Bone Marrow Niche Cells Research Articles

Selective Deletion of ATG5 in Bone Marrow Leptin-Receptor-Expressing Perivascular Stromal Cells Causes Leukopenia

Bone marrow niche cells, specifically the Leptin-receptor-expressing perivascular stromal cells (LepR+), CXCL12-abundant reticular cells (CAR), and endothelial cells play an important role in the maintenance and self-renewal functions of hematopoietic stem cells (HSCs). This maintenance function provided by the niche cells is mediated by a number of secretory-related molecules such as stem cell factor (SCF), CXCL12 and Angiopoietin. In addition, functional autophagy within HSCs is known to play a critical role for maintaining HSC homeostasis, as the loss of ATG7 in HSCs leads to loss of normal HSC functions and severe myeloproliferation. However, it remains unclear as to how hematopoiesis will be affected if autophagy is selectively blocked in bone marrow niche cells. To investigate the effect of autophagy deficiency within bone marrow niche cells on hematopoiesis, we generated a knockout mouse model allowing for a selective ablation of an autophagy-essential protein, ATG5, in LepR+ perivascular stromal cells. We have confirmed that the deletion of Atg5 is indeed restricted to LepR+ cells, and that the loss of ATG5 is sufficient to block autophagic function, as evident through the accumulation of p62 protein. We found that the white blood cell count (WBC) is significantly reduced in 16-week old male ATG5 KO mice compare to wild type (wt) littermate control mice (p<0.05). Further, the myeloid population (CD11b+ Ly6G+) in both bone marrow and peripheral blood is significantly decreased in ATG5 KO mice (p value < 0.05, ATG KO vs. wt control). WBC count continues to decline in 24-week old ATG5 KO male mice (p=0.02) but not in female mice (p=0.58). Both LSK (Lin- Sca-1+ c-Kit+) and long term HSC (LT-HSCs, CD150+ CD48- LSK) populations are decreased in both 16-week and 24-week old male mice; however, this trend did not reach statistical significance (p>0.05). Although the absolute cell count of lymphocytes, including B and T cells (CD19+ and CD3+, respectively) in peripheral blood, is significantly dropped in ATG5 KO mice compared to wt controls, there is no significant change of mature B and T cells in bone marrow. These preliminary results suggest that deletion of a key autophagy regulator in LepR+ bone marrow niche cells will cause leukopenia. The underlying mechanism is currently under investigation and it will help us to better understand the role of autophagy in hematopoiesis within the bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow (BM) Microenviroment Factors As Early Markers of Response in Patients with Newly Diagnosed Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Treated with Nilotinib

IntroductionTyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients.MethodsThe study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits.Results33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05).Conclusion/SummaryTaken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. DisclosuresSoverini:Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Fluorescence Lifetime Imaging of Apoptosis.

Genetically encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools for analyzing cell signaling and function at single-cell resolution in standard 2D cell cultures, but these reporters rarely have been applied to 3D environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in light absorption complicate this analysis method in 3D settings. Herein we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from 2D cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control the resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow.

Hematopoietic stem cells (HSCs) depend on the bone marrow (BM) niche for their maintenance, proliferation, and differentiation. The BM niche is composed of nonhematopoietic and mature hematopoietic cells, including megakaryocytes (Mks). Thrombopoietin (Thpo) is a crucial cytokine produced by BM niche cells. However, the cellular source of Thpo, upon which HSCs primarily depend, is unclear. Moreover, no specific molecular pathway for the regulation of Thpo production in the BM has been identified. Here, we demonstrate that the membrane protein C-type lectin-like receptor-2 (CLEC-2) mediates the production of Thpo and other factors in Mks. Mice conditionally deleted for CLEC-2 in Mks (Clec2(MkΔ/Δ)) produced lower levels of Thpo in Mks. CLEC-2-deficient Mks showed down-regulation of CLEC-2-related signaling molecules Syk, Lcp2, and Plcg2. Knockdown of these molecules in cultured Mks decreased expression of Thpo. Clec2(MkΔ/Δ) mice exhibited reduced BM HSC quiescence and repopulation potential, along with extramedullary hematopoiesis. The low level of Thpo production may account for the decline in HSC potential in Clec2(MkΔ/Δ) mice, as administration of recombinant Thpo to Clec2(MkΔ/Δ) mice restored stem cell potential. Our study identifies CLEC-2 signaling as a novel molecular mechanism mediating the production of Thpo and other factors for the maintenance of HSCs.

Hypoxia regulates the hematopoietic stem cell niche.

Bone marrow, the site of hematopoiesis throughout adulthood, is a physiologically hypoxic organ. Thus, various biological oxygen sensors and their signaling cascades play a pivotal role in hematopoietic systems in the bone marrow under both physiologic and pathologic conditions. Hypoxia-inducible factors (HIFs) are hypoxic stress sensor proteins that are stabilized under homeostatic or stress-induced hypoxia. In the hypoxic bone marrow, HIFs play crucial roles in hematopoietic stem cells (HSCs) and in the cells of the HSC niche. The signals downstream of the HIFs maintain HSC quiescence, survival, and metabolic homeostasis through both cell-autonomous and non-cell-autonomous mechanisms. Leukemic stem cells (LSCs) hijack these delicate hypoxia-sensing mechanisms to sustain their self-renewal potential, promoting disease progression and drug resistance even under normoxic conditions. This review focuses on HIF-mediated oxygen-sensing mechanisms of adult HSCs and LSCs and their niche cells in the hypoxic bone marrow.

Expression of Hedgehog Pathway Mediator GLI Represents a Negative Prognostic Marker in Human Acute Myeloid Leukemia and Its Inhibition Exerts Antileukemic Effects.

The Hedgehog pathway plays an important role in stem-cell biology and malignant transformation. Therefore, we investigated the expression and prognostic impact of Hedgehog pathway members in acute myeloid leukemia (AML). Pretreatment samples from 104 newly diagnosed AML patients (AMLSG 07-04 trial) were analyzed by qPCR, and expression of Hedgehog family members was correlated with clinical outcome. Inhibition of GLI by GANT61 or shRNA was investigated in AML cells in vitro and in vivo. Expression of receptors Smoothened and Patched-1 and their downstream mediators, GLI1, GLI2, and GLI3, was found in AML patients in contrast to Hedgehog ligands. GLI2 expression had a significant negative influence on event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS; P = 0.037, 0.026, and 0.013, respectively) and was correlated with FLT3 mutational status (P < 0.001). Analysis of a second, independent patient cohort confirmed the negative impact of GLI2 on EFS and OS (P = 0.007 and 0.003, respectively; n = 290). Within this cohort, GLI1 had a negative prognostic impact (P < 0.001 for both EFS and OS). Although AML cells did not express Hedgehog ligands by qPCR, AML patients had significantly increased Desert Hedgehog (DHH) plasma levels compared with healthy subjects (P = 0.002), in whom DHH was presumably provided by bone marrow niche cells. Moreover, the GLI inhibitor GANT61 or knockdown of GLI1/2 by shRNA caused antileukemic effects, including induction of apoptosis, reduced proliferation, and colony formation in AML cells, and a survival benefit in mice. GLI expression is a negative prognostic factor and might represent a novel druggable target in AML.

Pericytes: Properties, Functions and Applications in Tissue Engineering.

Mesenchymal stem cells (MSCs) are one of the most studied adult stem cells and in recent years. They have become attractive agents/cell source for cellular therapy and regenerative medicine applications. During investigations about their origin, researchers hypothesized that perivascular regions are the common anatomical regions where MSCs come from and perivascular cells like pericytes (PCs) (Rouget cells, mural cells) are in vivo counterparts of MSCs. Beside capillaries and microvessels as their most common locations, PCs are also found in large vessels (arteries and veins). They can be isolated from several tissues and organs particularly from retina and brain. There are different approaches about their isolation, characterization and culture but there has been no common protocol yet because of the lack of defined PC-specific marker. They make special contact with endothelial cells in the basement membrane and have very important functions in several tissues and organs. They participate in vascular development, stabilization, maturation, and remodeling, blood pressure control, endothelial cell proliferation and differentiation, contractility of vascular smooth muscle cells, wound healing, vasculogenesis and angiogenesis, long-term maintenance of hematopoietic stem cells in bone marrow niche. Their multipotential differentiation capacity and participation in many events in the body make PCs preferred cells in tissue engineering applications including 3D blood-brain barrier models, skeletal muscle constructs, bone tissue engineering and tissue-engineered vascular grafts.

Maintenance of leukemic and normal hematopoietic stem cells in bone marrow niches by EVI1-regulated GPR56

Acute myeloid leukemia (AML) with high ecotropic viral integration site-1 (EVI1) expression (EVI1high AML) is classified as a refractory leukemia with a poor prognosis. We identified G protein-coupled receptor 56 (GPR56) as a novel marker for EVI1high AML, which is an orphan adhesion G protein-coupled receptor (GPCR). GPR56 was found to be associated with high cell adhesion and anti-apoptotic activities in EVI1high AML through activation of RhoA signaling. Moreover, in Gpr56-/- mice, the number of hematopoietic stem cells (HSCs) in bone marrow was significantly decreased with proportional increases in the spleen and peripheral blood, reflecting extramedullary hematopoiesis. The number of Gpr56-/- HSC progenitor cells in the G0 phase was significantly reduced with impaired adhesion and the loss of GPR56 function, which diminished the in vivo repopulating ability of HSCs. In conclusion, GPR56 may represent an important GPCR for the maintenance of quiescence and cellular adhesion of EVI1high AML and HSCs in the bone marrow niche. Moreover, given that GPR56 expression in leukemia stem cells is much higher than that in HSCs, GPR56 is a candidate therapeutic target for leukemia stem cells in EVI1high AML.

The Dynamics of Stroma-Leukemia Interaction in the Hypoxic BM Niches in Vivo

We and others have previously reported that leukemia progression is associated with vast expansion of the hypoxic niches and stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in leukemic cells (Frolova et al. Cancer Biol Ther. 2012, 10:858; Benito et al. PLoS One 2011, 6(8); e23108:1). Interactions of leukemia and the bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells, and we have shown that HIF-1α stabilization in stromal cells of the microenvironment facilitates leukemia homing and progression (Chen et al. Blood 2012, 119:4971). In this study, we aimed to characterize the time-dependent progression of BM hypoxia involving both leukemia cells and components of the BM niche, using the multiphoton intravital microscopy (MP-IVM) technique.We first generated a transplantable, imageable leukemia model by retrovirally transducing C57Bl6-Ai14 murine BM cells that express red fluorescing tdTomato with the p190-Bcr/Abl oncogene. The resulting p190-Bcr/Abl tdTomato cells caused rapid development of acute lymphocytic leukemia (ALL) in un-irradiated C57Bl6 immunocompetent mice, manifested by infiltration of the spleen, liver, BM within long bones, skull, and central nervous system followed by death within 28 days. Leukemia cells collected from the BM (LBC) of these animals were transplantable into secondary recipients and triggered accelerated ALL development (14-16 days). Time-course analysis of skull and femur bones in the secondary recipients by MP-IVM demonstrated LBC lodging on day 1 after ALL cell injection, followed by rapid accumulation of leukemia cells localized predominantly within the sinusoidal spaces, which were visualized by injecting the vascular fluorescent dye BSA-647 (Fig. 1a).To detect in vivo hypoxia development, we utilized HS680 (HypoxiSense 680), a carbonic anhydrase IX (CAIX)–targeted fluorescent agent that can be used to image overexpression of CAIX, a direct HIF-1α target, in tumors in response to regional hypoxia. C57Bl6 mice were engrafted with 2 x 105 LBC , and HS680 was injected intravenously at serial intervals followed by MP-IVM. In two separate experiments, increased HS680 fluorescence was detected in bone-lining cells in the BM niches of mice harboring ALL on days 8 and 13, but not in their healthy littermates (Fig 1b). To obtain an independent confirmation of hypoxia, additional mice (n=3) at the same stage (day 14) of leukemia development were sacrificed 3 hr after injection of chemical hypoxia probe pimonidazole (Pimo), and hypoxic BM cells that bound the hypoxia probe were detected by immunohistochemistry. Pimo staining demonstrated vastly spread areas of hypoxia that enclosed both leukemia cells and BM niche cells (Fig 1c), consistent with our previously published observations in different leukemia models.In summary, these findings demonstrate rapid development of intra-BM hypoxia that parallels leukemia progression and involves not only leukemia cells, but also BM niche cells. The HS680 probe can detect hypoxia in vivo within niche cells but not in leukemia cells, likely because of differential expression of CAIX. Our ongoing studies will characterize the cellular origin of hypoxic niche cells by utilizing immunohistochemical techniques and Col2.3-GFPemd transgenic mice to visualize osteoblasts. We postulate that the tumor microenvironment altered with hypoxic niche cells will influence leukemia development or responses to therapy. To this end, we have generated mice with conditionally deleted HIF-1α within BM stromal cells and are investigating the differences in leukemia homing, progression, and chemoresistance between these mice and mice whose BM stromal cells express HIF-1a. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PGE2 Promotes BM Hematopoietic Stem Cell Retention Via Stromal Lactate Production­­­­, cAMP and CXCL12/CXCR4 Regulation

The CXCR4/CXCL12 axis is essential for retention and protection from DNA damage of quiescent hematopoietic stem cells (HSC) in their bone marrow (BM) niches. Murine CXCR4+ HSC tightly adhere to BM stromal cells which functionally express cell surface CXCL12. Stress induces secretion of CXCL12 by BM stromal cells and its release to the circulation, mediating hematopoietic stem and progenitor cell (HSPC) egress, recruitment and clinical mobilization. Previously, we reported that Prostaglandin E2 (PGE2), highly produced by COX-2+ BM αSMA+ monocyte/macrophages, upregulates surface CXCR4 expression on enriched human CD34+ HSPC and their CXCL12 induced motility via cAMP activation in vitro. PGE2 inhibits intracellular reactive oxygen species (ROS) generation in HSPC and also increases membrane bound CXCL12 expression by BM stromal cells leading to HSC adhesion to their niche supporting cells in vivo, overall contributing to BM stem cell retention. We also found that elevation in cAMP activation promotes CXCL12 secretion from BM stromal cells, and another report has recently shown that lactate signaling via its major receptor HCA-1 inhibits cAMP.Thus, we hypothesized that the major metabolite lactate, cAMP and PGE2 cross-regulate BM stem cell retention by modulating the CXCR4/CXCL12 axis. We found that both hematopoietic stem cells and BM stromal cells functionally express the lactate receptor HCA-1. Stimulation with PGE2 elevated lactate production by BM stromal cells and stimulation with a HCA-1 receptor agonist, or with lactate, both elevated membrane bound expression of CXCL12 on BM stromal cells. Moreover, since cAMP is elevated by PGE2 signaling whereas lactate signaling was shown to inhibit cAMP, we tested the role of cAMP in CXCL12 expression and secretion by BM stromal cells. We found that in vitro the cAMP enhancer forskolin increased CXCR4 expression by HSPC and in vivo forskolin administration reduced membrane bound CXCL12 levels and elevated CXCL12 secretion as expected. Conversely, in vivo forskolin co-administered with lactate, elevated membrane bound CXCL12 levels and reduced CXCL12 secretion, indicating that lactate limits cAMP elevation and promotes surface CXCL12 expression by BM stromal cells. In accordance,inhibition of cAMP under PGE2 stimulation both in vitro and in vivo, augmented membrane bound CXCL12 expression and inhibited CXCR4 upregulation, mimicking the effects of lactate. We found that PGE2 administration in vivo resulted in reduced CXCR4 expression on primitive BM HSPCs however in vitro PGE2 elevated CXCR4 expression on enriched HSPC. Our results suggest that PGE2 signaling in vivo induces secretion of the metabolite lactate by BM stromal cells, increasing membrane bound CXCL12 expression and reducing expression of CXCR4 on HSPC via cAMP inhibition.Importantly, repeated in vivo administration of PGE2, lactate or its receptor HCA-1 agonist (once daily for 2 days), all reduced CXCR4 expression and steady state egress of HSPC to the bloodcirculation. Thus, PGE2 via downstream lactate secretion acts as a BM stem cell retaining factor. In accordance, we found that in vivo inhibition of PGE2 production by repeated (once daily for five days) injections of COX-2 inhibitors, such as Meloxicam led to HSPC mobilization. This mobilization was abrogated by co-administration of lactate, suggesting that in vivo inhibition of meloxicam induced CXCL12 secretion and release by lactate prevents HSPC mobilization. We found that in vivo COX-2 inhibition reduced membrane expression of CXCL12 by BM stromal cells and elevated surface CXCR4 expression by BM HSPC in a ROS dependent manner. Moreover, neutralization of CXCR4 or CXCL12 by specific antibodies, or ROS by its scavenger NAC, all blocked meloxicam induced stem and progenitor cell mobilization. These results reveal that COX-2 inhibition increased BM CXCL12 secretion and its release to the blood, upregulated CXCR4 leading to HSPC mobilization in a ROS and CXCL12 dependent manner.In conclusion, our results reveal that PGE2 enhances both cAMP elevation and lactate secretion by BM stromal cells in the vicinity of hematopoietic stem cells. Lactate acts in an autocrine manner modulating surface CXCL12 expression by BM niche cells and reduced CXCR4 expression by hematopoietic stem cells via inhibition of cAMP, promoting retention and preservation of hematopoietic stem cells in their BM niches. DisclosuresNo relevant conflicts of interest to declare.

Macrophages and Bone Marrow Stem Cell Niches: The Roles of PGE2 and Coagulation

Blood-forming hematopoietic stem cells (HSC) functionally express the chemokine receptor CXCR4, which is required to control their directional motility, cell cycle, adhesion, and bone marrow (BM) repopulation. HSC are mostly BM-retained in a quiescent, non-motile mode via CXCR4-induced adhesion interactions with BM niche cells, which functionally express surface CXCL12. Surface CXCL12-mediated adhesion interactions with BM stromal cells protect quiescent CXCR4+ stem cells from DNA damaging agents while preserving their developmental potential. On the other hand, CXCL12 secretion by BM stromal cells and its release to the blood increase CXCR4 expression and signaling in hematopoietic stem and progenitor cells (HSPC), inducing their egress and clinical mobilization. Finally, CXCR4+ HSC also follow CXCL12 gradients to the BM when they home back from the blood. Intriguingly, dynamic CXCR4/CXCL12 signaling cascades control not only HSC retention in the BM, but also their homing to the BM and their release and mobilization to the blood. In addition to their central role in host defense, myeloid cells participate in organ homeostasis, including HSC localization. Monocyte-derived bone resorbing osteoclasts cleave and release endosteal membrane-bound CXCL12, stem cell factor, and osteopontin, factors needed for HSC adhesion and BM retention, leading to CXCL12/CXCR4-mediated HSPC mobilization. Recently, we identified a rare population of BM αSMA+ macrophages that highly express, which protects HSC from inflammatory insults via COX-2-mediated PGE2 secretion and reactive oxygen species inhibition. In vitro PGE2 upregulates CXCR4 on enriched human cord blood CD34+ and murine HSC via cAMP activation, leading to enhanced CXCL12-induced migration, homing and BM repopulation. However, preliminary results reveal that in vivo PGE2 treatment reduced CXCR4 expression on BM HSC by increasing surface CXCL12 expression by stromal cells. Surface CXCL12 upregulation is due to PGE2-mediated secretion and autocrine signaling of lactate, which is followed by cAMP inhibition in BM stromal cells. Indeed, antagonizing COX-2 or activating cAMP induced HSPC mobilization via BM CXCL12 secretion and increased CXCR4 expression and signaling in HSPC. Coagulation cascades also navigate HSC localization. We revealed that anticoagulant microenvironments in the BM mediate HSC adhesion and retention via inhibition of nitric oxide (NO) production and HSC migration. Physiologic stress induced extensive production of the pro-coagulant factor thrombin, which activates NO generation, CXCL12 secretion and enhanced CXCR4+ stem cell motility and mobilization. We found multinucleated pre-osteoclast cell clusters in femoral metaphysis expressing Tissue Factor (TF) a potent initiator of coagulation leading to thrombin generation. Osteoclast maturation and stress signals also activate TF induced pro-coagulation cascades, mediating HSPC egress and recruitment to the circulation. Daily light and darkness cues regulate many physiological processes, including osteoclast/osteoblast bone remodeling, BM CXCL12 production and secretion and CXCR4+ HSC egress. In addition to the previously identified morning peak of HSC egress to the blood, we identified two peaks of BM HSC proliferation: a morning peak in conjunction with stem cell egress and an evening peak of HSC expansion without egress. Higher BM levels of the HSC-protecting aSMA+ monocyte/macrophages documented in the evening and their associate “low-CXCR4/high CXCL12” BM microenvironment provide the mechanism for these fluctuating patterns. Finally, we found higher PGD2 levels and reduced PGE2 levels in monocyte/macrophages in the morning, whereas at night we documented the opposite patterns. PGD2 induced CXCL12 secretion and HSC egress, while PGE2 induced their retention. Our studies attribute central roles for BM myeloid cells in HSC regulation. Disclosures No relevant conflicts of interest to declare.

A Novel Humanized Bone Marrow Niche Xenotransplantation Model Allows Superior Engraftment of Human Normal and Malignant Hematopoietic Cells and Reveals Myelofibrosis-Initiating Cells in the HSC Compartment

Xenotransplantation into immunocompromised mice is a powerful tool for studying human normal and malignant hematopoiesis. However, xenotransplant models do not necessarily recapitulate human physiology since environmental factors provided by bone marrow (BM) niche cells may differ between mice and humans. Although existing mouse models allow for propagation of hematopoietic stem and progenitor cells (HSPCs) and aggressive acute leukemias, engraftment of more committed normal progenitor cells and many other hematopoietic malignancies is still lacking. Here, we report the development of a novel mouse model bearing a subcutaneously accessible human bone ossicle formed by in situ differentiation of BM-derived mesenchymal stromal cells (MSCs). These human ossicles contain a functional humanized BM niche that facilitates robust engraftment of normal human HSPC and exhibits superior engraftment and expansion of primary AML. Interestingly, this model allows robust engraftment of PML-RARA positive APL and JAK2 V617F+ PMF, both of which fail to engraft in conventional xenotransplantation models.Subcutaneous injection of human BM-derived MSCs resulted in the formation of human ossicles through endochondral ossification resulting in a bone marrow cavity harboring normal mouse hematopoietic elements. After conditioning irradiation, direct intraossicle injection of cord blood CD34+ HSPC (n=5) resulted in robust multi-lineage human engraftment including B cells, T cells, NK cells, mature neutrophils, eosinophils, monocytes, as well as red blood cell precursors and platelets, as assessed by flow cytometric analysis of aspirates taken directly from the ossicles. The self-renewal potential of engrafted HSPCs was demonstrated by re-establishing multi-lineage hematopoiesis in secondary recipients. Unlike conventional NSG mice, this xenograft model allowed engraftment of phenotypically defined L-MPP (n=7) and GMP (n=7). Engraftment analysis 2 weeks post transplantation showed human engraftment with a majority of CD33+ myeloid cells (LMPP: 77.3±14.4%; GMP: 97.3±1.8%) and intermediate (LMPP: 20.8±14.4%) to low (GMP: 1.8±1.8%) numbers of B lymphoid cells.In the investigation of AML, direct intraossicle transplantation of blasts (n=5) resulted in faster (as early as 6 weeks) and significantly higher leukemic engraftment in humanized niches compared to mouse BM. Furthermore, engraftment was also significantly higher in humanized niches after intravenous transplantation and demonstrated a paratrabecular engraftment pattern, suggesting preferential homing and expansion of AML cells in a human BM microenvironment. Limiting dilution analysis revealed a 10-250 fold higher leukemia-initiating cell frequency using direct intraossicle transplantation compared to intrafemoral injection into mouse BM. Intraossicle transplantation also allowed for engraftment and propagation of PML-RARA-positive APL blasts (n=3) resulting in almost 100% leukemic chimerism and granulocytic sarcoma formation 10-15 weeks post-transplantation. Finally, transplantation of PMF patient-derived CD34+ cells (n=5) into the humanized niche led to robust engraftment with up to 50% human chimerism, and further subpopulation studies demonstrated that PMF-engraftment was exclusively initiated from the HSC (CD34+/CD38-/CD90+) population. Engrafted cells showed marked myeloid skewing with JAK2 V617F-positive cells, and detection of only minimal JAK2 wild type lymphoid cells.In summary, we report the development of a novel xenotransplantation model using human bone ossicles formed by in situ differentiation of BM-derived MSCs that substantially improves the engraftment of normal and malignant hematopoietic populations, leading to the formal demonstration that PMF-initiating cells reside in the immunophenotypic HSC compartment. DisclosuresNo relevant conflicts of interest to declare.

Thrombopoietin Activates STAT2 Inducing Type I Interferon Effects and Gene Expression: Implications for in Vivo Tpo Treatment and for Myeloproliferative Neoplasms

Thrombopoietin (Tpo) acts via its cell surface receptor (MPL/TpoR) to promote megakaryocyte progenitor survival, proliferation and differentiation via activation of JAK2, STAT5, STAT3, STAT1, ERK1,2-MAPK-kinase and PI-3′-kinase/Akt. In addition, Tpo signaling in the bone marrow niche is required for maintaining hematopoietic stem cells (HSCs). Recently we showed that in cells expressing high JAK2, such as megakaryocytes or engineered cell lines, Tpo induced an anti-proliferative and pro-senescence physiologic effect. Down-modulation of TpoR in myeloproliferative neoplasms (MPNs) progenitors might confer resistance to such anti-proliferative effects in megakaryocytes, thus contributing to their uncontrolled proliferation.Here we report that Tpo activates tyrosine phosphorylation and transcriptional activation of STAT2, and of a complex containing STAT2, STAT1 and IRF-9. This is specific for type I interferon (IFN) and is associated with anti-proliferative and antiviral effects. We detected Tpo induced STAT2 activation in platelets of thrombocytopenia patients treated in vivo with Tpo or Tpo agonists as well as in human CD34+-derived megakaryocytes. In cell lines reconstituted with MPL/TpoR and JAK2, Tpo behaved like an IFN by protecting cells from subsequent viral infection and inducing IFN-stimulated gene expression. We asked whether Tpo-induced STAT2 activation depended on type I IFN receptors, on TYK2 or on STAT1. Using primary cells from mice where these genes have been knocked-out we show that Tpo indeed activates STAT2 via TpoR itself, and does neither require IFNAR1, IFNAR2 nor TYK2, demonstrating that this effect is direct.By using cell lines expressing different levels of JAK2 we show that Tpo exerts a bimodal effect. At low JAK2 levels Tpo induces mainly STAT5, STAT3 and STAT1 activation, while at high JAK2 levels (comparable to those in UT7 cells or primary megakaryocytes) Tpo induces STAT2/STAT1/IRF9 activation, in addition to STAT5/STAT3/STAT1 activation. Interestingly, the STAT2 signaling is dominant and induces an anti-proliferative effect, which can be inhibited by shRNA targeting STAT2, or by mutation of distal receptor tyrosines. Moreover, using STAT2-/- mice we show that STAT2 activation by Tpo is relevant for the steady-state levels of platelets, with knock-out mice having significantly higher platelet numbers. Recovery studies after 5-FU injection and examination of HSCs and early progenitors showed that STAT2 acts as a negative regulator of the stimulatory effects of Tpo on myelopoiesis.We recently identified inactive and active dimeric conformations of TpoR. Here we tested which dimeric receptor conformation activates STAT2 using dimeric fusion proteins where a dimeric coiled coil (Put3) was fused to the transmembrane segment. While cc-TpoR-I was found to recapitulate the classical stimulatory effect of Tpo on STAT5, STAT3 and STAT1 and platelet formation in vivo, here we show that STAT2 activation is induced by another dimeric conformation, represented by cc-TpoR-IV, which is the strongest JAK2 activator. cc-TpoR-IV also induces strong cell-to-cell adhesion possibly reminiscent of the intercellular HSC interactions to the bone marrow niche cells. Increasing JAK2 protein levels leads to stabilization of a Tpo-activated TpoR dimer that assumes a conformation that corresponds to cc-TpoR-IV. Thus, JAK2 levels act on fine-tuning the dimeric conformation of the TpoR dimer, switching it from a classical proliferative cytokine to an IFN-like cytokine, which is anti-proliferative.The new Tpo-induced signaling pathway we describe is of very high relevance for MPN pathogenesis and treatment with IFN-I. Our data indicate that HSCs and megakaryocytes where TpoR-JAK2 V617F signal would be primed for IFN action, and might explain why IFN-I targets for inhibition the mutated HSCs, eventually decreasing the allele burden. Last but not least, autoimmune and viral-induced thrombocytopenia patients are treated with Tpo mimetics, and we could detect STAT2 activation in certain patients after in vivo Tpo treatment. Differences in the ratios of classical versus STAT2 activation might be at the basis of differences in response to Tpo of such patients. DisclosuresConstantinescu:Shire: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Personal Genetics: Scientific Adv Board, Scientific Adv Board Other, Speakers Bureau; Dafra Pharma: Consultancy, Scientific Advisory Board Other.

Abstract 4865: Differential survival and self-renewal of LSC and HSC in aged and leukemic niches

Abstract Background: Acute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Evidence indicates that in the development of AML deregulation of stem cells activity is as important as deregulation of the microenvironment. We have recently reported that inflammatory changes in the aging bone marrow (BM) niche via aberrant RNA editing and splicing may predispose patients to leukemic transformation. However, little is known about the functional effects of niche driven aberrant RNA processing in the AML pathogenesis, as well as of role of aberrant RNA editing and splicing in human aged stem cell fate, dormancy and regeneration in the context of microenvironment. Here we investigate: 1/ the LSC impacts to the BM niche and 2/ changes in the activity of BM niche cells contribute to AML pathogenesis. Methods: BM CD34- cell from normal donors and AML patients were used for the development of the primary human stromal monolayers : young (y-BM, n=3); old (a-BM, n=3) and AML (n=3). Human CD34+ cells were selected from AML primary samples (n=6). As a control, CD34+ cells from cord blood (CB, n=5), y-BM (n=3) or a-BM (n=3) were used for the co-culture experiments and then plated in survival and self-renewal assays. Results: AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood HSC (n=5) lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. Aged BM also impaired survival and self-renewal of cord blood HSC (n=3) in stromal co-culture models. In similar experiments HSC from a-BM (n=3) demonstrated significantly higher survival and self-renewal capacity then co-cultured with y-BM stroma (n=3) compared to a-BM stroma (n=3). Conditioned media (CM) both from-, or co-culture with-, old or AML stroma impaired HSC survival and self-renewal. Notably, co-culture conditions resulted in a greater reduction in survival and self- renewal capacity, suggesting that cell-cell contact or unstable secreted factors exacerbate the effects. Cord blood HSC were also co-cultured with AML-derived stroma or CM , and this severely impaired their survival and self-renewal. Moreover, pre-treatment of the HS-5 cells with CM from a-BM or AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood HSC (n=3) survival and self-renewal. Conclusions: Together these data indicate that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic and aged stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of AML. Citation Format: Larisa Balaian, Leslie Crews, Anna Kulidjian, Edward Ball, Catriona Jamieson. Differential survival and self-renewal of LSC and HSC in aged and leukemic niches. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4865. doi:10.1158/1538-7445.AM2014-4865

Chronic variable stress activates hematopoietic stem cells.

Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis1,2. While incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known crosstalk between the brain and immune system includes the hypothalamic–pituitary–adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic–adrenal–medullary axis, which controls stress–induced catecholamine release in support of the fight–or–flight reflex3,4. It remains unknown however if chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive progenitors, giving rise to higher levels of disease–promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Sympathetic nerve fibers release surplus noradrenaline, which uses the β3 adrenergic receptor to signal bone marrow niche cells to decrease CXCL12 levels. Consequently, elevated hematopoietic stem cell proliferation increases output of neutrophils and inflammatory monocytes. When atherosclerosis–prone ApoE−/− mice encounter chronic stress, accelerated hematopoiesis promotes plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.

Pathogenesis of cell death in lupus bone marrow (BA12P.112)

Abstract Pristane-treated mice develop lupus mediated by TLR7-driven production of IFNα (autoantibodies, nephritis) and TNFα (anemia). Anemia is IFN-independent, is caused by TNFα produced by bone marrow (BM) neutrophils, and is associated with a striking accumulation of dead BM niche cells and erythroid precursors in the BM. TNFα and dead cells also were found in BM from anemic SLE patients. The dead cells may drive TNFα/IFNα production by releasing TLR7 ligands. Fas-deficient (B6/lpr) mice were completely refractory to induction of pristane-lupus implicating cell death in its pathogenesis. Interestingly, accumulation of dead cells was abolished in BM from B6 TNFα-/- mice, suggesting that they were killed by TNFα. Signaling through both Fas and the TNF receptor I can activate caspase-3 and processing of the BCL-2 family member BID, which is involved in a mitochondrial amplification loop for Fas-mediated apoptosis. However, pristane injection in BID-/- mice induced TNFα and IFNα production as well as autoantibody production comparable to wild type B6 mice. We conclude that the requirement for Fas signaling in pristane-lupus involves the death of “Type I” cells, which undergo Fas-mediated apoptosis without a requirement for truncated BID and its crosstalk with the mitochondrial cell death pathway.

Expression Of Hedgehog Pathway Mediator Gli2 Represents a Clinically Negative Prognostic Marker In Acute Myeloid Leukemia and Its Inhibitor GANT61 Exerts Anti-Leukemic Effects In Vitro

▪ BackgroundLeukemic stem cells depend on signals provided by bone marrow (BM) niche cells. Due to its function in stem cell biology, we investigated expression and prognostic impact of the Hedgehog pathway in acute myeloid leukemia (AML). Methods and resultsPre-treatment samples from 104 patients with newly diagnosed AML (AMLSG 07-04 trial) were analyzed by quantitative PCR. Expression of receptors Smoothened and Patched and downstream transcription factors Gli1, Gli2 and Gli3 was found in 69%, 41%, 73%, 20% and 26% of cases, respectively. However, no expression of Hedgehog ligands was observed. Gli2 expression had a significant influence on event-free, relapse-free and overall survival (p=0.037, p=0.026 and p=0.013, respectively) and was furthermore correlated to FLT3 mutational status (p<0.001).To verify our findings in a second, independent patient cohort, microarray-based gene expression data of patients published by Valk et.al.(N Engl J Med. 2004 Apr 15;350(16):1617-28) was used. Clinical data of 291 AML patients from this cohort were available and the negative prognostic impact of Gli2 expression on overall and event-free survival could be confirmed (p=0.003 for overall and p=0.007 for event-free survival, respectively; no data for relapse-free survival available). Additionally, the correlation of Gli2 expression to FLT3 mutational status was also observed in this patient cohort (p=0.003).Although Hedgehog ligands were not expressed in AML bone marrow aspirates, Desert Hedgehog (DHH) plasma levels were significantly increased in AML patients compared to healthy subjects (p=0.002). Provision of Desert Hedgehog could be ascribed to BM niche cells including endothelial cells and osteoblasts, since DHH expression was found in outgrowth endothelial cells (OECs) and osteoblasts in vitro and on immunohistochemistry of leukemic bone marrow samples in patients.Next, we investigated the effect of Gli inhibition in vitro. AML cell lines KG-1, OCI-AML5 and UKE-1 as well as freshly isolated primary AML cells were treated with the specific Gli inhibitor GANT61. GANT61 dose-dependently induced apoptosis in all three cell lines investigated. Furthermore, GANT61 treatment resulted in significantly increased apoptosis in primary AML cells upon treatment with 10 µM, 30 µM and 90 µM GANT61 (n=10 pts). Additionally, a pronounced growth inhibitory effect on primary AML cells was observed with a significant reduction in cell count to 82 ± 13%, to 54 ± 11% and to 47 ± 13% upon treatment with 10 µM, 30 µM and 90 µM of GANT61, respectively (n=9 pts). Incubation of KG-1, UKE-1 and OCI-AML5 cells for 7 days with 30 µM GANT61 resulted in reductions in cell numbers compared to the control to 14 ± 2%, to 44 ± 6% and to 31 ± 0%, respectively. Moreover, GANT61 mediated a strong and significant inhibitory effect on the colony formation capacity of AML cell lines and primary AML cells. The average number of colonies was reduced to 44 ± 16% and 4 ± 4% upon treatment with 10 µM and 30 µM GANT61, respectively, while no colony formation was observed with a concentration of 90 µM GANT61 in primary AML cells (n=4 pts). ConclusionIn conclusion, Gli2 expression is a negative prognostic factor in AML that mirrors activated Hedgehog signaling induced by paracrine stimulation through BM niche cells. Inhibition of Hedgehog signaling by blocking Gli activity might represent a promising therapeutic approach for AML treatment with special regard to patients carrying a mutated FLT3. Disclosures:Fiedler:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Acute Myeloid Leukemia Alters The Mesenchymal Stem Cell Potential Of The HSC Niche: Evidence For Modulation By β-Adrenergic Signals

Hematopoietic stem cells (HSC) reside in specific bone marrow niches comprised of perisinusoidal Nestin-GFP+ (Nes-GFP+) and leptin receptor (LepR)+ stromal cells which highly overlap with each other, as well as osteolineage and endothelial cells. These different cellular constituents regulate HSC maintenance and retention in the bone marrow (BM). Recently, our laboratory further identified rare periarteriolar Nes-GFPbright cells which have been identified to be pericytes and functionally crucial for HSC quiescence and maintenance, from the more abundant reticular Nes-GFPdim population (Kunisaki et al, unpublished data). The function of Nes-GFP+ niche cells is tightly regulated by the sympathetic nervous system (SNS) via β2- and β3-adrenergic receptors. In acute myeloid leukemia (AML), BM infiltration by leukemic blasts is known to lead to hematopoietic failure. Although cytopenias are thought to result from the BM occupation by AML, the mechanisms remain unclear. In this study, we investigated the impact of AML on BM niche constituents, and evaluated the influence of SNS signals in AML progression using a syngeneic murine MLL-AF9 transplantation model. We observed a ∼4.4-fold expansion of Nes-GFP+ stromal cells (p<0.05) in leukemic BM, which positively correlated with the degree of leukemic marrow infiltration (r=0.49, p<0.01). However, while we observed a massive expansion of reticular Nes-GFPdim cells, we detected a ∼2.2-fold reduction in pericytic Nes-GFPbright cells (p<0.05). Phenotypically, Nes-GFPdim cells showed similar expression of LepR, PDGFRα and CD51 to healthy controls, markers enriching for BM mesenchymal stem cells. Interestingly, Nes-GFP+ cells in leukemic BM had a ∼1.5-fold higher colony-forming units-fibroblast (CFU-F) and ∼2.6-fold higher CFU-Osteoblast capacity. The gene expression profile of Nes-GFP+ cells using Affymetrix Gene 1.0 ST microarrays revealed a profound dysregulation when the BM was infiltrated with AML, including most notably activated TGFβ signaling pathways and upregulation in the expression of inflammatory response genes. Nes-GFP+ niche cells were previously shown to be highly enriched for HSC-maintenance gene expression. In leukemic BM, Nes-GFP+ cells showed a significant reduction in the expression of Cxcl12, Scf and Vcam1 (p<0.01), while no significant differences were observed for Opn. In line with these data, we observed a significant reduction of HSC-enriched Lin−Sca1+c-Kit+Flt3− and progenitor cells in the BM (p<0.05, except for granulocyte-macrophage progenitors) and their mobilization into peripheral blood and spleen (p<0.01). To further analyze if Nes-GFP+ niche cells in leukemic BM still depended on adrenergic signals, we performed in vivo chemical sympathetic denervation with 6-hydroxydopamine. Sympathectomized leukemic mice showed a ∼1.5-fold increase in Nes-GFP+ cells, a ∼1.8-fold increase of IL-7R-Lin-c-KithiCD34+FcγRII/IIIhi leukemic stem cells (LSC, p<0.05) which was associated with significantly shorter survival (p=0.017). Denervated mice did not show differences in homing, proliferation or apoptosis of leukemic cells. We confirmed our findings in a xenograft model, in which sympathectomized mice were transplanted with primary human AML samples and showed a higher leukemic BM infiltration (p<0.05). Further analyses using specific adrenergic β2- and β3-receptor antagonists (ICI118,551 hydrochloride and SR59230A) suggested that adrenergic signals were predominantly mediated by the β2-adrenergic receptor. Preliminary data indicate that activation of the β2-adrenergic receptor leads to decreased leukemia burden. In summary, our data identify that leukemic cells severely transform the BM niche by gradually expanding and directing mesenchymal stem cell differentiation into the osteoblastic lineage and simultaneously decreasing numbers of pericytic niche cells. Ultimately, this creates a microenvironment with impaired HSC maintenance capacity and favors myeloid expansion. This transformed niche, however, remains regulated by signals from the SNS which in turn modulates leukemic BM infiltration. These results thus uncover a novel approach for niche-targeted therapeutic strategies in AML. Disclosures:Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Metabolic Shift In Hypoxia-Adapted CML Cells and Mechanisms Of Acquired Dasatinib Resistance Uncovered By Proteomic iTRAQ Profiling

Hypoxia is one of the essential components of the leukemia bone marrow (BM) microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito, PlosOne 2011). Tyrosine kinase inhibitors (TKIs) do not eradicate the total mass of chronic myeloid leukemia (CML) cells including primitive and quiescent cells. Persistence of CML cells in the hypoxic BM niche after cessation of TKI therapy may result in disease relapse. We have reported that hypoxia adapted CML cells acquire TKI resistance associated with higher glyoxalase-1 (Glo-1) enzyme activity which detoxifies methylglyoxal, a cytotoxic by-product of glycolysis (Takeuchi, Cell Death Differ 2010).In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to investigate additional molecular mechanisms of CML adaptation to hypoxia and acquired resistance to dasatinib under hypoxic conditions. The specific pathway alterations were identified by KEGG(Kyoto University) and MetaCore (GeneGo). We utilized two hypoxia-adapted (HA) subclones of CML cell lines, KCL22-HA and KBM5-HA cells, which were selected over a month under 1.0 % oxygen culture conditions. The growth rate of both HA-CML cell lines was slower than that of the corresponding parental cells (ratio of incremental increase in cell numbers, 0.39 for KCL22-HA/KCL22, 0.54 for KBM5-HA/KBM5 at 48hrs). Although parental KCL22 cells were sensitive to dasatinib, KCL22-HA cultured under hypoxia acquired resistance to dasatinib (IC50: KCL22 0.1 nM, KCL22-HA >20nM, at 48hrs by MTT). In contrast, dasatinib induced more prominent cell growth inhibition in KBM5-HA cells cultured under hypoxia compared to KBM5 parental cells (IC50: KBM5 1.3 nM, KBM-5/HA 0.3 nM). Dasatinib effectively downregulated the phosphorylation levels of Stat5 and ERK in parental KCL22 and KBM5 cells. Notably, the baseline levels of p-Stat5 and p-ERK were markedly diminished in both KCL22-HA and KBM5-HA cells. We next performed iTRAQ proteomic analysis and detected more than 1,300 proteins in each cell type. Comparison of the basal proteome of KCL22 vs KCL22-HA and KBM5 vs KBM5-HA cells showed differential expression of 54 proteins in KCL22 isogenic cells (37 upregulated, 17 downregulated) and of 159 proteins in KBM5 cells (56 upregulated, 103 downregulated). These alterations included consistent activation of glycolysis and gluconeogenesis pathway (p<0.0001) along with increased ATP synthase (p=0.02) and hypoxia induced HIF1 activation (p<0.0001) in both cells adapted to grow under hypoxia. In dasatinib-resistant KCL-HA cells, these changes were partially reversed by dasatinib which affected expression of 19 proteins (5 up-regulated / 14 down-regulated) with significant decrease in oxidative phosphorylation (p=0.01), ATP synthase (p=0.02), spliceosome (p=0.02) and Cytochrome C (p=0.04). On the contrary, in dasatinib-sensitive KBM5-HA cells dasatinib upregulated 296 proteins including 39 apoptogenic proteins such as Cytochrome C (p<0.001) and apoptosis related mitochondrial chaperones HSP10 and HSP60 (p<0.001), along with further upregulation of the enzymes involved in the glycolytic energy production (p<0.001) and stimulation of nuclear mRNA splicing via spliceosome (p<0.001), known to play a crucial role in the control of gene expression of the apoptosis-regulating genes. We observed the consistent upregulation of glycolysis and increases of ribosome and spliceosome after dasatinib treatment in dasatinib-sensitive parental KCL22 and KBM5 cells. Interestingly, dasatinib-resistant KBM5-STI cells harboring T315 Bcr-Abl mutation exhibited significantly lower levels of baseline gluconeogenesis (p<0.0001) and translation regulation of translation initiation pathways (p<0.0001) compared to the parental KBM5 cells.In summary, these findings demonstrate the dramatic metabolic changes in hypoxia adjusted CML cells and indicate that hypoxia adapted / dasatinib resistant CML cells repress their stimulated glycolic metabolic state and oxidative phosphorylation along with spliceosomal mRNA splicing after dasatinib exposure, which allow them to enter quiescent stage and escape from apoptosis. Altogether these data indicate novel mechanisms of acquired bone marrow microenvironment-mediated resistance to TKI in CML. Disclosures:Tabe:Bristol-Myers Squibb: Research Funding.