesis. These diverse types of stroma cells are mesenchymal in origin (MSCs) and not only produce but also balance the turnover of ECM components and maintain the structural and functional integrity of the BM microenvironment. Recent work indicates that AML cells closely interact with bone marrow stroma cells in a paracrine manner, which increases AML cell survival and resistance to chemotherapy. In vitro data show that, by producing IL-10 and GM-CSF, AML cells augment secretion of growth arrest-specific gene 6 (Gas6) by MSCs. Gas6 in turn promotes AML cell survival and
chemoresistance. Interestingly, approximately 50% of AML patients express the Gas6 receptor Axl, and high Axl expression was independently associated with worse prognosis in a cohort of AML patients[26]. In another cohort of AML patients, high Gas6 rather than Axl expression predicted for worse prognosis[27]. While patient outcomes may be due to many factors including heterogeneity in the populations studied at both clinical and molecular level, these findings suggest that AML cells may depend on Gas6-Axl signaling. Gain- and loss-of-function experiments confirmed that Axl promotes AML cell survival and proliferation. Blockade of Axl by the small molecule inhibitor BGB324 or by genetic means using shRNA approaches inhibited growth of AML xenografts and prolonged survival of mice in a HoxA9-Meis1 model[26, 28]. In addition, the alternative Gas6 receptor Mer has also been identified as potential novel target in AML[29]. The therapeutic potential of Gas6targeting approaches including novel high affinity extracellular domains of the Axl receptor acting as Gas6 traps still needs to be determined[30] but these results propose Gas6-Axl as a novel therapeutic target in AML. AML cells bind to BM mesenchymal elements and extracellular matrix via β1- and β2integrins[31] which increases their proliferation and decreases apoptosis. In addition, leukemia cells induce fibroblast-dependent production of IL8 to stimulate angiogenesis[32] and release of matrix metalloproteinases to degrade extracellular structures thereby stimulating AML progression[33]. This bidirectional crosstalk not only reinforces the malignant hematopoietic clone but also induces genetic instability in MSCs[34]. For instance, leukemia cells activate Hedgehog signaling in the surrounding MSCs[35] to induce their own proliferation. Furthermore, direct contact via VLA-4/VCAM mediated adhesion induces NFkB activation in MSC[36] which in turn up regulate anti-apoptotic pathways and resistance to chemotherapy in AML cells[37]. More so, direct contact of focal adhesion
kinase (FAK) expressing LSCs with MSCs induces the production of AML growth promoting cytokines[38]. At a cellular level, dysregulation of the entire bone marrow microenvironment foster leukemogenesis. For instance, a retinoic receptor (RAR) γ deficient microenvironment results in a myeloproliferative (MPN) like disease[39]. While these mice have elevated TNFα levels, it is tempting to hypothesize that the altered HSC behavior may be due to a dysequilibrium in retinoid function in the stem cell niche since cell extrinsic expression of CYP26, a RAR target gene, controls the balance between HSC proliferation and differentiation[40]. Similarly, deregulated balance between proliferation and differentiation of HSC with resultant MPN can be induced by concommittent loss of Retinoblastoma gene in myeloidderived and osteoblastic microenvironment[41].
2.3.2 Normal myeloid cells interact with AML cells A variety of mononuclear hematopoietic cells, including macrophages and mature neutrophils have been described as regulators of normal BM microenvironment. For instance, macrophages regulate nestin positive MSCs to retain normal HSCs in the BM[42] which may explain why depletion of phagocytic cells is associated with reduction of CXCL12 levels and HSC egress from the BM[43]. Leukemia cells alter macrophage function and promote disease progression either by releasing arginase II in the microenvironment and promoting an immunosuppressive M2 phenotype[44] or by directly inhibiting their phacytosis via expression of CD47[45]. Defective innate immunity might persist in AML in remission because immature multi-lobed granulocyte progenitor cells can be identified[46].
Thus, absence of normal hematopoiesis in patients with AML may alter BM microenvironment and promote niches that favor leukemia homeostasis.
3. CLINICAL RELEVANCE OF LEUKEMIA-BONE MARROW CROSSTALK
One of the most succesful stories in AML remains the use of ATRA in acute promyelocytic leukemia (APL). In addition to being the first targeted therapy in oncology, this approach also unvailed minimal residual disease as the culprit for relapse as single agent ATRA induces remission without cure in APL and PML-RARα fussion transcripts can still be detactable in remission[47]. This work transformed the clinical management of APL such that cure can now be achieved without traditional chemotherapy[48]. Unfortunately, outside APL, there are only sparse reports of benefit from ATRA in AML, particularly NPM1 mutated AML[49]. Preclinical work suggest that in APL, as well as non-APL AML, the BM microenvironment is able to metabolize ATRA and thus protect AML cells from systemic retinoids[50]. Targeting such mechanisms may expand the use of ATRA in non-APL AML. Similar to the inactivation of retinoids, mesenchymal stroma was found to express a variety of drug metabolizing enzymes[51] bringing into quention if systemic drug levels correlate with levels in the stem cell niche (Table 1).
It is increasingly clear that both leukemia and normal HSCs depend on constant interaction with their microenvironment. Given the fact that the malignant clone dominates the BM landscape at diagnosis, there is a common assumption that LSCs “hijack” the stem cell niche and create a self-enforcing microenvironment. In xeno-transplantation models of primary human AML, normal cord blood HSCs appear able to outcompete LSCs when these are displaced from their niche[52]. Thus, targeting such disruptive niches may open a therapeutic
window to eliminate LSCs while preserving HSCs. To this end, inhibition of CXCR4-SDF1α axis in xenograft models of AML results in decreased tumor burden while similar treatments did not affect normal HSCs[53]. Similarly combination of CXCR4 inhibitor with chemotherapy was shown to be safe in patients with relapse refractory AML and had a remission rate of 46% in a phase 1/2 single institution study[54]. This may be due to higher levels of CXCR4 required by the AML blasts to be anchored in the BM microenvironment[55]. While the reduction in disease burden and achievement of remission in these very difficult patients is welcomed, there is no evidence so far that this translates into elimination of LSCs and improved cure rate. A number of clinical trials combining targeting CXCR4-SDF1α axis with chemotherapy and either GCSF (NCT00906945) or targeted agents (NCT01236144) are currently underway and they provide further evidence into the clinical potential of this approach.
The promising results of combination VEGF inhibitor with chemotherapy[21] also generated awareness as to the therapeutic role of targeting the vascular niche in AML. While microvessel density and plasma VEGF levels in these patients were monitored, it remains unclear to what extent Bevacizumab therapeutic benefit came from directly targeting AML and how much from modulating the BM microenvironment. Nevertheless, antiangiogenic drugs are currently being tested in combination with chemotherapy in AML. Drugs such as Lenalidomide (NCT00885508) and Pomalidomide (NCT02029950), while used as immune mediators are also powerful antiangiogenic drugs. Correlative studies on these patients coupled with results from studies of novel angiogenic inhibitors (i.e. AZD2171, NCT00321581) should provide valuable input on vascular niche remodeling and effects on LSCs and MRD.
Targeting AML paracrine interactions with the stromal compartment are currently entering the clinics with the first-in-patient Phase 1a/b trial with the Axl blocking compound BGB324 (BGBC003). Previously, BGB324 was safely applied to healthy volunteers[56]. BGB324 will be applied as a monotherapy to refractory AML patients and as frontline therapy to elderly patients non-eligible for intensive chemotherapy alone and in combination with cytarabine.
Provocative studies on the role of Hedgehog in AML have led to initiation of a variety of clinical trials using Hedgehog inhibitors either as single agents or in combination with demethylating
agents
or
low
dose
Cytarabine
(NCT00953758,
NCT01841333,
NCT02038777, NCT01842646, NCT01546038). Most recent data shows that in addition to a cell-intrinsic role, paracrine Hedgehog signaling also contributes to leukemia homeostasis and may actualy represent a more „rational target“ in AML[57, 58].
Lastly, the lack of normal hematopoiesis rather than the disease burden dictates the vast majority of morbidity and mortality in AML. While in most cases of AML the malignant clone dominates the BM microenvironment at diagnosis, enough normal HSCs are present to reconstitute a quasi-normal hematopoiesis when the patient achieves complete remission. Until now, distinguishing normal HSCs from LSCs in patients with AML has been impossible. Recent work has shown that in some AML patients (i.e. in core binding factor mutated AMLs), aldehyde dehydrogenase activity can separate LSCs (CD34+CD38ALDHintermediate) from normal HSCs (CD34+CD38-ALDHbright) at diagnosis[2]. These results allow functional characterization of normal and malignant hematopoiesis in patients with AML at diagnosis and upon entering remission. The hope is that such studies may provide tools to boost normal hematopoiesis in patients with AML and, while not curing the disease alleviate morbidity and improve mortality.
4. CONCLUSION Drug resistance in AML has been a source of constant frustration for leukemia clinicians and despair for our patients. Studies of the malignant hematopoietic clone furthered our understanding of cellular and molecular biology of leukemia and made possible progress in management of this disease. Thanks to this approach, we are now using targeted therapies in APL (ATRA), BCR-ABL/FLT3 ITD leukemia (TKIs) and most recently, IDH1 inhibitors. The question remains, how come we can eliminate the bulk of the tumor but retain some minimal residual disease? Several potential explanations have been proposed: a) some leukemia cells are intrinsically resistant to chemotherapy (LSCs) and LSC directed therapies are currently under investigation (see anti-CD123 clinical trial NCT01632852) or b) some leukemia cells are in a microenvironment that provides signals such as Gas6 that makes them resistant to chemotherapy (see above). These two possibillities are not mutually exclusive. In fact, it is likely that they act in complementary and/or cooperative fashions. Targeting such microenvironments should eliminate MRD, decrease relapse rate and improve outcome in AML.
5. Acknowledgement We thank Dr. Judy Karp for critical review and discussions on this manuscript. G. Ghiaur was supported by the Passano Foundation, Clinician Scientist Award and the Leukemia Lymphoma Society, Translational Research Program. S. Loges was supported by the Max-Eder group leader program from Deutsche Krebshilfe, the Deutsche Forschungsgemeinschaft (Grant #LO1863/3-1), the Jose Carreras Stiftung (DJCLS R14/06), the Roggenbuck Stiftung, the Hamburger Krebsgesellschaft, the Medical
Faculty of the University of Hamburg (FFM program) and the Hamburger Exzellenzinitiative (LEXI program).
6. Conflicts of interests G. Ghiaur received Advisory Board Honoraria from Spectrum Pharmaceuticals, Inc.
S. Loges received Advisory Board Honoraria, Travel Support and Research Support from BerGenBio.
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FIGURE LEGENDS:
Figure 1: AML and its microenvironment. Interaction between leukemic cells and the bone marrow microenvironment occurs with in three major compartments. (A) In the vascular niche AML cells induce vessel proliferation via CXCL8 and possibly fuse with endothelial cells whereas endothelial cells induce quiescence in AML blasts that leads to increased chemoresistance. (B) Mesenchymal stroma cells: i) get educated to produce the mitogen Gas6 wich in turn induces proliferation and chemoresistance in AML cells; ii) release matrix metallo proteinases (MMP) that liberate matrix-bound growth factors (green dots) which fuel AML growth; iii) express retinoid-inactivating CYP26 and creat a retinoid low niche to protect AML cells. In addition, direct interactions of AML cells with MSC and/or ECM promote leukemia resistance to chemotherapy and apoptosis. (C) Within the endosteal niche, AML cells interfere with osteoblast differentiation and redirect this process towards immature osteoblasts. Instead of maintaining HSC-quiescence immature osteoblasts produce the mitogen TGFβ2 which leads to AML proliferation and egression of hematopoietic stem cells from the bone marrow.
TABLES
Table 1. Agents targeting the bone marrow microenvironment.
Target in the Niche
Agents
Study in AML
Drug metabolism
CYP26 inhibitors (R115866)
Preclinical
CYP3A4 inhibitors
Preclinical
(Ketoconazole) CXCR4-SDF1α
AMD3100
Preclinical, Phase I/II
VLA4-fibronectin
AS101
Preclinical, Phase II
Antiangiogenesis
Anti-VEGF antibodies
Phase II
(Bevacizumab)
Phase II
VEGFR inhibitor (AZD2171)
Phase II
IMiDs (Lenalidomide, Pomalidomide) Gas6-Axl
Hedgehog signaling
BGB324
Phase Ib
TP0903
Preclinical
PF-04449913
Phase Ib/II
Leukemic cells HSC Mature osteoblasts Immature osteoblasts
Vascular niche
EEndosteal d t l niche i h
Mesenchymal cells ECM
Inducon of quiescence MSC
V-AML CXCL8 CXCL L8
β1 - / β2 integrins
VCAM/VLA4
AML-ECfusion
IL10 L10 MCSF GMCSF
MSC
Gas 6 Ga
MMPs MM MMP MPs
CYP26
Vascular endothelium
A
BM stroma
B
C