Leukemic Bone Marrow Cells Research Articles

Leukemic Stem Cells Co-Opt Normal Bone Marrow Niches As a Source of Energy and Antioxidant Defence

Modulation of oxidative stress as well as metabolic adjustments are key aspects in cancer stem cell-derived diseases, such as acute myelogenous leukemia (AML). Metabolic reprogramming in leukemia seems to be influenced by the crosstalk of malignant cells with their surrounding microenvironment. Reactive oxygen species (ROS), mainly generated by the mitochondria, are relatively high in malignant cells, without reaching a cytotoxic level that would compromise their survival. In addition, chemotherapy efficacy lies on increased ROS-mediated cellular damage. Cumulative evidence shows that AML often exhibits poor response to chemotherapy partly due to the support of leukemic stem cells (LSCs) by the bone marrow (BM) microenvironment, which includes BM mesenchymal stem cells (BMSCs). However, how exactly this happens, i.e. the specific mechanism(s) and cell populations implicated, remains incompletely understood.Hematopoietic stem cell (HSC)-niche forming BMSCs can be identified by the expression of the intermediate filament protein nestin and can be isolated and propagated as non-adherent ‘mesenspheres’, which can self-renew and support HSCs ex vivo.Here we have studied the contribution of nestin+ BMSCs to MLL-AF9-driven AML development and resistance.Immunohistochemistry for nestin on BM samples from AML patients (N=35) showed that the number of nestin+ niches per square millimeter was 2.4-fold higher as compared to control samples (N=12). To study the role of BMSCs in AML, we used a doxycycline-inducible iMLL-AF9 mouse strain that closely mimics the human disease (Stavropoulou V et al., Cancer Cell 2016;30:43-58). Development of AML in iMLL-AF9 mice caused a significant reduction of stromal cells in the BM but it did not reduce the number of BM nestin+ cells. To study the possible contribution of nestin+ cells to leukemia development, we partially depleted nestin+ cells by inducing diphtheria toxin expression in these cells using Nestin-creERT2 ; iDTA mice previously transplanted in a competitive fashion with pre-leukemic and normal BM cells. Nestin+ cell depletion selectively diminished leukemia burden in BM, spleen and blood, and reduced the number of primitive leukemic cells, without affecting normal residual hematopoiesis. Combined nestin+ cell depletion and standard chemotherapy further exaggerated the elimination of BM leukemic cells, suggesting that nestin+ cells contribute to chemotherapy resistance.To investigate the crosstalk of BMSCs and leukemic blasts, we developed a novel in vitro co-culture system using BM mesenspheres and iMLL-AF9+ leukemic blasts. Co-cultures with nestin+ mesenspheres promoted leukemic blast survival under metabolic stress (FBS deprivation) and chemotherapy (cytarabine, Ara-C). Increased survival correlated with a strong protection against excessive ROS levels and lipid peroxidation. Particularly, reduced glutathione (GSH), a key antioxidant element, increased in leukemic blasts co-cultured with nestin+ mesenspheres.Mitochondrial transfer from BMSCs has also recently been proposed as a metabolic-related mechanism contributing to chemoresistance in AML (Moschoi R et al. Blood 2016;128:253-64). Using the Seahorse Extracellular Flux Analyzer, we actually revealed that leukemic blast bioenergetics increased in co-culture with mesenspheres. Surprisingly, we found that chemotherapy-induced transfer of mitochondria from BMSCs to AML cells correlated with decreased ROS levels in the leukemic blasts, pointing out mitochondria transfer as an important ROS detoxifying mechanism to allow chemoresistance. We demonstrated that cell-to-cell contact could be required for Ara-C-mediated protective effects of mesenspheres on leukemic blasts and their mitochondria transfer. Accordingly, either the inhibition of GSH generation or mitochondrial transfer (by avoiding cell contact) may reduce BMSCs survival support after chemotherapy.Altogether, these results suggest that simultaneous co-opting of energy sources and anti-oxidant mechanisms of BMSCs contribute to chemoresistance in AML. DisclosuresNo relevant conflicts of interest to declare.

Combination of PP242 and dasatinib suppresses the progression of acute myeloid leukemia in a mouse model

Objective: To establish the acute myeloid leukemia (AML) mouse model, and to preliminarily investigate the efficiency of dasatinib, a tryosine kinase inhibitor, and PP242, an inhibitor of PI3K/Akt/mTOR signaling pathway in the development of AML. Methods: The lineage(-) (Lin(-)) cells of C57BL/6J were transduced with retrovirus carrying MSCV-MLL-AF9-IRES-GFP fusion gene. The transduced cells were transplanted into lethally irradiated recipient mice to induce AML, and then the AML mouse model were established. The leukemia mice were treated with vehicle, dasatinib, PP242, dasatinib+ PP242, separately. The survival of the recipient mice was observed and the percentage of leukemia cells in peripheral blood (PB) and bone marrow (BM) was examined every four days. The apoptosis rates and cell cycle status of leukemia cells were also examined by FLOW. The leukemia cells in different group were sorted, the mRNA of these leukemia were extracted and reverse transcripted for related gene expression by qRT-PCR. The molecular mechanism of supression of leukemia cells growth was studied via RNA-Seq experiments. Results: Compared with control group, either PP242 or dasatinib could prolong the survival rate of recipient mice, however, the combination treatment AML mice with PP242 and dasatinib prolonged the life span of AML mice more significantly. The combination of PP242 and dasatinib could decrease the percentage of leukemia cells in PB and BM more significantly, arresting more leukemia cells in G0 phase, inducing more apoptosis of leukemia cells. Conclusion: Combination of tryosine kinase inhibitor-dasatinib and PI3K/Akt/mTOR signaling pathway inhibitor- PP242 could delay the progression of AML by inducing more leukemia to apoptosis and arrest more leukemia cells in the cell cycle G0 phase, it may be provied a new method for clinical therapy.

UNC2025, a MERTK Small-Molecule Inhibitor, Is Therapeutically Effective Alone and in Combination with Methotrexate in Leukemia Models.

Purpose: MERTK tyrosine kinase is ectopically expressed in 30% to 50% of acute lymphoblastic leukemias (ALL) and more than 80% of acute myeloid leukemias (AML) and is a potential therapeutic target. Here, we evaluated the utility of UNC2025, a MERTK tyrosine kinase inhibitor, for treatment of acute leukemia.Experimental Design: Preclinical in vitro and in vivo assays using cell lines and primary leukemia patient samples were used to evaluate antileukemic effects of UNC2025.Results: UNC2025 potently inhibited prosurvival signaling, induced apoptosis, and reduced proliferation and colony formation in MERTK-expressing ALL and AML cell lines and patient samples. Approximately 30% of primary leukemia patient samples (78 of 261 total) were sensitive to UNC2025. Sensitive samples were most prevalent in the AML, T-ALL, and minimally differentiated (M0) AML subsets. UNC2025 inhibited MERTK in bone marrow leukemia cells and had significant therapeutic effects in xenograft models, with dose-dependent decreases in tumor burden and consistent two-fold increases in median survival, irrespective of starting disease burden. In a patient-derived AML xenograft model, treatment with UNC2025 induced disease regression. In addition, UNC2025 increased sensitivity to methotrexate in vivo, suggesting that addition of MERTK-targeted therapy to current cytotoxic regimens may be particularly effective and/or allow for chemotherapy dose reduction.Conclusions: The broad-spectrum activity mediated by UNC2025 in leukemia patient samples and xenograft models, alone or in combination with cytotoxic chemotherapy, supports continued development of MERTK inhibitors for treatment of leukemia. Clin Cancer Res; 23(6); 1481-92. ©2016 AACR.

Erratum to: Bortezomib interferes with adhesion of B cell precursor acute lymphoblastic leukemia cells through SPARC up-regulation in human bone marrow mesenchymal stromal/stem cells.

The poor prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is attributed to leukemia cells that are protected by the bone marrow (BM) microenvironment. In the present study, we explored the pharmacological targeting of mesenchymal stromal/stem cells in BM (BM-MSCs) to eliminate chemoresistant BCP-ALL cells. Human BCP-ALL cells (NALM-6 cells) that adhered to human BM-MSCs (NALM-6/Ad) were highly resistant to multiple anti-cancer drugs, and exhibited pro-survival characteristics, such as an enhanced Akt/Bcl-2 pathway and increased populations in the G0 and G2/S/M cell cycle stages. Bortezomib, a proteasome inhibitor, interfered with adhesion between BM-MSCs and NALM-6 cells and up-regulated the matricellular protein SPARC (secreted protein acidic and rich in cysteine) in BM-MSCs, thereby reducing the NALM-6/Ad population. Inhibition of SPARC expression in BM-MSCs using a small interfering RNA enhanced adhesion of NALM-6 cells. Conversely, recombinant SPARC protein interfered with adhesion of NALM-6 cells. These results suggest that SPARC disrupts adhesion between BM-MSCs and NALM-6 cells. Co-treatment with bortezomib and doxorubicin prolonged the survival of BCP-ALL xenograft mice, with a significant reduction of leukemia cells in BM. Our findings demonstrate that bortezomib contributes to the elimination of BCP-ALL cells through disruption of their adhesion to BM-MSCs, and offer a novel therapeutic strategy for BCP-ALL through targeting of BM-MSCs.

Bortezomib interferes with adhesion of B cell precursor acute lymphoblastic leukemia cells through SPARC up-regulation in human bone marrow mesenchymal stromal/stem cells.

The poor prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is attributed to leukemia cells that are protected by the bone marrow (BM) microenvironment. In the present study, we explored the pharmacological targeting of mesenchymal stromal/stem cells in BM (BM-MSCs) to eliminate chemoresistant BCP-ALL cells. Human BCP-ALL cells (NALM-6 cells) that adhered to human BM-MSCs (NALM-6/Ad) were highly resistant to multiple anti-cancer drugs, and exhibited pro-survival characteristics, such as an enhanced Akt/Bcl-2 pathway and increased populations in the G0 and G2/S/M cell cycle stages. Bortezomib, a proteasome inhibitor, interfered with adhesion between BM-MSCs and NALM-6 cells and up-regulated the matricellular protein SPARC (secreted protein acidic and rich in cysteine) in BM-MSCs, thereby reducing the NALM-6/Ad population. Inhibition of SPARC expression in BM-MSCs using a small interfering RNA enhanced adhesion of NALM-6 cells. Conversely, recombinant SPARC protein interfered with adhesion of NALM-6 cells. These results suggest that SPARC disrupts adhesion between BM-MSCs and NALM-6 cells. Co-treatment with bortezomib and doxorubicin prolonged the survival of BCP-ALL xenograft mice, with a significant reduction of leukemia cells in BM. Our findings demonstrate that bortezomib contributes to the elimination of BCP-ALL cells through disruption of their adhesion to BM-MSCs, and offer a novel therapeutic strategy for BCP-ALL through targeting of BM-MSCs.

Leukemia Stem Cells Microenvironment.

The dynamic interactions between leukemic cells and bone marrow (BM) cells in the leukemia BM microenvironment regulate leukemia stem cell (LSC) properties including localization, self-renewal, differentiation, and proliferation. Recent research of normal and leukemia BM microenvironments has revealed several key components of specific niches that provide a sanctuary where subpopulations of leukemia cells evade chemotherapy-induced death and acquire a drug-resistant phenotype, as well as the molecular pathways critical for microenvironment/leukemia interactions. Although the biology of LSCs shares many similarities with that of normal hematopoietic stem cells (HSCs), LSCs are able to outcompete HSCs and hijack BM niches. Increasing evidence indicates that these niches fuel the growth of leukemia cells and contribute to therapeutic resistance and the metastatic potential of leukemia cells by shielding LSCs. Not only "microenvironment-induced oncogenesis," but also a "malignancy-induced microenvironment" have been proposed. In this chapter, the key components and regulation of BM niches in leukemic BM is described. In addition, metabolic changes in LSCs, which are currently a subject of intense investigation, will also be discussed to understand LSC survival.

Drug Resistance and Dormancy Represent Reversible Characteristics in Patients' ALL Cells Growing in Mice

IntroductionDrug resistant cells represent a major threat for tumor patients as they might induce relapse and severely decrease disease outcome. Relapse represents a major drawback in patients with acute lymphoblastic leukemia (ALL), the single most frequent malignancy in children. Novel treatment options are intensively desired to remove drug resistant cells, which often additionally display dormancy.AimWe aimed at unraveling basic mechanisms determining drug resistance and dormancy, as basis for developing novel treatment strategies to prevent relapse.MethodsUsing cutting edge in vivo technology, we performed genetic engineering in the individualized xenograft mouse model of ALL. Primary patients' ALL cells were amplified in mice to generate patient-derived xenograft (PDX) cells. ALL PDX cells were lentivirally transduced to express transgenes. Recombinant luciferase allowed highly sensitive and reliable follow-up of leukemia growth and treatment. Recombinant surface markers enabled an unbiased approach to reliably and effectively enriching minute numbers of PDX cells from mouse bone marrow. Two independent, complementary innovative preclinical in vivo mouse models were established.In the first model, proliferation sensitive dyes allowed identifying and enriching in vivo long-term dormant PDX ALL cells.In the second model, the clinically highly relevant and challenging situation of MRD was mimicked in mice. PDX ALL cells were grown to advanced leukemia stages of above 30 % human blasts in bone marrow, when systemic chemotherapy with conventional cytotoxic drugs was initiated for prolonged periods of time, similar as applied in ALL patients. Chemotherapy reduced advanced leukemia down to 0,1 % or 10-3 leukemia cells in bone marrow, resembling not only complete morphologic remission, but even molecular remission.This novel preclinical model allows for the first time to characterize patients' dormant and MRD cells in detail including functional in vivo assays.ResultsUsing our innovative preclinical model of dormancy, we identified a novel, distinct, rare subpopulation of PDX ALL cells that displayed long term dormancy in vivo. Long-term dormant cells showed significant resistance against drug treatment in vivo, as therapy nearly exclusively targeted proliferating cells. Dormant cells showed stem cell behavior as they initiated leukemia upon re-transplantation into further recipient mice. Long-term dormant cells thus combined the three challenging characteristics of relapse-inducing cells dormancy, drug resistance and stemness with re-growth upon withdrawal of treatment pressure.Using our second novel preclinical model, we isolated a pure, vivid fraction of rare MRD cells. These cells showed drug resistance in vivo and stemness features.We used single cell RNA sequencing to compare the transcriptomes of dormant and MRD populations and found that they were highly similar. Both populations had further similarities with primary high-risk ALL cells and dormant sub-fractions in patients' leukemia samples.Of high relevance for future treatment strategies, both, dormancy and drug resistance revealed transient characteristics in PDX ALL cells. When PDX long-term dormant ALL cells were distracted from their in vivo environment, they started proliferating similarly as their previously highly proliferative counterparts. When in vivo drug resistant PDX ALL cells were retrieved from murine bone marrow, they showed similar drug sensitivity in vitro as their sensitive counterparts.Summary/ConclusionThus, both in vivo dormancy and drug resistance represent reversible characteristics in ALL cells which might result from the localization of ALL cells in the bone marrow niche. Dissolving ALL cells from their in vivo environment might sensitize them towards treatment. Addressing and inhibiting the interaction between ALL cells and their bone marrow niche might represent an attractive future therapeutic strategy to prevent ALL relapse. DisclosuresNo relevant conflicts of interest to declare.

Infusion of Umbilical Cord Blood-Derived Natural Killer Cells in Immune-Deficient Mice for the Treatment of Resistant Acute Myeloid Leukaemia

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against acute myeloid leukaemia (AML). Success of this NK cell immunotherapy is dependent on obtaining high numbers of functional NK cells that have the potential to survive in vivo. The use of umbilical cord blood (UCB) CD34+ cells as a source of allogenic NK cells is an interesting method that can generate a readily available, non-invasive, off the shelf cellular product.We developed a cytokine-based culture method for the generation of NK cell products derived from CD34+hematopoietic progenitor cells (HPC) isolated from fresh UCB units. Immuno-phenotyping of ex vivo expanded NK cells showed typical inhibitory and activating NK receptors except for CD16 and the KIR receptors.UCB-derived NK cells displayed good cytolytic activity against NK-sensitive K562 cells with a percentage of specific lysis varying from 30 to 50%. Cytolysis was directly correlated to CD94 expression since CD94-sorted NK cells were responsible for all the in vitro cytolytic function of differentiated NKs against K562 cells. There was an inconstant susceptibility of patient-derived primary AML cells to UCB-derived NK lysis in vitro with a specific lysis ranging from 0 to 25%.We further characterized UCB-derived NK cells by investigating their toxicity, biodistribution, and anti-leukemic potential in vivo. As adoptive transfer of NK cells is an attractive approach for treating refractory leukemia, immune deficient mice were engrafted with a patient derived AML strain resistant to NK-mediated lysis and doxorubicin.After successful engraftment and randomization, leukemic mice were injected with either UCB- derived NK cells or NK cells from healthy donors (NKhds) or doxorubicin, with one control group that didn't receive any treatment. Mice were sacrificed after 2 weeks of treatment and leukemia load along with NK distribution were evaluated by flow cytometry in the blood, bone marrow (BM) and spleen.There was no evidence of toxicity of UCB-derived or healthy donors NK cells in mice. Both types of cells were preferentially found in the blood and in the spleen, even though NKhds reached much higher levels than UCB-derived NKs.As for treatment efficacy, none of our treatment showed anti-leukemic potential based on the absence of decrease of leukemic cells in BM, blood, and spleen. In vivo microenvironment didn't overcome resistance of the patient derived AML cell to NK lysis or to doxorubicin.Remarkably, all of the UCB derived NK cells founded in vivo expressed the CD94 whereas not more than 20% of the injected cells were positive for this marker. Whether it was by in vivo selection or by in vivo differentiation must be investigated.Interestingly, a small cell population with CD56 and CD34 double staining was distinguished in UCB-derived NK and NK healthy donor treated leukemic mice suggesting in vivo interaction between leukemic and NK cells. Further characterization of this population may help to understand the molecular mechanism of leukemic recognition by NK cells and resistance of leukemic cells to cytolysis.In conclusion, UCB-derived NK generation is feasible. Investigation of the role of CD94 in these cells is needed, as cell sorting by CD94 selection in addition to the CD56 could be an interesting approach in the future to select highly functional expanded NK cells before therapeutic use. Furthermore, infusion of UCB-derived NK cells into immune-deficient mice is achievable and non-toxic. However, in vivo environment didn't overcome primary in vitro resistance of AML cells despite an established interaction. Additional elucidation of AML resistance mechanisms to NK lysis is mandatory before therapeutic application. DisclosuresNo relevant conflicts of interest to declare.

FLT4 Expressing CD34+CD38- Leukemic Stem Cells in Bone Marrow of Refractory Patients are Protected by Receptor Internalization under Abundant VEGF-C Condition

So far, the cause of relapse or refractoriness in acute myeloid leukemia (AML) is regarded as the persistence of chemoresistant leukemic stem cells (LSCs). Even VEGF-C/FLT4 axis mediates blast proliferation and survival, understanding of the dynamic behavior of LSCs in bone marrow remains elusive. Especially, FLT4 endocytosis by VEGF-C is related to various biologic functions including signal activation. Based on our previous study, which addressed the potential of FLT4 as a marker for LSCs, we showed that surface FLT4 can be internalized by VEGF-C exposure in bone marrow (BM) derived LSCs. It resulted in resistant to chemotherapy, suggesting LSCs protection. In addition, data for apoptosis revealed that inhibition of FLT4 using MAZ51 with cytosine arabinoside (Ara-C) can effectually increase cell death under abundant VEGF-C in refractory patients. It indicated that targeting FLT4 allows investigators to establish advanced therapeutic strategy with conventional Ara-C in refractory patients with high VEGF-C. In clinic, low level of surface FLT4 in de novo AML-BM LSCs, but not PB cells were inclined to undergo refractory status after induction chemotherapy. To investigate whether FLT4 expressing CD34+CD38- LSCs can be protected by VEGF-C via internalization, FACS analysis, western blotting and immunocytochemistry were performed. Meanwhile, total 66 newly diagnosed (ND) AML patients were used to analyze for clinical correlation. (BM, n=37; PB, n=19). FACS analysis showed that surface FLT4 expressing CD34+CD38- LSCs in BM, but not PB, were significantly decreased by VEGF-C (In BM; without VEGF-C & with VEGF-C, 38.7±8.1% & 16.0±5.1%). Similar with surface FLT4 in PB cells, both intracellular FLT4 in BM and PB was highly sustained, regardless of VEGF-C (In BM; without VEGF-C & with VEGF-C, 62.9±17.2% & 69.7±14.9%, In PB; without VEGF-C & with VEGF-C, 77.4±15.6% & 70.4±21.8%). Immunocytochemistry and western blotting also showed internalization of surface FLT4 by VEGF-C treatment. FLT4+CD34+CD38- cells both in BM and PB were significantly higher in refractory patients than in post allogeneic stem cell transplantation (SCT) group, implying clinical correlation of FLT4 with refractory patients. Consistent with findings in ND-AML and refractory groups, FLT4+CD34+CD38- cells were highly sustained in complete remission group, showing drug resistant population (ND-AML, n=30; refractory AML, n=9; Complete remission AML, n=18; post-allogeneic stem cell transplantation, n=9). High level of VEGF-C was detected in refractory patients, compared to that of normal donors (Refractory AML, n=9, normal donors, n=7). Apoptosis results showed that high number of apoptotic CD45dimCD34+CD38- cells in MAZ51 (FLT4 antagonist) and Ara-C dual treatment under VEGF-C exposure, compared to no treatment of both MAZ51 and Ara-C (without VEGF-C; 0.5-1.3 folds, with VEGF-C; 2.7-43.3 folds) or single exposure either to MAZ51 or Ara-C (In Ara-C only; without VEGF-C; 1.0-9.5 folds, with VEGF-C; 0.1- 1.3 folds, In MAZ51 only; without VEGF-C; 1.1-1.7 folds, with VEGF-C; 1.3-39.3 folds), suggesting that Ara-C induced-blast apoptosis can be augmented by FLT4 inhibition even in the presence of VEGF-C in a sub-group of either refractory or relapsed AML patients. VEGF-C/FLT4 axis in AML is involved in PI3K and AKT pathway.Collectively, we demonstrated that FLT4 internalization under VEGF-C in BM leads to protection of FLT4 expressing LSCs and is clinically relevant to refractory subgroup. This data could suggest some clues to develop therapeutic strategies in targeting FLT4 expressing refractory LSCs. DisclosuresNo relevant conflicts of interest to declare.

Vcam1 Is a "Don't-Eat-Me" Signal on Healthy Hematopoietic and Leukemic Stem Cells

▪Hematopoietic stem cells (HSCs) possess the ability to maintain the entire population of blood cells throughout life and to replenish the hematopoietic system after transplantation into marrow-ablated recipients. In mammalian adults, HSCs predominantly reside in the bone marrow to generate and maintain all blood cell types. HSCs, however, retain their ability to migrate to ectopic niches via the bloodstream, and traffic back to the bone marrow microenvironment via in part the interactions of alpha4 integrins with Vascular Cell Adhesion Molecule-1 (Vcam1, also known as CD106), constitutively expressed by endothelial and stromal cells. In the course of studies to investigate the role of Vcam1 in the macrophage erythroblastic niche, we have found using Vcam1-floxed mice crossed with a Csf1r-iCre (hereafter Vcam1Δ/Δ) that Vcam1 and Cre, using this transgenic line, were expressed on Lineage− (Lin−) c-Kit+ Sca1+ CD48− CD150+ HSCs. Although Vcam1Δ/Δ mice show no significant steady-state hematopoietic defect, transplantation of Vcam1Δ/Δ bone marrow, both in competitive and non-competitive settings, failed to engraft irradiated wild-type recipients. Indeed, ~80% of animals transplanted with Vcam1Δ/Δ cells died, and the few that survived failed to successfully engraft secondary recipient mice. Vcam1Δ/Δ HSC/progenitors homed to bone marrow as well as wild-type counterparts and showed no detectable change in apoptosis in the bone marrow. However, syngeneic Vcam1Δ/Δ HSC/progenitors were selectively cleared in vivo by bone marrow macrophages over 4 days. The in vivo Vcam1Δ/Δ HSC/progenitor clearance did not require irradiation-induced damage since in steady-state parabiosis (Vcam1Δ/Δ and wild-type co-joined pairs), Vcam1Δ/Δ HSCs did not engraft the partner after G-CSF-induced mobilization whereas wild-type HSCs engrafted the parabiont partner. In addition, in vitro incubation of bone marrow-derived macrophages with Lin− cells revealed rapid phagocytosis by macrophages of Vcam1Δ/Δ, but not wild-type, cells and phagocytosis was also induced when the integrin alpha4 beta1 (also known as VLA-4) was blocked in wild-type Lin− cells. Thus, these results suggest that Vcam1 expressed on HSCs serves as a novel don’t-eat-me signal, controlling the migration in the bone marrow by a macrophage-enabled checkpoint. Since we found that Vcam1 is expressed at higher levels on acute myelogenous leukemia (AML) cells than on healthy HSCs, and high VCAM1 expression correlates with poor prognosis in human AML patients (Ley et. al. NEJM 2013), we examined whether Vcam1 expression by AML cell could affect leukemogenesis in vivo. To test this idea, we generated AML-Vcam1Δ/Δ and AML-Control cells by transduction of bone marrow Lin− c-Kit+ Sca1+ cells with the pMSCV-MLL-AF9-GFP oncogene. Strikingly, FACS analysis of primary AML recipient bone marrow revealed a marked reduction (>99%) in the number and percentage of phenotypic Lin− IL7Rα− Sca1− MLL-AF9 GFP+ c-Kithigh CD34low FcγRII/IIIhigh leukemic stem cells in AML-Vcam1Δ/Δ mice compared to control. These results were confirmed by imaging analyses, which showed a marked reduction in leukemic infiltration in AML-Vcam1Δ/Δ mice compared to control. Kaplan-Meier survival analysis of secondary recipient mice receiving 20,000 GFP+ leukemic cells revealed a significantly greater survival of mice harboring AML-Vcam1Δ/Δ cells relative to AML-Control (P<0.0001). To assess in a pre-clinical experimental setting whether Vcam1 inhibition can alter the course of AML, we established AML in immunocompetent C57BL/6 recipients and started therapy of moribund leukemic mice (>50% circulating AML-GFP+ cells) with a daily injection of saline, IgG control, anti-Vcam1 blocking antibody (100 μg/day), cytarabine (100 mg/kg), or a combination of anti-Vcam1/cytarabine for 5 days. Remarkably, this short treatment with anti-Vcam1 blocking antibody was able to preferentially and significantly reduce the frequency and absolute number of phenotypic bone marrow leukemic stem cells in vivo. In addition, our results suggest that using a blocking anti-Vcam1 antibody synergizes with cytarabine treatment to decrease leukemic burden in vivo. These studies lay the groundwork for the development of a new therapeutic strategy for eliminating leukemia stem cells by modulating the innate immune response. DisclosuresFrenette:GSK: Research Funding; Pfizer: Consultancy; PHD Biosciences: Research Funding.

Up-Regulated Mir-146a Expression in Leukemia Cells Induced By G-CSF Correlates with Enhanced Sensitivity to Cytarabine Treatment: A Putative Novel Predictive Biomarker for Chemotherapy

BackgroundsLeukemic stem cells (LSCs) have emerged as one of reasons for relapse to chemotherapy. To eradicate these quiescent LSCs is of great importance. Administration of granulocyte colony stimulating factor (G-CSF) with low-dose chemotherapy, as a priming strategy, has shown satisfactory effect in acute myelocytic leukemia treatment, especially in elder patients. It is important to investigate the specific signaling pathways and explore useful biomarker to monitor clinical response to therapy. MiR-146a and its downstream putative targets CXCR4 and Smad4 may sustain quiescent LSCs in bone marrow niches. The objective of this study is to further explore the possibility of miR-146a as a marker for sensitivity detection to G-CSF priming chemotherapy.Patients and MethodsMiR-146a over-expression (OE), knock-down (KD) and empty virus (EV) HL-60 cells were constructedin vitro. Leukemia cells were pre-treated with G-CSF for 48h or not. MiR-146a, p50, p65, CXCR4 and Smad4 expression levels were detected. Human stromal cell line HS-5 and HL-60 cells were co-cultured to simulate interaction between bone marrow microenvironment and leukemia cells. Chemotaxis, cell viability, cell cycle and apoptosis were estimated individually. Relationship between miR-146a expression and prognostic factors were analyzed in 46 new diagnosis AML patients, including overall survival (OS) rates, relapsed-free survival (RFS) rates and complete remission (CR) rates.ResultMiR-146a-OE cells showed lower CXCR4 and Smad4 levels than miR-146a-EV cells (p=0.011 and 0.01), while miR-146a-KD cells showed increased CXCR4 and Smad4 expression (p=0.0002 and 0.001), both in mRNA and total protein levels. Higher miR-146a expression was observed in G-CSF pre-treated group. p50 and p65 mRNA and total protein levels were both elevated in response to G-CSF treatment (p<0.01). Meanwhile, the amount of cells migrating to medium containing SDF-1a or HS-5 cells decreased significantly. More cells turned into G1 phase with accelerated proliferative rates, and more apoptotic cells were detected in response to cytarabine treatment.Taking median miR-146a value (3.085) as boundaries, patients with high miR-146a expression at diagnosis (n=23) had longer 2-year OS rates (56.5% vs 26.1%, p=0.042), and longer 2-year RFS rates (52.2% vs 13%, p=0.044) than low-expression group (n=23) (Fig 1). After first course of CAG regimen treatment, sensitive respondents with significantly increased miR-146a (n=24) showed longer OS (83.3% vs 40.9%, p<0.01), longer RFS (62.5% vs 22.7%, p=0.032), and higher CR rates (70.8% vs 40.9%, p=0.042) than those with no molecular response (n=22) (Fig 2). According to their response to G-CSF treatment, we divided patients with low miR-146a expression at diagnosis into 2 subgroups. It was proved that significant miR-146a up-regulation after G-CSF treatment promoted more patients into CR courses with longer OS (80% vs 23.1%, p<0.01) and RFS (50% vs 15.4%, p=0.023).Conclusions1. CXCR4 and Smad4 are both downstream targets of miR-146a.2. G-CSF up-regulates miR-146a expression via activating upstream NF-KB signaling pathway, thus altering a series of biological processes including migration, proliferation and cell cycle of leukemia cells, and eventually sensitized them to chemotherapy.3. High miR-146a levels at diagnosis might predict better outcomes.4. Patients with significantly increased miR-146a levels after G-CSF treatment often have longer OS and RFS rates.5. MiR-146a might be a novel biomarker for evaluating clinical prognosis and treatment effect. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The PARP Inhibitor Olaparib Antagonizes Leukemic Growth Induced By TET1 Overexpression in AML1-ETO Positive Acute Myeloid Leukemia

DNA methylation patterns are highly deregulated in human acute myeloid leukemia (AML) cases and stratify AML patient samples into different subgroup. AML1-ETO is the most commonly occurring fusion gene in AML and these AML cases exhibit an aberrant and distinct methylation pattern. So far, the underlying mechanisms for this are only poorly understood. The TET1 dioxygenase has recently emerged as an important epigenetic modifier: by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) TET1 plays an important role in active demethylation, thereby regulating a variety of biological processes. It was linked to tumorigenesis based on the observation that its expression is frequently deregulated in solid cancer. However, the role of TET1 in AML1-ETO+ (AE+)human AML cases is yet unexplored. Using quantitative real time (qRT)- PCR we now show that AE+ AML is characterized by high and aberrant expression of TET1: the gene was significantly higher expressed in the majority of AE+ patients (n=7, p<0.01) compared to other AML subtypes such as inv(16) (n=11), PML-RARα+ (n=31), cytogenetically normal (CN)-AML patients (n=33) and CD34+ normal BM cells (n=4). This observation was consistent with published cDNA microarray data on large patient cohorts (Haferlach et al., JCO 2010, p<0.008 t-test, p<0.01 Anova) and recently published transcriptome data (TCGA) of AML patients. In contrast to TET1, TET2 and TET3 did not show significant higher expression in AE+ patients compared to other AML subtypes. In line with patient data, TET1 was highest expressed in the AE+ AML cell line KASUMI-1 and SKNO-1 compared to other AML cell lines (p<0.05 and n=3). Compared to normal CD34+ and myeloid (CD33+, CD15+ and CD14+) cells (n=3), TET1 was 10-fold and 16-fold higher expressed in AE+ patient samples (n= 7). Aberrant expression of TET1 in AE+ leukemic cells was associated with hypomethylation of its promoter and enrichment for H3K4me3 euchromatic marks at its promoter as determined by LC/MS and ChIP-qPCR respectively. Knockdown (KD) of TET1 mRNA using two short hairpin RNAs (shRNAs) in AE+ AML cell lines impaired their cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). shRNA mediated depletion of TET1 did not impact the cell growth and clonogenicity of the TET1 negative cell line RAJI, ruling out off target effects of the shRNAs (n=3). In mice, KD of Tet1 in leukemic bone marrow cells expressing the truncated leukemogenic AML1-ETO9a (AE9a) fusion, dramatically inhibited cell growth (>60% compared to scrambled, n=3, p<0.01), clonogenicity (>50-70% reduction in primary CFCs, p<0.01, n=3) and importantly delayed onset of leukemia in vivo (median survival 35 days for scr vs 80 days for shRNA mice, n=4/arm, p<0.03). Tet1-knock-out c-kit+ hematopoietic stem and progenitor cells (HSPCs) transduced with AE9a showed reduced primary colony formation and impaired serial replanting capacity in vitro compared to AE9a transduced Tet1-wild-type HSPCs (>50% and >70%, respectively; p<0.001, n=3). Global analysis of 5hmC and 5mC levels using hMeDIP/MeDIP-seq performed on TET1 depleted KASUMI-1 cells revealed lower global 5hmC levels and increase in 5mC as compared to cells transduced with scrambled control (n=2). 3324 promoter regions lost 5hmC and gained 5mC upon TET1 depletion (-5kTSS, Fold enrichment cut off <2-fold, q-value<1e5). Recent studies have shown that PARP activity induces TET1 expression by regulating its promoter epigenetically. We could show that aberrant TET1 expression could be antagonized by the PARP inhibitor olaparib in AE+ leukemic cell lines. Furthermore, olaparib treatment decreased 5hmC levels and reduced cell growth and clonogenicity of human AE+ cell lines and of the murine AE9a+ leukemic cell line in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1 expression contributes to the growth of AE+ AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML. DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

The N6-Adenine Methyltransferase METTL14 Plays an Oncogenic Role in Acute Myeloid Leukemia

N6-methyladenosine (m6A) modification is the most abundant internal RNA modification in eukaryotes. Recent studies have shown that the dynamic and reversible regulation of m6A modifications in mRNAs or non-coding RNAs plays critical roles in tissue development, stem cell self-renewal and differentiation, control of heat shock response, and circadian clock controlling, as well as in RNA metabolism and processing. However, little is known about the functions of m6A and m6A regulators in malignant hematopoiesis.METTL14 is a major m6A writer which together with METTL3 forms the core of the methyltransferase complex that catalyzes the conversion of adenosine (A) to m6A. Through qPCR assays, we found that METTL14 was aberrantly up-regulated in mononuclear cells (MNC) from acute myeloid leukemia (AML) patients with t(11q23), t(15;17), or t(8;21) relative to those from healthy donors.To investigate the pathological role of METTL14 in AML, we transduced lineage negative (Lin-) bone marrow (BM) progenitor cells from Mettl14fl/flCreERT mice with MLL-AF9, AML1-ETO9a, or PML-RARa fusion genes and performed colony-forming/replating assays with or without addition of 4-hydroxytamoxifen (4-OHT). Induction of genetic knockout of Mettl14 by 4-OHT treatment remarkably impaired the colony-forming ability of all these AML-related fusion genes after replating. After the first round of plating, we harvested MLL-AF9-transduced cells that were not treated with 4-OHT and transplanted them into lethally irradiated recipient mice. As expected, tamoxifen (TAM) treatment of transplanted mice significantly delayed leukemogenesis compared to mice treated with vehicle (MLL-AF9+TAM, with median survival of 91 days; MLL-AF9+vehicle, with median survival of 71 days; P=0.0012) (Fig.1A). In addition, specific knockdown of Mettl14 with shRNAs showed similar patterns to Mettl14 knockout. Thus our data demonstrate that Mettl14 is crucial for cell transformation and leukemogenesis. Further, to determine the role of Mettl14 in the maintenance of leukemia, we transduced leukemic BM cells from primary MLL-AF9 leukemic mice with shRNAs against Mettl14 or scramble shRNA and transplanted these cells into lethally irradiated recipient mice. Again, a significantly prolonged survival was observed in Mettl14 knockdown groups compared to that in the control group (MLL-AF9+shRNA1, with median survival of 32 days; MLL-AF9+shRNA2, with median survival of 32 days; MLL-AF9+shScramble, with median survival of 23.5 days; P< 0.001 for both knockdown groups) (Fig.1B). Noticeable, mice in Mettl14 knockdown groups showed less c-kit+ cells in BM than mice in the control group (Fig.1C).In addition to the mouse model, we used human leukemia cell lines to investigate the function of METTL14 in human AML cells. Silencing of METTL14 with shRNAs significantly inhibited cell viability, induced apoptosis as well as terminal differentiation of MONOMAC6 and NB4 cell lines (Fig.1D, E, F). Moreover, xenograft model showed that repression of METTL14 significantly inhibited the engraftment of MONOMAC6 cells and thus delayed the onset of leukemia in NSG-SGM3 (NSGS) immunodeficient mice (Fig.1G). Furthermore, knockdown of METTL14 sensitized MONOMAC cells to ATRA or PMA-induced differentiation. Taken together, our results support the oncogenic role of METTL14 in AML and highlight METTL14 as a novel therapeutic target in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Loss of Function RUNX1 Mutations Restrict Protein Biosynthesis during Pre-Leukemia and MDS Transition but Not after Leukemic Transformation

Runx1, a DNA binding subunit of core binding factors, is found frequently mutated in hematological malignancies. Runx1 mutation can be an early event in leukemogenesis endowing pre-leukemic stem cells with a selective advantage in the bone marrow, and is associated with an unfavorable outcome. In mouse models, loss of function (LOF) Runx1 mutations cause a broad decrease of ribosome biogenesis in hematopoietic stem and progenitor cells (HSPCs) by directly binding to ribosomal related genes essential for protein synthesis, and confers resistance to genotoxic stress (Cai et al. 2015 Cell Stem Cell 17(2):165-77). Paradoxially, leukemia cells generally require higher biosynthetic activity, and AML patients with LOF Runx1 mutations show upregulated ribosome signatures compared with those without Runx1 mutations (Silva et al., 2009 Blood 114:3001-3007). It remains unclear whether RUNX1 plays a role in regulating protein synthesis in leukemogenesis as in normal HSPCs, and if LOF Runx1 mutations are important for leukemia initiation, transformation and/or maintenance. To examine such mechanistic roles of RUNX1 in AML progression, we have used a previously reported MLL-PTD; Mx Cre; Runx1 Flox/Flox (Double mutant -DM) mouse model (Hayashi et al., 2015 Blood 126:303 ) that allows experimental tracking of the step-wise transition of HSPCs from pre-disease stage to a MDS-like stage, prior to full blown AML. Various subpopulations of the HSPCs, including genotypic HSCs, MPP, GMP, CMP, MEP, were isolated from the mice at pre-disease, MDS-like, and AML full-blown stages, and were assayed for protein synthesis rates by O-propargyl puromycin incorporation, DNA synthesis rates by BrdU labeling, and FACS analysis. At the pre-disease state, DM HSCs, as well as all the progenitor populations, had lower protein biosynthesis activity compared with similar populations of wild-type control or MLL-PTD mutant mice, consistent with LOF Runx1 mutations providing stress-resistance and survival advantage. As disease progressed, the DM mice developed MDS-like phenotypes including severe anemia and bone marrow fibrosis, with the HSCs (LSK CD34-Flt3- cells) showing increased protein synthesis rate compared with the pre-disease DM mice. Upon the onset of full-blown leukemia, the protein translation rates in all subpopulations of DM HSPCs were significantly faster than the control non-leukemic cells, regardless of the Runx1 mutant status. Importantly, preliminary analyses of two human AML samples found that CD34+ cells with LOF Runx1 mutations displayed a similarly enhanced protein synthesis rates than CD34+ leukemia bone marrow cells carrying wild type Runx1, as seen in the mouse model. Our results show that at early initiation, LOF Runx1 mutation supresses protein biosynthesis; during transition to MDS, the inhibitory regulation was bypassed in LT-HSCs (LSK CD34-Flt3-), suggesting that Runx1-controlled protein translation is involved in the early clonal selection of disease progression. In full-blown leukemia cells including the primitive subpopulations, however the protein synthesis rate appears to become uncoupled from Runx1 regulation possibly due to an activation of compensatory machineries. This study of the role of Runx1 mutation in pre-leukemia cell progression to full blown leukemia raises the question that while some tumor initiating mutations such as LOF Runx1 mutations contribute to the tumor initiation and transformation process, they may not be essential for maintaining certain crucial leukemia cell phenotypes such as protein biosynthesis. DisclosuresNo relevant conflicts of interest to declare.

In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells.

Farnesyltransferase inhibitors (FTIs) are a class of oral anti-cancer drugs currently tested in phase I-II clinical trials for treatment of hematological malignancies. The in vitro effects of various FTIs (alpha-hydroxyfarnesylphosphonic acid, manumycin-A and SCH66336) were tested on CD34+ KG1a cell line and in primary acute myeloid leukemia (AML) cells from 64 patients. By cell viability and clonogeneic methylcellulose assays, FTIs showed a significant inhibitory activity in CD34+ KG1a and primary bone marrow (BM) leukemic cells from 56% of AML patients. FTIs also induced activation of caspase-3 and Fas-independent apoptosis, confirmed by the finding that inhibition of caspase-8 was not associated with the rescue of FTI-treated cells. We concluded that other cellular events induced by FTIs may trigger activation of caspase-3 and subsequent apoptosis, but the expression of proapoptotic molecules, as Bcl-2 and Bcl-XL, and antiapoptotic, as Bcl-X(s), were not modified by FTIs. By contrast, expression of inducible nitric oxide synthase (iNOS) was increased in FTI-treated AML cells. Our results suggest a very complex mechanism of action of FTIs that require more studies for a better clinical use of the drugs alone or in combination in the treatment of hematological malignancies.

Expression and Clinical Significance of N-cadherin in Bone Marrow Leukemic Cells Derived from Patients with Acute Leukemia

To investigate the expression of N-cadherin in bone marrow leukemic cells derived from acute leukemia patients and its clinical significances. A total of 113 patients with acute leukemia were enrolled in this study. Flow cytometry was employed to detect the expression of N-Cadherin in bone marrow leukemic cells from acute leukemia patients and the relationships between the N-cadherin expression and the clinical characteristics of patients with acute leukemia were analyzed. The expression of N-Cadherin in bone marrow leukemic cells deriveted from patients with acute leukemia was variable with 0%-99.7%. For adult AML patients, the positive rate of CD34 in N-cadherin+ group was significantly higher than that in N-cadherin- group(67.39% vs 33.33%)(P=0.013), while the differences of total CR rate and rate of CR after 1 cycle of induction treatment were not significant between these 2 groups(P>0.05). As to ALL patients, N-cadherin+ group had significant lower WBC count (21.31±7.07 vs 51.10±23.69)(P=0.008) and lower percentage of peripheral blood blast (43.22±5.75% vs 66.45±5.65%)(P=0.015). The CR rate after 1 cycle of induction treatment and rate of overall CR were lower and the relapse rate was higher in N-cadherin+ ALL group than those in N-cadherin- ALL group, but the differences were not significant (P>0.05). For childhood ALL, the positive rate of CD33 in N-cadherin+ group was significantly higher than that in N-cadherin- group(47.62% vs 0%)(P=0.012). The relapse rate was higher in N-cadherin+ group than that in N-cadherin- group (30.00% vs 0%)(P=0.115). The median survival time, 3-year overall OS rate and 3-year relapse-free survival rate in N-cadherin- groups of adult AML, non-M3 AML, ALL and chidhood ALL paients were superior to N-cadherin+ groups, but the differences were not significant. The expression of N-cadherin in bone marrow leukemic cells relates to some clinical features of patients with acute leukemia and to some extent has inferior effect on survival of patients with acute leukemia.

Cytoprotective autophagy maintains leukemia-initiating cells in murine myeloid leukemia

AbstractDespite advances in the treatment of acute myeloid leukemia (AML), relapse and drug resistance frequently occur. Therefore, detailed mechanisms of refractoriness, including leukemia-initiating cell (LIC) biology, should be elucidated to treat AML. The self-degradative property of cytosolic macromolecules is central to autophagy and can contribute to homeostasis and stress response. Recent reports suggest the importance of autophagy in hematopoietic stem cells and various tumors. Thus, this study investigated the functional role of autophagy in AML maintenance and drug resistance using tamoxifen-inducible conditional knockout mice of Atg5 or Atg7, which are essential genes for autophagy, combined with an mixed lineage leukemia–eleven nineteen leukemia–induced murine AML model. Inactivation of autophagy by deletion of Atg5 or Atg7 prolonged survival in leukemic mice and reduced functional LICs. Atg7-deficient LICs displayed enhanced mitochondrial activity and reactive oxygen species production together with increased cell death. In addition, Atg7 deletion markedly decreased peripheral blood leukemia cells, concurrent with increased apoptosis, suggesting a higher dependency on autophagy compared with bone marrow leukemia cells. Finally, cytarabine (AraC) treatment activated autophagy in LICs, and Atg7 deletion potentiated the therapeutic effects of AraC, which included decreased LICs and prolonged survival, suggesting that autophagy contributes to AraC resistance. Our results highlight the intratumoral heterogeneity related to autophagy in AML and the unique role of autophagy in leukemia development and drug resistance.

Growth differentiation factor 15 contributes to cancer-associated fibroblasts-mediated chemo-protection of AML cells.

BackgroundChemo-resistance is still a major obstacle in efforts to overcome acute myeloid leukemia (AML). An emerging concept has proposed that interactions between the bone marrow (BM) microenvironment and leukemia cells reduce the sensitivity of the leukemia cells to chemotherapy. As an important element of the tumor microenvironment, the cancer-associated fibroblasts (CAFs) are considered to be activated modulators in the chemo-resistance of many solid tumors. But their contribution to AML has yet to be fully understood. Here we report a critical role for CAFs which were thought to be a survival and chemo-protective factor for leukemia cells.MethodsA retrospective study on the BM biopsies from 63 primary AML patients and 59 normal controls was applied to quantitative analysis the fiber stroma in the BM sections. Then immunohistochemistry on the BM biopsies were used to detect the makers of the CAFs. Their effects on drug resistance of leukemia cells were further to be assessed by co-cultured experiments in vitro. Moreover, the possible mechanisms involved in CAF-mediated chemo-protection of AML cells was investigated by antibody neutralization and siRNA knockdown experiments, with particular emphasis on the role of GDF15.ResultsIn our study, excessive reticular fibers in the BM led to higher frequency of relapse and mortality in primary AML patients, bringing the inspiration for us to investigate the functional roles of the fiber-devied cells. We declared that the CAF cells which expressed higher levels of FSP1, α-SMA or FAP protein were widely distributed in the marrow of AML. Then in vitro co-cultured tests showed that these CAFs could protect leukemia cell lines (THP-1/K562) from chemotherapy. Interestingly, this effect could be decreased by either treatment with a neutralizing anti-GDF15 antibody or knockdown GDF15 (with siGDF15) in CAFs. Furthermore, we also confirmed that the GDF15+ cells mainly co-localized with FAP, which was identified as the typical phenotype of CAFs in the BM stroma.ConclusionsWe firstly demonstrate that the functional CAFs are widespread within the BM of AML patients and should be a critical chemo-protective element for AML cells by producing amount of GDF15.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0405-0) contains supplementary material, which is available to authorized users.

RUNX3 plays an important role in As2O3‑induced apoptosis and allows cells to overcome MSC‑mediated drug resistance.

The interaction between bone marrow stromal cells and leukemia cells is critical for the persistence and progression of leukemia, and this interaction may account for residual disease. However, the link between leukemia cells and their environment is still poorly understood. In our study, runt‑related transcription factor3(RUNX3) was identified as a novel target gene affected by As2O3 and involved in mesenchymal stem cell(MSC)‑mediated protection of leukemia cells from As2O3‑induced apoptosis. We observed induction of RUNX3 expression and the translocation of RUNX3 into the nucleus after As2O3 treatment in leukemia cells. In K562 chronic myeloid leukemia cells, downregulation of endogenous RUNX3 compromised As2O3‑induced growth inhibition, cell cycle arrest, and apoptosis. In the presence of MSC, As2O3‑induced expression of RUNX3 was reduced significantly and this reduction was modulated by CXCL12/CXCR4 signaling. Furthermore, overexpression of RUNX3 restored, at least in part, the sensitivity of leukemic cells to As2O3. We conclude that RUNX3 plays an important role in As2O3‑induced cellular responses and allows cells to overcome MSC‑mediated drug resistance. Therefore, RUNX3 is a promising target for therapeutic approaches to overcome MSC‑mediated drug resistance.

Abstract 3284: Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model

Abstract FLT3 internal tandem duplication (ITD) mutations are the most common alterations in acute myeloid leukemia (AML) and are associated with poor disease prognosis. Targeted therapy using FLT3-ITD inhibitors showed limited effect in reducing leukemia blasts in bone marrow (BM) than that in peripheral blood. The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents. Therefore, blocking CXCR4 and E-selectin concomitantly with FLT3 inhibition could theoretically eliminate the protection in FLT3-mutant AML patients. We recently reported that targeting CXCR4/E-selectin with the dual inhibitor GMI-1359 showed significant prolongation of survival of mice engrafted with FLT3-ITD mutant MV4-11 leukemia cells. In the present study, we further investigated anti-leukemia effects of dual CXCR4/E-selectin inhibition. Results indicate efficient mobilization of leukemia cells into the circulation by GMI-1359 in a MOLM14-engrafted murine model 2h after drug treatment, which was 3.3-fold (± 0.3) higher compared with the CXCR4 antagonist plerixafor, and 7.4-fold (± 2.7) higher compared with controls. In addition, GMI-1359 also mobilized normal murine leukocytes from the BM, suggesting that GMI-1359 may block interactions of leukemia cells with various BM components. Combination therapy of GMI-1359 and sorafenib significantly reduced leukemia burden (1.9e7 vs. 2.3e9, 1.0e9 and 8.5e7 photons/sec in the combination group versus control, GMI-1359 and sorafenib groups, respectively, at day 20 as determined by Xenogen IVIS bioluminescence Imaging; p &amp;lt; 0.001). Moreover, the combination treatment profoundly reduced leukemia burden in the BM of leukemic mice after a 15-day treatment (1.7e7 vs. 1.6e9, 6.3e8 and 7.0e7 photons/sec, p &amp;lt; 0.01; and 1.76% vs. 70.96%, 72.8% and 33.8%.leukemia cell engraftment measured as GFP positive leukemic cells in BM by flow cytometry, in the combination group vs. vehicle, GMI-1359 and Sorafenib groups, respectively, p &amp;lt; 0.01). Of note, the combination therapy profoundly extended the median survival of the leukemia-bearing mice from 27 days in the control group to 46.5 days in the combination treatment groups (p &amp;lt; 0.001). Mechanistically, we demonstrated that direct blockade of the interaction of E-selectin-CXCR4/SDF-1α with GMI-1359 was critical for leukemic cell mobilization via the disruption of adhesion to stromal cells. Furthermore, the presence of GMI-1359 enhanced sorafenib-induced apoptosis in FLT3-ITD mutated leukemic cells co-cultured with stromal cells. This effect occurred even under hypoxia, which is characteristic for the leukemic BM microenvironment. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3-mutant AML. Citation Format: Weiguo Zhang, Hong Mu, Qi Zhang, Nalini B. Patel, William E. Fogler, John L. Magnani, Michael Andreeff. Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3284.