Leukemic Niche Research Articles

Treatment-induced arteriolar revascularization and miR-126 enhancement in bone marrow niche protect leukemic stem cells in AML

BackgroundDuring acute myeloid leukemia (AML) growth, the bone marrow (BM) niche acquires significant vascular changes that can be offset by therapeutic blast cytoreduction. The molecular mechanisms of this vascular plasticity remain to be fully elucidated. Herein, we report on the changes that occur in the vascular compartment of the FLT3-ITD+ AML BM niche pre and post treatment and their impact on leukemic stem cells (LSCs).MethodsBM vasculature was evaluated in FLT3-ITD+ AML models (MllPTD/WT/Flt3ITD/ITD mouse and patient-derived xenograft) by 3D confocal imaging of long bones, calvarium vascular permeability assays, and flow cytometry analysis. Cytokine levels were measured by Luminex assay and miR-126 levels evaluated by Q-RT-PCR and miRNA staining. Wild-type (wt) and MllPTD/WT/Flt3ITD/ITD mice with endothelial cell (EC) miR-126 knockout or overexpression served as controls. The impact of treatment-induced BM vascular changes on LSC activity was evaluated by secondary transplantation of BM cells after administration of tyrosine kinase inhibitors (TKIs) to MllPTD/WT/Flt3ITD/ITD mice with/without either EC miR-126 KO or co-treatment with tumor necrosis factor alpha (TNFα) or anti-miR-126 miRisten.ResultsIn the normal BM niche, CD31+Sca-1high ECs lining arterioles have miR-126 levels higher than CD31+Sca-1low ECs lining sinusoids. We noted that during FLT3-ITD+ AML growth, the BM niche lost arterioles and gained sinusoids. These changes were mediated by TNFα, a cytokine produced by AML blasts, which induced EC miR-126 downregulation and caused depletion of CD31+Sca-1high ECs and gain in CD31+Sca-1low ECs. Loss of miR-126high ECs led to a decreased EC miR-126 supply to LSCs, which then entered the cell cycle and promoted leukemia growth. Accordingly, antileukemic treatment with TKI decreased the BM blast-produced TNFα and increased miR-126high ECs and the EC miR-126 supply to LSCs. High miR-126 levels safeguarded LSCs, as shown by more severe disease in secondary transplanted mice. Conversely, EC miR-126 deprivation via genetic or pharmacological EC miR-126 knock-down prevented treatment-induced BM miR-126high EC expansion and in turn LSC protection.ConclusionsTreatment-induced CD31+Sca-1high EC re-vascularization of the leukemic BM niche may represent a LSC extrinsic mechanism of treatment resistance that can be overcome with therapeutic EC miR-126 deprivation.Graphic abstract

Leukemia-Induced Cellular Senescence and Stemness Alterations in Mesenchymal Stem Cells Are Reversible upon Withdrawal of B-Cell Acute Lymphoblastic Leukemia Cells.

Leukemic cell growth in the bone marrow (BM) induces a very stressful condition. Mesenchymal stem cells (MSC), a key component of this BM niche, are affected in several ways with unfavorable consequences on hematopoietic stem cells favoring leukemic cells. These alterations in MSC during B-cell acute lymphoblastic leukemia (B-ALL) have not been fully studied. In this work, we have compared the modifications that occur in an in vitro leukemic niche (LN) with those observed in MSC isolated from B-ALL patients. MSC in this LN niche showed features of a senescence process, i.e., altered morphology, increased senescence-associated β-Galactosidase (SA-βGAL) activity, and upregulation of p53 and p21 (without p16 expression), cell-cycle arrest, reduced clonogenicity, and some moderated changes in stemness properties. Importantly, almost all of these features were found in MSC isolated from B-ALL patients. These alterations rendered B-ALL cells susceptible to the chemotherapeutic agent dexamethasone. The senescent process seems to be transient since when leukemic cells are removed, normal MSC morphology is re-established, SA-βGAL expression is diminished, and MSC are capable of re-entering cell cycle. In addition, few cells showed low γH2AX phosphorylation that was reduced to basal levels upon cultivation. The reversibility of the senescent process in MSC must impinge important biological and clinical significance depending on cell interactions in the bone marrow at different stages of disease progression in B-ALL.

AML Differentially Alters Fate and Function of Mesenchymal Stem- and Osteolineage Cells in the Leukemic Niche

Acute Myeloid Leukemia (AML) alters the composition of the supportive bone marrow (BM) stroma, leading to a leukemia-supportive microenvironment that promotes extrinsic chemotherapy resistance. We previously reported functional and gene expression changes in patient derived mesenchymal stem cells (MSC) that included the increased secretion of chemoprotective cytokines, sustained in the absence of AML cells through serial passage ex vivo . Other groups demonstrated altered DNA methylation patterns in patient MSCs that correlated with increased extrinsic drug resistance in vitro . However, a more rigorous analysis of changes in stromal fate and function requires longitudinal- including pre diagnostic- observations, impossible to obtain from patients and further complicated by patient-patient heterogeneity as well as expansion culture induced in vitro artifacts. Here, we developed a protocol for flow-cytometric isolation and culture- free analysis of stringently purified endosteal MSCs (CD45-, CD31-, CD51+, SCA-1+) and osteoblastic progenitor cells (OPCs; CD45-, CD31-, CD51+, SCA-1-). This provides a robust platform in our established AML NSG xenograft model (using Molm14, HL60 or U937 cell lines) for a direct analysis of two principle mesenchymal populations known to be involved in leukemia drug resistance. At a leukemic burden of 60-90% in the BM, we initially observed a significant decline in the proportion of OPC in xenograft-bearing mice compared to healthy controls. We hypothesized that this could be caused by a block in osteogenic differentiation in the MSCs, or an increase in turnover of the OPCs. Surprisingly, we found an increase in osteoblastic differentiation in MSCs derived from xenograft-bearing mice, evidenced by strong induction of Osterix, Runx2, and other regulators of osteogenesis. We also found a concomitant increase in Annexin V and phosphorylated P53 within the OPC population, indicating significant apoptotic turnover, whereas no apoptosis in MSCs was observed. It was recently demonstrated that AML blasts can induce an unfolded protein response (UPR) in bystander cells. Here, we found that while both MSCs and OPCs demonstrate increased expression of Bip, Chop, and Xbp1, OPCs appear to be more sensitive to AML-induced UPR stress, an observation entirely consistent with their increased secretory function. These studies also provide a potential mechanism to explain experimental and clinical observations of osteoblastic cell loss as an aspect of disease progression in the AML BM. The induction of UPR stress in the expanding MSCs in the AML niche along with prior evidence of persistent functional changes led us to determine if an epigenetic mechanism was involved. First, in a candidate screen we discovered that AML significantly dysregulates the expression of key regulators of DNA methylation patterning in MSCs. Notably, the de novo methyltransferases Dnmt3a and 3b, as well as Tet2, were significantly upregulated in MSCs from all three AML xenografts. This highly reproducible in vivo observation prompted us to perform whole genome bisulfite sequencing on MSCs derived from our Molm-14 xenograft model, revealing substantial changes between AML xenografts and control MSCs in the DNA methylation signatures of CpG islands within annotated promoter regions. Gene ontology analyses of these promoter regions identified significant AML induces changes in apoptotic, UPR stress, and differentiation programs, consistent with both our transcriptional profiling and the change in MSC/ OPC composition in the niche. In sum, this work provides a link to explain prior studies in patients and demonstrates the power of xenograft models to experimentally dissect the complex and dynamic relationship between leukemia and discrete stromal populations in the AML niche. We provide direct evidence, free of tissue culture bias, that AML effects changes in the BM niche that differentially affect two key cellular components responsible for extrinsic drug resistance and relapse in AML. DisclosuresNo relevant conflicts of interest to declare.

Hyper-Activation of the Adenosinergic Axis in the Eμ-TCL1 Chronic Lymphocytic Leukemia Mouse Model Offers Therapeutic Opportunities

Background. Tumor immunosuppression is a major cause for treatment failure and disease relapse, both in solid tumors and leukemia. Several indications suggest that increased extracellular adenosine levels, which potently suppress T cell responses and skew macrophages towards an M2 phenotype, characterize the tumor microenvironment. Hence, there is intense investigation to identify drugs that target this axis.We previously showed that CLL cells, particularly those endowed in the proliferation centers, express CD39 and CD73, the enzymes that lead to extracellular adenosine production. We also showed that the A2A adenosine receptor is expressed at high levels in CLL lymph nodes, likely due to the effect of hypoxia in positively modulating adenosine production.Aim of the work. The main aim of this work is to study the expression and the role of the adenosinergic axis in vivo, determining the impact of this signaling pathway in CLL progression and modulation of immune responses. To this purpose, we exploited the Eμ-TCL1 (TCL1) mouse model based on the adoptive transfer of spleen-purified leukemic cells in naïve C57BL/6 immunocompetent recipients (WT).Results. Firstly, expression of the adenosinergic axis (CD39, CD73, CD26 and A2A) both on the leukemic side and on bystander immune cells was assessed by comparing cells obtained from the spleen, the bone marrow (BM) and peripheral blood (PB) of TCL1 and WT mice. Flow cytometry analysis of B220+/CD5+ leukemic cells showed that CD39 was constantly expressed at high levels, in contrast to normal circulating B cells from naïve mice. CD73 expression was more heterogeneous, with both CD73+ and CD73- leukemic clones. These data recapitulate the phenotype observed in CLL patients and sustain the hypothesis of variable levels of adenosine in tissues colonized by leukemic cells. In contrast, CD26, the adenosine deaminase-anchoring protein was poorly detected, as opposed to WT B cells. Real-time PCR data obtained from purified cell populations confirmed this expression pattern and also indicated dramatic up-regulation in the expression of the A2A receptor, suggesting that leukemia development is accompanied by increased ability to respond to extracellular adenosine. The development of CLL is usually associated with specific T cell and myeloid subset changes. Multiparametric flow cytometry analyses, with selective markers, were adopted to identify naïve, memory, central memory, effector memory and effector T cells, as well as inflammatory, intermediate or patrolling monocytes. Data obtained confirm that in TCL1 mice the naïve T cell subset was dramatically reduced compared to WT mice, with the simultaneous increase in memory, exhausted and regulatory T cell sub-populations. Both CD4+ and CD8+ lymphocyte subsets in TCL1 mice were highly CD39+ compared to the normal counterpart, as opposed to CD73 that was expressed in distinct subpopulation of cells, including regulatory T cells. No significant differences were highlighted for CD26, in all the tissues considered, while the A2A receptor became expressed at very high levels in all cellular components of the leukemic niche. In the monocyte compartment, we observed a reduction of the inflammatory subset, with a parallel increase of patrolling population, functionally similar to M2 tumor-supportive macrophages.Given the role of low oxygen tension as a critical modulator of the adenosinergic system, hypoxia levels were determined, comparing tumor-bearing and WT mice. Mice were treated with pimonidazole hydrochloride, a hypoxia marker, 2 hours prior euthanasia. Flow cytometry and immuno-histochemistry analyses indicated that leukemic engraftment is paralleled by increased hypoxia levels in the lymphoid tissues, such as spleen or BM.Conclusions.Taken together, these results show that the adenosinergic axis is over-expressed in the TCL1 mouse model and confirm that the presence of leukemic cells polarizes the host immune cells, favoring the establishment of a tumor supportive environment. This could be due, at least partly, to the presence of a highly hypoxic leukemic niche. These observations confirm what described for CLL patients, indicating that this animal model can be used to test the impact of adenosine-targeting therapies, in combination with drugs that directly target the clone. DisclosuresNo relevant conflicts of interest to declare.

Biological Properties of Mesenchymal Stromal Cells Obtained from Paediatric Patients with B-Cell Precursor Acute Lymphoblastic Leukemia

PURPOSEBone Marrow-derived Mesenchymal Stromal Cells (BM-MSCs) are the main component of the bone marrow microenvironment, where they regulate differentiation, proliferation and survival of adjacent hematopoietic precursors, by cell-to-cell contact and soluble factors. In addition, it has been demonstrated that BM-MSCs play a crucial role also within the leukemic bone marrow niche by regulating disease development and progression. The purpose of our work was to isolate and characterize morphologically, phenotypically and functionally MSCs derived from paediatric patients with B-Cell-Precursor Acute Lymphoblastic Leukemia (BCP-ALL) at disease onset. METHODSSeveral BM-MSC lines have been generated from BM aspirates of Healthy Donors (HD) (n=16) as well as BCP-ALL patients (n=16) and stored at “M. Tettamanti” Res. Center. MSCs were characterized at fourth passage in terms of morphology, immunophenotype (FACS analysis) and in vitro adipogenic and osteogenic differentiation potential. Chromosomal translocations detected in leukemia cells were investigated in BCP-ALL-MSCs by fluorescence in situ hybridizations (FISH) or polymerase chain reaction (PCR). MSCs were also analysed in terms of secretoma (BioRad Protein Array). RESULTSBoth HD-MSCs and BCP-ALL-MSCs resulted comparable in terms of morphology. They both expressed the typical MSC markers CD73, CD90 and CD105, while were negative for the hematopoietic markers CD14, CD34, CD45 and MHC-II. In addition, MSCs from all investigated BCP-ALL patients did not present the chromosomal translocations that had been detected in leukemia cells (1 patient BCR-ABL p210, 5 patients TEL-AML1). To identify possible differences, BM-MSCs isolated from HD and BCP-ALL patients were compared for their principal biological properties. We found no significant differences in terms of proliferation and differentiation into adipogeneic and osteogeneic lineages between the two different groups. Moreover, the secretome profile of BCP-ALL-MSCs and HD-MSCs resulted comparable. CONCLUSIONSWe found that BCP-ALL-MSCs were similar in terms of morphology, phenotype and differentiation ability to HD-MSCs. Furthermore, MSCs from patients did not reveal the chromosomal translocations present in leukemia blasts. Further assays aimed at investigating MSC production of soluble molecules, migration, adhesion to different substrates and Extracellular Matrix (ECM) deposition are currently under investigation. DisclosuresNo relevant conflicts of interest to declare.

The Meninges Enhance Acute Lymphoblastic Leukemia Chemoresistance in the Central Nervous System

▪Central nervous system (CNS) relapse is a principal cause of treatment failure among pediatric patients with acute lymphoblastic leukemia (ALL). Furthermore, available CNS-directed therapies are associated with significant short and long-term morbidities. While extensive research has demonstrated a critical role of the bone marrow microenvironment in leukemia biology, how the CNS regulates ALL survival and chemoresistance has yet to be fully determined.To identify where leukemia cells home within the CNS, we transplanted multiple human leukemia cell lines into immune-compromised mice. After systemic leukemia development that included the CNS, the mice were euthanized, fixed, and examined by histopathology and immunohistochemistry. We identified both the meninges and, to a lesser extent, the choroid plexus as the predominant CNS sites that harbor leukemia cells both before and after treatment with systemic cytarabine (Figure 1A).In contrast, parenchymal involvement by leukemia was a rare, and often late, finding.This pattern of CNS involvement by leukemia also recapitulates the histopathology of human CNS leukemia.We then used ex vivo co-culture approaches to focus more specifically on the effects of the meninges on leukemia biology. We found that leukemia cells adhered to meningeal cells in a co-culture system that can substitute human cerebral spinal fluid for tissue culture media. Moreover, co-cultured leukemia cells were significantly more resistant to cytarabine-induced apoptosis, as measured by annexin-V staining, relative to the same leukemia cells grown in suspension or adherent to either fibronectin-treated plates or a neural precursor cell line (Figure 1B). Extending these results, meningeal-mediated leukemia chemoresistance was also observed with methotrexate as well as when measured by caspase 3/7 & 8 activity and mitochondrial polarization by TMRE staining. Moreover, primary ALL cells exhibited significantly increased apoptosis when cultured in suspension relative to when co-cultured with primary meningeal cells (60.4% vs. 8.9%, P <0.0001), further supporting the pro-survival effect of meningeal cells on leukemia cells. Finally, while leukemia cells co-cultured with meningeal cells exhibited striking chemoresistance, leukemia cells cultured in meningeal conditioned media also exhibited moderate chemoresistance (Figure 1B). Together, these results suggest that both a mechanism dependent upon direct cell contact and a soluble factor(s) secreted by meningeal cells contribute to meningeal-mediated leukemia chemoresistance.In order to better understand the mechanism of meningeal-mediated leukemia chemoresistance, we examined the effect of co-culture on the apoptosis pathway in leukemia cells. An apoptosis antibodyarray showed changes in the expression of multiple apoptosis family proteins (including caspase 3, caspase 8, Bax, Bad, BID, BIM, BCL-2, SMAC and survivin) in leukemia cells co-cultured with meningeal cells relative to leukemia cells in suspension. However, assessing how the dynamic levels, activities, and complex interactions of these and other BCL-2 family of pro-and anti-apoptotic proteins (BH3 proteins) integrate to regulate the overall apoptotic balance is experimentally challenging. To address this issue, we utilized a functional apoptosis assay, BH3 profiling, which predicts cellular responses to stimuli based on measuring the response of mitochondria to perturbation by a panel of BH3 domain peptides. BH3 profiling of leukemia cells in suspension or co-cultured with meningeal cells showed that leukemia cells co-cultured with meningeal cells are significantly less primed to undergo apoptosis relative to leukemia cells in suspension.In summary, these results show that the meninges provide a unique CNS leukemia niche that regulates the apoptotic balance in leukemia cells in order to enhance leukemia chemoresistance and contribute to relapse. This work also suggests that CNS leukemia persistence and relapse is not only related to poor penetration of some chemotherapy agents across the blood-brain barrier and into the CNS. Further characterization of the cellular and molecular mechanisms of meningeal-mediated leukemia chemoresistance will provide new insights into the pathophysiology of CNS leukemia as well as inform novel therapeutic strategies to target leukemia cells in the CNS niche. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Driving CAR T Stem Cell Targeting in Acute Myeloid Leukemia: The Roads to Success.

Simple SummaryChimeric antigen receptor (CAR) T-cells are powerful therapeutic tools that have revolutionized the treatment of several hematological malignancies. However, their therapeutic application in acute myeloid leukemia (AML) remains challenging. In this review, the authors aimed to dissect how AML-leukemic stem cell and AML-bone marrow niche features can impact on the success of CAR T-cell therapy. The clinical implementation of some of the newly developed approaches discussed in this review may lead to the development of safe and effective CAR T-cell strategies for AML, accounting for the disease heterogeneity.Current treatment outcome for acute myeloid leukemia (AML) patients is unsatisfactory and characterized by high rates of relapse and poor overall survival. Increasing evidence points to a crucial role of leukemic stem cells (LSC) and the bone marrow (BM) leukemic niche, in which they reside, in AML evolution and chemoresistance. Thus, future strategies aiming at improving AML therapeutic protocols are likely to be directed against LSC and their niche. Chimeric antigen receptor (CAR) T-cells have been extremely successful in the treatment of relapsed/refractory acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma and comparable results in AML are highly desirable. At present, we are at the dawn of CAR T-cell application in AML, with several preclinical studies and few early phase clinical trials. However, the lack of leukemia-specific targets and the genetic and phenotypic heterogeneity of the disease combined with the leukemia-induced remodeling of the BM microenvironment are limiting CAR T-cell exploitation in AML. Here, we reviewed AML-LSC and AML-BM niche features in the context of their therapeutic targeting using CAR T-cells. We summarized recent progress in CAR T-cell application to the treatment of AML, and we discussed the remaining therapeutic challenges and promising novel strategies to overcome them.

Targeting the plasticity of mesenchymal stromal cells to reroute the course of acute myeloid leukemia

AbstractBone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell–cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but >30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

The Bone Marrow Niche in B-Cell Acute Lymphoblastic Leukemia: The Role of Microenvironment from Pre-Leukemia to Overt Leukemia.

Genetic lesions predisposing to pediatric B-cell acute lymphoblastic leukemia (B-ALL) arise in utero, generating a clinically silent pre-leukemic phase. We here reviewed the role of the surrounding bone marrow (BM) microenvironment in the persistence and transformation of pre-leukemic clones into fully leukemic cells. In this context, inflammation has been highlighted as a crucial microenvironmental stimulus able to promote genetic instability, leading to the disease manifestation. Moreover, we focused on the cross-talk between the bulk of leukemic cells with the surrounding microenvironment, which creates a “corrupted” BM malignant niche, unfavorable for healthy hematopoietic precursors. In detail, several cell subsets, including stromal, endothelial cells, osteoblasts and immune cells, composing the peculiar leukemic niche, can actively interact with B-ALL blasts. Through deregulated molecular pathways they are able to influence leukemia development, survival, chemoresistance, migratory and invasive properties. The concept that the pre-leukemic and leukemic cell survival and evolution are strictly dependent both on genetic lesions and on the external signals coming from the microenvironment paves the way to a new idea of dual targeting therapeutic strategy.

Clinical Implications of Inflammation in Acute Myeloid Leukemia.

Recent advances in the description of the tumor microenvironment of acute myeloid leukemia, including the comprehensive analysis of the leukemic stem cell niche and clonal evolution, indicate that inflammation may play a major role in many aspects of acute myeloid leukemia (AML) such as disease progression, chemoresistance, and myelosuppression. Studies on the mechanisms of resistance to chemotherapy or tyrosine kinase inhibitors along with high-throughput drug screening have underpinned the potential role of glucocorticoids in this disease classically described as steroid-resistant in contrast to acute lymphoblastic leukemia. Moreover, some mutated oncogenes such as RUNX1, NPM1, or SRSF2 transcriptionally modulate cell state in a manner that primes leukemic cells for glucocorticoid sensitivity. In clinical practice, inflammatory markers such as serum ferritin or IL-6 have a strong prognostic impact and may directly affect disease progression, whereas interesting preliminary data suggested that dexamethasone may improve the outcome for AML patients with a high white blood cell count, which paves the way to develop prospective clinical trials that evaluate the role of glucocorticoids in AML.

Protein kinase C-β-dependent changes in the glucose metabolism of bone marrow stromal cells of chronic lymphocytic leukemia

Survival of chronic lymphocytic leukemia (CLL) cells critically depends on the support of an adapted and therefore appropriate tumor microenvironment. Increasing evidence suggests that B-cell receptor-associated kinases such as protein kinase C-β (PKCβ) or Lyn kinase are essential for the formation of a microenvironment supporting leukemic growth. Here, we describe the impact of PKCβ on the glucose metabolism in bone marrow stromal cells (BMSC) upon CLL contact. BMSC get activated by CLL contact expressing stromal PKCβ that diminishes mitochondrial stress and apoptosis in CLL cells by stimulating glucose uptake. In BMSC, the upregulation of PKCβ results in increased mitochondrial depolarization and leads to a metabolic switch toward oxidative phosphorylation. In addition, PKCβ-deficient BMSC regulates the expression of Hnf1 promoting stromal insulin signaling after CLL contact. Our data suggest that targeting PKCβ and the glucose metabolism of the leukemic niche could be a potential therapeutic strategy to overcome stroma-mediated drug resistance.

Understanding of the crosstalk between normal residual hematopoietic stem cells and the leukemic niche in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease, yet clinically most patients present with pancytopenia resulting from bone marrow failure, predisposing them to life-threatening infections and bleeding. The mechanisms by which AML mediates hematopoietic suppression is not well known. Indeed, much effort has so far been focused on how AML remodels the bone marrow niche to make it a more permissive environment, with less focus on how the remodeled niche affects normal hematopoietic cells. In this perspective, we present evidence of the key role of the bone marrow niche in suppressing hematopoietic stem cells (HSCs) during leukemic progression and provide perspectives on how future research on this topic may be exploited to provide treatments for one of the key complications of AML.

Hypoxia favors chemoresistance in T-ALL through an HIF1α-mediated mTORC1 inhibition loop

AbstractResistance to chemotherapy, a major therapeutic challenge in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), can be driven by interactions between leukemic cells and the microenvironment that promote survival of leukemic cells. The bone marrow, an important leukemia niche, has low oxygen partial pressures that highly participate in the regulation of normal hematopoiesis. Here we show that hypoxia inhibits T-ALL cell growth by slowing down cell cycle progression, decreasing mitochondria activity, and increasing glycolysis, making them less sensitive to antileukemic drugs and preserving their ability to initiate leukemia after treatment. Activation of the mammalian target of rapamycin (mTOR) was diminished in hypoxic leukemic cells, and treatment of T-ALL with the mTOR inhibitor rapamycin in normoxia mimicked the hypoxia effects, namely decreased cell growth and increased quiescence and drug resistance. Knocking down (KD) hypoxia-induced factor 1α (HIF-1α), a key regulator of the cellular response to hypoxia, antagonized the effects observed in hypoxic T-ALL and restored chemosensitivity. HIF-1α KD also restored mTOR activation in low O2 concentrations, and inhibiting mTOR in HIF1α KD T-ALL protected leukemic cells from chemotherapy. Thus, hypoxic niches play a protective role of T-ALL during treatments. Inhibition of HIF-1α and activation of the mTORC1 pathway may help suppress the drug resistance of T-ALL in hypoxic niches.

The Role of Notch and Wnt Signaling in MSC Communication in Normal and Leukemic Bone Marrow Niche.

Notch and Wnt signaling are highly conserved intercellular communication pathways involved in developmental processes, such as hematopoiesis. Even though data from literature support a role for these two pathways in both physiological hematopoiesis and leukemia, there are still many controversies concerning the nature of their contribution. Early studies, strengthened by findings from T-cell acute lymphoblastic leukemia (T-ALL), have focused their investigation on the mutations in genes encoding for components of the pathways, with limited results except for B-cell chronic lymphocytic leukemia (CLL); in because in other leukemia the two pathways could be hyper-expressed without genetic abnormalities. As normal and malignant hematopoiesis require close and complex interactions between hematopoietic cells and specialized bone marrow (BM) niche cells, recent studies have focused on the role of Notch and Wnt signaling in the context of normal crosstalk between hematopoietic/leukemia cells and stromal components. Amongst the latter, mesenchymal stromal/stem cells (MSCs) play a pivotal role as multipotent non-hematopoietic cells capable of giving rise to most of the BM niche stromal cells, including fibroblasts, adipocytes, and osteocytes. Indeed, MSCs express and secrete a broad pattern of bioactive molecules, including Notch and Wnt molecules, that support all the phases of the hematopoiesis, including self-renewal, proliferation and differentiation. Herein, we provide an overview on recent advances on the contribution of MSC-derived Notch and Wnt signaling to hematopoiesis and leukemia development.

Toward Therapeutic Targeting of Bone Marrow Leukemic Niche Protective Signals in B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) represents the malignant counterpart of bone marrow (BM) differentiating B cells and occurs most frequently in children. While new combinations of chemotherapeutic agents have dramatically improved the prognosis for young patients, disease outcome remains poor after relapse or in adult patients. This is likely due to heterogeneity of B-ALL response to treatment which relies not only on intrinsic properties of leukemic cells, but also on extrinsic protective cues transmitted by the tumor cell microenvironment. Alternatively, leukemic cells have the capacity to shape their microenvironment towards their needs. Most knowledge on the role of protective niches has emerged from the identification of mesenchymal and endothelial cells controlling hematopoietic stem cell self-renewal or B cell differentiation. In this review, we discuss the current knowledge about B-ALL protective niches and the development of therapies targeting the crosstalk between leukemic cells and their microenvironment.

Involvement of GPx-3 in the Reciprocal Control of Redox Metabolism in the Leukemic Niche.

The bone marrow (BM) microenvironment plays a crucial role in the development and progression of leukemia (AML). Intracellular reactive oxygen species (ROS) are involved in the regulation of the biology of leukemia-initiating cells, where the antioxidant enzyme GPx-3 could be involved as a determinant of cellular self-renewal. Little is known however about the role of the microenvironment in the control of the oxidative metabolism of AML cells. In the present study, a coculture model of BM mesenchymal stromal cells (MSCs) and AML cells (KG1a cell-line and primary BM blasts) was used to explore this metabolic pathway. MSC-contact, rather than culture with MSC-conditioned medium, decreases ROS levels and inhibits the Nrf-2 pathway through overexpression of GPx3 in AML cells. The decrease of ROS levels also inactivates p38MAPK and reduces the proliferation of AML cells. Conversely, contact with AML cells modifies MSCs in that they display an increased oxidative stress and Nrf-2 activation, together with a concomitant lowered expression of GPx-3. Altogether, these experiments suggest that a reciprocal control of oxidative metabolism is initiated by direct cell–cell contact between MSCs and AML cells. GPx-3 expression appears to play a crucial role in this cross-talk and could be involved in the regulation of leukemogenesis.

Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant clonal disease derived from hematopoietic stem/progenitor cell. Leukemia blasts cause extensive hypoxia of bone marrow (BM), which lead to disorder and remodeling of BM niche, thereby becoming "leukemic niche" to support the development and drug-resistance of AML as well as the maintenance of normal hematopoietic stem cells. In this study, the biological characteristics (such as self-renewal, apoptosis, migration, autocrine) and function (vascularization) of mesenchymal stem cells (MSCs) and human umbilical artery endothelial cells (HUAECs) that make up BM arteriolar niche in simulated hypoxia AML context were investigated. It was found that moderate hypoxia enhanced the viability of the arteriolar niche cells, but severe hypoxia of AML BM resulted in the damage of arteriolar niche cells and the disorder of vascular cytokines C-X-C motif chemokine ligand 6 (CXCL6). The dynamic changes of CXCL6 in the system as well as its anti-apoptotic and promoting angiogenic effects suggested that CXCL6 played an important role in the remodeling of BM arteriolar niche in AML. Taking advantage of CXCL6 can save the damaged MSCs and HUAECs, which is the hope of rescuing arteriolar niche. It is suggested that CXCL6 may be an assistant strategy for microenvironment targeted therapy of AML.

Microrna-155 Trafficking Incites Compartmental Inflammation in the Leukemic Niche

Compartmental bone marrow (BM) inflammation has been linked to acute myeloid leukemia (AML) progression and hematopoietic dysfunction, yet the underlying mechanisms remain unclear. MicroRNAs (miRs) are capable of broadly deregulating cellular gene expression programs through simultaneous, dose-dependent action on a panel of target genes. MicroRNA-155, a widely known proinflammatory miRNA is overexpressed in AML patients, in particular those with Flt3-ITD. We recently observed that miR-155 is highly abundant in AML patient plasma-derived extracellular vesicles (AML-EV) and identified inflammation as one of the most upregulated gene ontology (GO) categories in a proteomic screen of hematopoietic stem and progenitor cells (HSPCs) exposed to AML-EV. Here, we investigate whether AML-derived miR-155 coordinately regulates inflammatory signaling in the leukemic niche. To better model compartmental inflammation in AML, we first established a highly penetrant, immunocompetent, fully congenic system. Using the monoblastic AML cell line C1498 (CD45.2+), we were able to reliably graft animals without myeloablation, thereby avoiding unintended changes in BM niche function. Across multiple experimental cohorts, we then analyzed the BM plasma secretome and observed dynamic changes in the level of several inflammatory chemokines and cytokines. At sacrifice, flow cytometric analysis of the residual HSPC (CD45.1+) showed concurrent upregulation of key inflammatory response genes (ISG15, STAT1, CXCL10) and evidence of replicative stress (γ-H2AX) in residual HSPC. Consistent with potential trafficking of miR-155 via AML-EV, we next confirmed the enrichment of miR-155 in C1498-EVs, observed gains in miR-155 in BM plasma EVs and demonstrated increased levels in HSPCs following EV uptake. To determine if miR-155 could function as a regulatory hub under inflammatory conditions we utilized four separate target prediction databases (miRDB, TargetScan, miRWalk, and miRanda) to generate miR-155 target gene set. With an initial list of 360 potential gene targets that met a minimum threshold prediction score, we queried six publicly available RNA-Seq datasets to focus on genes belonging to inflammation or DNA damage repair GO groups highly expressed in HSPCs. This yielded a final set of 9 candidate targets (Wee1, E2f2, Rps6ka3, Rps6ka5, Csflr, Mgme1, Rad51, Rad51b, Ercc1). Reasoning that miR-155 target sets are involved in niche inflammation and genotoxicity in AML, these computationally predicted targets will undergo testing and validation for involvement in inflammatory and genotoxic stress. Chronic inflammation in the AML niche contributes to disease persistence and failure of residual hematopoiesis. Our study will determine the role of miR-155 as a regulatory hub for compartmental inflammatory signaling, and simultaneously serve as a potential opportunity for novel therapeutic target discovery in leukemic BM. Disclosures No relevant conflicts of interest to declare.

B-Cell Precursor Acute Lymphoblastic Leukemia Instructs Mesenchymal Stromal Cells to Alter Interferon-Related Gene Expression and Cyto/Chemokine Secretion

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells that are present in the bone marrow microenvironment are able to hijack the normal hematopoietic stem cell niches to create a leukemic niche. The importance of this microenvironment for leukemic cells is demonstrated by the protection that the niche provides against chemotherapeutic agents. Patient-derived mesenchymal stromal cells (MSCs) mimic this protective effect in vitro. Unraveling the mechanism of protection is important to provide potential novel options for therapeutic intervention. Therefore, our research is focused on the interaction between BCP-ALL cells and MSCs. We aimed to determine gene expression changes in BCP-ALL cells and MSCs after co-culture compared to mono-culture, and to investigate which cyto- and chemokines are differentially secreted upon contact between BCP-ALL cells and MSCs. Methods: We performed co-cultures of primary MSCs and BCP-ALL cells, and mono-cultures of MSCs or BCP-ALL cells for 40 hours to determine gene expression changes. Viable cells were sorted by FACS and RNA was isolated. Total-RNA sequencing data (Illumina) were analyzed using R. Supernatant was saved to determine cyto/chemokine profiles by Luminex technology, and to investigate the effect of these cyto/chemokines on the survival and migration of the leukemic cells. Moreover, migration experiments using transwells (3.0µm pore size) were performed to determine the level of BCP-ALL migration towards cyto/chemokines of interest. Results: RNA sequencing data from 15 independent co-culture experiments revealed that interferon (IFN)-related genes, such as IFI6, MX1, IFI27, and OAS1, were 2.5 to 3.1-fold upregulated in the MSCs after co-culture with BCP-ALL compared to MSC mono-culture. The type of upregulated pro-inflammatory genes and amount of upregulation varied between BCP-ALL patients. However, the observed changes were always similar when an ALL case was co-cultured with different MSC samples. This suggests that the observed changes are induced by the leukemic cells and that leukemic cells manipulate MSCs. Survival benefit (0.3 - 37.9%) was observed in BCP-ALL cells after co-culture with MSCs compared to BCP-ALL mono-culture. Moreover, pro-inflammatory cytokines, and several migration-related chemokines such as CCL2, CXCL8, and CXCL10/IP-10 were upregulated in 5 out of 15 co-cultures compared to the sum of the separate mono-cultures of BCP-ALL and MSCs. A gradient of CXCL10/IP-10 in transwell experiments showed that this chemokine did not enhance the migration of primary BCP-ALL cells, suggesting that the ALL-induced secretion of this chemokine serves a different role in BCP-ALL. The role of CXCL10/IP-10 and other cyto/chemokines in immune regulation at the time of overt leukemia is part of ongoing studies. Conclusion: Our data show that IFN-related genes, pro-inflammatory cytokines and migration-related chemokines become upregulated in bone marrow stromal cells upon exposure to BCP-ALL. These induced changes may be important for BCP-ALL cell survival, affecting the mobility of other immune cells, and/or ensure that leukemic cells remain in close contact with MSCs. We postulate that interference with these affected genes and cyto/chemokines may disrupt the direct contact between leukemic cells and their niche, and may provide an alternative way to eliminate leukemic cells more efficaciously. Functional studies addressing this concept are currently being executed and the results will be presented during the meeting. Disclosures No relevant conflicts of interest to declare.

The IL1-IL1RAP axis plays an important role in the inflammatory leukemic niche that favors acute myeloid leukemia proliferation over normal hematopoiesis

Upregulation of the plasma membrane receptor IL1RAP in acute myeloid leukemia (AML) has been reported but its role in the context of the leukemic bone marrow niche is unclear. Here, we studied the signaling events downstream of IL1RAP in relation to leukemogenesis and normal hematopoiesis. High IL1RAP expression was associated with a leukemic GMPlike state, and knockdown of IL1RAP in AML reduced colony-forming capacity. Stimulation with IL1b resulted in the induction of multiple chemokines and an inflammatory secretome via the p38 MAPK and NFkB signaling pathways in IL1RAP-expressing AML cells, but IL1b-induced signaling was dispensable for AML cell proliferation and NFkB-driven survival. IL1RAP was also expressed in stromal cells where IL1b induced expression of inflammatory chemokines and cytokines as well. Intriguingly, the IL1b-induced inflammatory secretome of IL1RAP-expressing AML cells grown on a stromal layer of mesenchymal stem cells affected normal hematopoiesis including hematopoietic stem/progenitor cells while AML cell proliferation was not affected. The addition of Anakinra, an Food and Drug Aministration-approved IL1 receptor antagonist, could reverse this effect. Therefore, blocking the IL1-IL1RAP signaling axis might be a good therapeutic approach to reduce inflammation in the bone marrow niche and thereby promote normal hematopoietic recovery over AML proliferation after chemotherapy.