Normal Human CD34 Research Articles

Abstract 3904: Inhibition of the ERK5 pathway as a novel approach to target human chronic myeloid leukemia stem cells

Abstract Tyrosine kinase inhibitors (TKi) targeting BCR/ABL are very effective for the treatment of Chronic Myeloid Leukaemia (CML). However, resistance mechanisms or their inefficacy on CML leukaemia stem cells (LSC) may lead to relapse. Therefore, there is urgent need to identify new molecular targets. The Extracellular signal-Regulated Kinase 5 (ERK5) is a Mitogen-Activated Protein Kinase involved in cancer. Our aim was to study a possible role of the ERK5 pathway in CML LSC. Cells used were: human CML cell lines K562 and KCL22 that express constitutively active ERK5; CML patient-derived cells and CD34+ cells from heathy donors (after informed consent had been obtained). Cells were incubated in normoxic (routine) or hypoxic (0.2% O2) primary cultures (LC1) in the presence or the absence of drugs. Day-7 LC1 cells were transferred to drug-free, non-selective normoxic secondary cultures (LC2), to measure LC2 repopulation as a read-out of progenitor/stem cell potential (CRA assay). Compounds: XMD8-92 (ERK5 inhibitor) and BIX02189 (MEK5 inhibitor); imatinib (BCR/ABL inhibitor, IM). We previously showed that stem cell potential of CML LSC is maintained in severe hypoxia. In K562 and KCL22 cells and in primary cells derived from 9 CML patients, the treatment in hypoxic LC1 with XMD8-92 or BIX02189, but not with IM, impaired progenitor/stem cell potential. The same results were obtained by combined treatment of XMD892 with IM. Importantly, XMD8-92 did not affect progenitor/stem cell potential of CD34+ cells from heathy donors. In colony formation ability assays ERK5 inhibition decreased colony formation by human primary CML cells to a higher extent than that by normal human CD34+ hematopoietic cells. Interestingly, in hypoxia, combined treatment XMD8-92/IM decreased the expression of genes relevant for stem cell maintenance such as p21, nanog and c-myc and the expression of CD26, a CML LSC marker. Moreover, combined XMD8- 92/IM maintained low the expression of p27, another gene involved in stem cell maintenance, that is increased by either drug when administered alone. This indicate that the ERK5 pathway inhibitors impaired LSC maintenance of CML cell lines and primary CML cells. Citation Format: Ignazia Tusa, Giulia Cheloni, Nathanael Gray, Antonella Gozzini, Persio Dello Sbarba, Elisabetta Rovida. Inhibition of the ERK5 pathway as a novel approach to target human chronic myeloid leukemia stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3904. doi:10.1158/1538-7445.AM2017-3904

Enhanced and Accelerated Repopulation of Mutant C-Kit Immunodeficient Mice By Transplants of Primary Chronic and Acute Myeloid Leukemic Human Cells

Background: Assessment of the growth and differentiation of human hematopoietic cells transplanted into immunodeficient mice has become an important method for evaluating the numbers and properties of cells with engraftment potential and the progeny they can generate in an in vivo setting. Such xenograft models have undergone numerous modifications and have also been applied with increasing success to primary sources of many types of malignant as well as normal human hematopoietic cells. These advances include the incorporation of cDNAs encoding various human-specific growth factor genes into the genomes of the widely used and long-lived NOD-scid-IL2Rγcnull (NSG) mouse, the creation of regenerated human bone ossicles within the mice to more closely mimic the human marrow microenvironment, recognition of the superior supportive activity of female mice, and more recently of NSG mice additionally compromised by their genetic acquisition of a c-kit deficiency. Exploitation of these modifications to recipients of human hematopoietic cells has greatly increased our understanding of the complex composition of both normal and leukemic populations and their responses to various treatments. Nevertheless, many examples remain of both acute and chronic human myeloid leukemias that do not engraft or engraft very poorly, even in the most permissive mice thus far tested. These include samples of many cases of chronic phase chronic myeloid leukemia (CP-CML), myelodysplastic syndromes (MDS), and approximately one third of all cases of acute myeloid leukemia (AML). We have now created 2 new strains of immunodeficient mice that have the B, T, NK immunodeficiency determined by a SirpαNOD-Rag1-/--IL2Rγc-/- genotype (which is equivalent to that of NSG mice) and a c-kit deficiency obtained by replacement of the wild-type c-kit gene with a homozygous W41/W41genotype (SRG-W41 mice). The second strain has the same SRG-W41 genotype but is also transgenic for human IL3, GM-CSF, and SCF production (SRG-W41-3GS). NRG/SRG mice were chosen as the parental strain because they support similar levels of chimerism as NSG mice when given a radiobiologically equivalent dose of radiation, but can benefit from split or low dose rate irradiation protocols.Results: We first showed that leukemic cells from patients with AML and CP-CML produce progeny more rapidly in sub-lethally irradiated parental SRG-3GS mice than in SRG mice, and that SRG-W41 mice can support higher levels of human myeloid and B-lymphoid chimerism by normal human CD34+ cord blood (CB) and adult bone marrow (BM) cells than similarly suppressed or non-irradiated SRG controls. We then compared the chimerism obtained in different strains of mice transplanted each with 6x106 CD34+ cells from 1 CP-CML patient or ~106 CD34+ cells from 5 different AML patients pre-selected for their previously demonstrated poor or inability to engraft NSG-3GS mice. Within a few weeks, the level of the predominantly myeloid chimerism in 2 non-irradiated male SRG-W41-3GS mice and 2 sub-lethally irradiated female SRG-3GS recipients became equivalent and this equivalence was sustained for over a year. Only 3 of 5 AML samples tested engrafted either sex of sub-lethally irradiated SRG-W41-3GS or SRG-3GS mice, and in each case, the speed and level of engraftment was greatly enhanced in the SRG-W41-3GS mice as compared to sex-matched SRG-3GS hosts (with 20-30-fold and 2-3-fold higher levels at week 6 and 10 respectively in female as compared to male recipients of either strain). All the engrafted SRG-W41-3GS female hosts became moribund within 20 weeks post-transplant with a predominance of human leukemic cells in their BM and enlarged spleens filled with leukemic cells. In contrast, the SRG-3GS recipients of the same AML cells survived more than 20 weeks with a more delayed appearance of leukemic cells. Sublethally irradiated female SRG-W41-3GS recipients of 0.1-0.2x106 CD34+ cells from 3 of 3 chronic myelomonocytic leukemia (CMML) patients tested to date have also shown engraftment at either week 3 or 11 and, in one case, up to 17 weeks post-transplant.Conclusion: The introduction of a c-kit deficiency into highly immunodeficient SRG mice producing human IL3, GM-CSF and SCF transgenically offers new opportunities for examining the growth potential and sensitivity to treatment of a broader spectrum of primary human leukemias and neoplastic conditions than has been previously possible. DisclosuresDeininger:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The Nitric Oxide-Generating Prodrug Js-K Is Selectively Toxic Towards AML Compared to Normal Human Hematopoietic Cells

Nitric oxide (NO) induces apoptosis and differentiation in acute myeloid leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a lead agent of the arylated diazeniumdiolate class. JS-K is active in vitro and in vivo against AML, multiple myeloma, and several solid tumors. JS-K is directly cytotoxic to malignant cells and inhibits angiogenesis in vitro and in vivo. We have previously shown that JS-K is not toxic towards normal murine hematopoietic cells.Aiming at its clinical application to treat AML, we have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic®P123 polymers. The formulation (P123/DMSO) consists of 2.25% P123 and 2% dimethyl sulfoxide in phosphate buffered saline. P123/JS-K is currently at an advanced stage of pre-clinical development.Here, we compared the cytotoxicity of JS-K and its formulation between normal hematopoietic and AML cells. HL-60 AML cells or normal human CD34+ cells isolated from 2 different cord blood units were treated for 24 hours with P123/DMSO or P123/JS-K before plating in semi-solid media. Colonies were scored at 10 days. P123/DMSO alone had no effect on colony formation by HL-60 or normal CD34+ cells. P123/JS-K at concentrations of 0.1, 0.25, and 0.5 μM inhibited HL-60 colony growth by 27, 74, and 100%, respectively (P < 0.05 for the comparison between 0.25, 0.5 μM and controls).At the same concentrations, P123/JS-K inhibited the growth of erythroid colonies from the first cord blood sample, by 26, 43, and 61%, respectively. At a concentration of 0.1 μM, P123/JS-K did not inhibit the growth of erythroid colonies from the second cord blood sample but inhibited erythroid colony growth by 18, and 27% at concentrations of 0.25 and 0.5 μM, respectively. However, the differences with untreated controls were not statistically significant for either cord blood sample.At the same concentrations P123/JS-K inhibited the growth of myeloid colonies from the first cord blood sample, by 8, 3, and 37%, respectively. At a concentration of 0.1 μM, P123/JS-K did not inhibit the growth of myeloid colonies from the second cord blood sample but inhibited myeloid colony growth by 2, and 6% at concentrations of 0.25 and 0.5 μM, respectively. However, the differences with untreated controls were not statistically significant for either cord blood sample.With clinical development in mind, we also sought to determine whether the P123/DMSO formulation affects platelet function. P123/DMSO was added to normal donor blood obtained in either EDTA or citrate anticoagulant. With either anticoagulant, P123/DMSO did not affect the measured hemoglobin, white blood cell, or platelet counts. Inspection of peripheral blood smears (Wright stain) obtained with either anticoagulant revealed no platelet clumping. We then studied the effect of P123/DMSO on platelet aggregation in vitro in response to ADP, collagen, ristocetin, or arachidonic acid. Light transmission platelet aggregometry was performed using platelet-rich plasma from a healthy volunteer with normal platelet counts and function. In order to determine whether P123/DMSO could enhance or inhibit platelet aggregation, we chose a range of agonist concentrations from sub-threshold to concentrations expected to induce complete aggregation. P123/DMSO was added at a concentration expected to be equal to peak in vivolevels based on a prior dog toxicology study. Normal aggregation responses were observed in the saline control. There were no significant differences between this control and responses observed in the presence of P123/DMSO. Specifically, significantly decreased aggregation in P123/DMSO-treated platelets was not observed in response to agonists expected to elicit complete aggregation responses. Likewise, enhanced aggregation was not observed in response to sub-threshold agonist concentrations.Our results confirm the potent anti-leukemic activity and limited toxicity against normal human hematopoietic cells of JS-K formulated in P123 micelles. Furthermore, the formulation does not affect platelet aggregation. We therefore expect that P123/JS-K will have a favorable hematologic toxicity profile and as such, will constitute an important addition to our armamentarium for the treatment of AML. DisclosuresDeininger:Gilead: Research Funding; BMS: Consultancy, Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Shami:JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Metformin Induces FOXO3-Dependent Fetal Hemoglobin Production in Primary Erythroid Cells

Elevated fetal hemoglobin (HbF, α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by diluting HbS and inhibiting its polymerization in patient red blood cells. Hydroxyurea (HU) the only approved drug for SCD acts in part by raising HbF levels. However, up to 50% of adult SCD patients treated with hydroxyurea, do not have a clinically meaningful response, illustrating the need for new improved agents. We identified FOXO3, a potentially "druggable" transcription factor, as a candidate HbF inducer through analysis of rare variants from whole exome sequence data from SCD patients. Specifically, numerous rare variants that potentially impair FOXO3 activity and/or expression were associated with reduced HbF levels. FOXO3 was previously shown to facilitate erythroid precursor survival, maturation and resistance to oxidative stress.To investigate our findings from genomic studies further, we manipulated FOXO3 expression in normal human CD34+ hematopoietic stem and progenitor cells (HSPCs) induced to undergo erythroid differentiation and examined the effects on γ-globin RNA levels and HbF production. Transfection of CD34+ HSPCs with lentivirus expressing FOXO3 shRNA that reduced levels of the corresponding RNA by 80% reduced γ-globin (HBG1 and HBG2) mRNA by 60% compared to control cultures expressing scrambled shRNA (n= 3 separate experiments, p < 0.0005). A second non overlapping FOXO3 shRNA reduced the corresponding mRNA by 50% and reduced γ-globin by 31% (n=3 separate experiments, p < 0.0001). Reduction of FOXO3 mRNA was accompanied by delay in differentiation (11% GPA+ cells with 80%FOXO3 kd by day 14 compared to 72% GPA+ cells in the scramble kd).Metformin, an FDA-approved drug used for type 2 diabetes, is known to increase FOXO3 expression and therefore, we tested its effects on HbF expression in CD34+ HSPC-derived erythroblasts by treating with 30, 50 and 100 µM drug, beginning at day 5 of culture. At day 14 of culture, 59% late basophilic erythroblasts were present in control cultures, as evidenced by band3 and α4 integrin expression. In contrast, maturation was accelerated in metformin treated cultures, with 70% late basophilic erythroblasts, consistent with previous studies. Remarkably, metformin treatment also caused a dose dependent increase in HbF-immunostained cells ("F-cells", Figure 1) and in % HbF, as measured by HPLC (Figure 2). HPLC analysis at d14 showed that %HbF (HbF/(HbF + HbA) rose from 10.3% at baseline, to 19.8% and 30% at 50 and 100 µM metformin (n= 3, p=0.04 and 0.005). For dosage comparison patients on standard metformin therapy for diabetes typically exhibit plasma drug levels of 70 µM. When cultures were treated with metformin (100µM) and HU (30µM), %HbF was increased to 45% compared to 26% and 30% in cultures treated with HU or metformin respectively as single agents (n=3, p=<0.0001, p=0.0016, and p=0.005).Thus, HU and metformin show additive effects, on HbF induction, suggesting that combination therapy in patients may be more effective than standard therapy of HU alone. In addition, metformin may be a superior agent than HU as single therapy because it does not cause myelosuppression, and therefore requires minimal laboratory monitoring, and does not arrest erythropoiesis, making it a potentially effective agent for quantitative beta hemoglobinopathies. Taken together, our results indicate that FOXO3 is a positive regulator of γ-globin expression and a potential therapeutic target for HbF induction. Given our current results and the excellent safety profile of metformin, a pilot clinical trial of the drug (1500-2500 mg/day) alone or combined with hydroxyurea at stable maximum tolerated dose, in patients aged 16-40 years with sickle cell anemia or non-transfusion dependent beta thalassemia has been approved by the Baylor College of Medicine IRB. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Methylcytosine Dioxygenase TET3 Is Aberrantly Expressed in Acute Myeloid Leukemia and Promotes AML Growth

The epigenetic regulator dioxygenase Ten-eleven translocation 3 (TET3) catalyzes the oxidation of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and plays an important role in histone modification, thereby regulating the gene expression. Epigenetic aberrations play a key role in the pathophysiology of acute myeloid leukemia (AML), however the reasons for the highly distorted epigenome in AML are poorly known. Though in particular TET2 is widely studied in normal and malignant hematopoiesis, the role of TET3 in human myeloid leukemia is still unknown. First we quantified the expression of TET3 in normal murine and human hematopoiesis by quantitative real-time (qRT)-PCR. Among the Tet genes Tet3 showed highest expression in murine hematopoietic stem cells (HSCs) (2-fold and 3-fold compared to Tet1 and Tet2 respectively, p=0.01, p=0.006, n=3). Within the hematopoietic hierarchy highest expression of Tet3 was found in HSCs and lymphoid committed progenitors (CLPs) compared to committed myeloid progenitors (CMPs) (up to ~3.5-fold, p=0.003, n=3). In humans TET3 was higher expressed in CD34+ bone marrow (BM) cells compared to total BM cells (p=0.024, n=3) and within the lineage positive compartment the TET3 was higher expressed in CD33+ myeloid cells compared to lymphoid cells (CD19+ B cells, p=0.008 and CD3+ T cells, p=0.02, n=3). Our qRT-PCR expression data were in agreement with published microarray data demonstrating that TET3 is significantly higher expressed in myeloid cells compared to lymphoid cells (p<0.001, n=5) (Bagger FO et. al., Nucleic Acids Res. 2013). In AML patients TET3 showed a broad range of expression (ΔCT: -1.4 ± 1.4 SD, n=80), but was aberrantly expressed in majority of the AML patients compared to normal total BM (n=3) and CD33+ BM myeloid cells (n=3) (60% and 81% respectively, cut-off for higher expression: 1.5-fold SD of the median). TET3 was higher expressed in 30% of the cases in cytogenetically normal (CN)-AML (n=50) and in 95% of cases in PML-RARa+ AML (n=21) compared to total BM and CD33+ myeloid cells. The AML cell lines OCI-AML3 (NPM1 mutation+), NB4 (PML-RARa+)and KASUMI-1 (AML1-ETO+) showed higher expression of TET3 compared to other AML cell lines (EOL1, Molm 13, MONO-MAC-6 and THP-1) (p<0.001, n=3). shRNA mediated knockdown (KD) of TET3 in human AML cell lines (OCI-AML3, NB4 and KASUMI-1) adversely affected their cell growth (45%-80% reduction compared to scrambled (scr) control, p<0.001, n=3) and clonogenicity (57%-76% reduction, n=3) in vitro. Furthermore, TET3 KD significantly reduced the leukemic engraftment (median 32±17 and 5.6±6 for scr and shRNA respectively) of NB4 and OCI-AML3 in NSG mice (shRNA n=12; scr n=11, p<0.001). Histopathology showed absence or reduced infiltration of leukemic blast cells in different organs of mice transplanted with shRNA expressing AML cells. In contrast, shRNA mediated KD of TET3 in human normal CD34+ cord blood (CB) cells did not impact cell growth in vitro (n=3). TET3 depletion decreased total 5hmC level in the AML cell line NB4 and in parallel changed expression of genes involved in regulation of cell proliferation, MAP kinase activity and cell migration as determined by microarray gene expression analysis (n=3). Constitutive overexpression of wild-type TET3 in human AML cell lines significantly further enhanced their clonogenicity compared to empty vector control (p<0.05, n=3). Interestingly, overexpression of wild-type TET3 or the TET3-CDconstruct containing only the catalytic domain (CD) inhibited cell growth of human CD34+ CB cells and impaired myeloid differentiation in the CFC assays compared to empty vector (EV) (number of colonies EV=68± 5.7SD, TET3=23±1, TET3-CD=36±10, p<0.001, n=3) whereas erythroid colony formation was not affected. These results were also confirmed by flow cytometry with a decrease in the number of CD11b+CD14+ myeloid cells in the colonies (>70% reduction, n=3). In conclusion, our data indicates that ordered expression levels of TET3 are necessary for normal human HSC proliferation and differentiation. The growth and engraftment potential of AML cells are depending on high TET3 expression levels. DisclosuresAkashi:Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Celgene: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol Meyers Squibb: Research Funding; Sunitomo Dainippon Pharma: Consultancy. Buske:Celltrion, Inc.: Consultancy, Honoraria.

Molecular Analysis and In Vivo Efficacy Studies on a Novel Chemical-Series of FLT3 Inhibitors in Human FLT3-ITD AML

Approximately 25% of all acute myeloid leukemias (AMLs) contain an internal tandem duplication (ITD) in the juxtamembranal region of FLT3. Even with standard chemotherapies, FLT3-ITD AML is associated with poor prognosis, with a five-year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. Therefore, FLT3 inhibitors have been developed as a targeted treatment approach. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming, with few complete remissions and a high rate of relapse and resistance. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse in FLT3-ITD AML patients.Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. We have previously shown that our current lead compound (CTDS-004) has excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 <5 x 10-10 M). Additionally, CTDS-004 potently inhibits proliferation and progenitor function, and induces apoptosis of FLT3-ITD positive human AML cell lines in comparison to the second generation FLT3 inhibitors, AC220 and Crenolanib. Importantly, CTDS-004 exhibits minimal effects on the function and viability of normal human CD34+ BM cells. The most important distinction between CTDS-004 and current FLT3-ITD inhibitors is that CTDS-004 retains its efficacy and prevents subclonal recovery of FLT3-ITD AML cells that have become refractory to AC220 or Crenolanib treatment. To gain insight into the mechanistic basis for the superior response of FLT3-TID AML to CTDS-004 as compared to AC220, we performed RNA sequencing of MLL-AF9 FLT3-ITD cells after 6 and 12 hours of treatment with AC220 or CTDS-004 (IC10: 0.3 and 0.1 nM, respectively). Although both compounds target FLT3-ITD, there were striking distinctions in gene expression changes after treatment with AC220 and CTDS-004, highlighting the differences in the biological impact and cytotoxic effect of these compounds.Our lead compound has promising pharmacokinetic and pharmacodynamics properties in vivo, therefore, we tested CTDS-004 in a xenograft model of human AML. NRGS mice (NOD.Cg-Rag1tm1MomIL2rgtm1WjlTg[CMV-IL3,CSF2,KITLG]1EAv/J) were transplanted with human MLL-AF9 FLT3-ITD leukemic cells (2 x 105 cells/mouse) via tail vein injection. Starting on day 10 post-engraftment, the mice were treated with CTDS-004 (30 mg/kg; n=5), AC220 (15 mg/kg; n=5), or PBS (n=5) via intraperitoneal injection on a cycle of five days of daily injection followed by two days of rest for the duration of the experiment. Kaplan-Meier survival analysis revealed that CTDS-004 is extremely effective at prolonging survival in the human FLT3-ITD AML xenograft model. There was a significant increase in median survival for CTDS-004 and AC220 treatment groups compared to the PBS control group (Median survival post-xenograft: PBS: 28 days, AC220: 54 days, CTDS-004: 53 days; p=0.0002). Taken together, these findings suggest our novel FLT3 inhibitor shows promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Identification of ASCT1 As a Candidate Molecule Enhancing Antioxidant Activity in Primary Human AML Cells

Introduction:Recent studies have shown that the specific alteration of metabolic pathways are involved in the regulation of function of normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, little is known about the features of metabolic activity in human HSCs and LSCs. To reveal the metabolic pathway alterations in primary AML cells, we performed the comprehensive metabolome analysis by comparing normal human CD34+ hematopoietic stem/progenitor cells (HSPCs)(n=5) and CD34+ primitive AML cells containing LSCs (n=16) using highly sensitive CE-tandem mass spectrometry.Method:Metabolome analysisMetabolites were extracted from primitive CD34+ AML cells (n=16) and normal CD34+ bone marrow cells (n=4) and cord blood cells (n=1). Metabolome analysis was conducted by the C-SCOPE package of HMT using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for cation analysis, and capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) for anion analysis. 116 metabolites were targeted for analysis in this study.Oxygen consumption rates and extracellular acidification rateO2 consumption rates (OCR) and extracellular acidification rate (ECAR) were measured by the Seahorse XF96 extracellular flux analyzer. Three replicate wells of 400,000 leukemic or normal cells per well were seeded in 96-well XF96 well plates coated with BD Cell-Tak (BD Biosciences) in serum-free unbuffered DMEM. Analyses were performed both at basal conditions and after injection of OLI (1 mg/ml), FCCP (1 mM), Antimycin A (5 mM).Result:We detected 101 metabolites involved in central carbon and energy metabolism. In glucose metabolism, the level of lactate, an end-product of aerobic glycolysis, were lower in CD34+ AML cells than normal HSPCs, whereas the level of pyruvate, a precursor of lactate, was not different. Thus, CD34+ AML cells had a significantly high pyruvate/lactate ratio as compared to normal HSPCs, suggesting that aerobic respiration is preferentially utilized in CD34+ AML cells. To confirm this observation, we directly measured the O2 consumption rate (OCR) and extracellular acidification rate (ECAR) of CD34+ AML cells (n=4) and normal HSPCs (n=5) by XF96 extracellular flux analyzer. OCR reflects the activity of aerobic respiration, whereas ECAR reflects lactate generation and correlates with anaerobic respiration activity. Therefore, OCR/ECAR ratio is a good index for the discrimination of aerobic and anaerobic respiration pattern. We found that the OCR/ECAR ratio of CD34+ primitive AML cells was significantly high as compared to that of HSPCs, suggesting that CD34+ AML cells predominantly utilized aerobic respiration.Although the aerobic respiration resulted in the production of reactive oxygen species (ROS), the intracellular ROS level was not different between CD34+ AML (n=7) cells and normal HSPCs (n=3), suggesting that the antioxidant activity should be strongly enhanced in CD34+ AML cells. Consistent with the observation, we found that CD34+ AML cells had a much higher level of glutathione (GSH), a primary intracellular antioxidant, than normal HSPCs.To clarify the molecular mechanisms how CD34+ primitive AML cells could maintain high GSH level, we analyzed the expression of cysteine transporters, because cysteine uptake is the rate-limiting step of GSH synthesis. In human, three amino acid transporters including ASCT1, ASC1 and xCT are known as cystine/cysteine transporters. Interestingly, ASCT1 was significantly highly expressed in CD34+ AML cells (n=10) as compared to normal CD34+ HSPCs (n=3). Of note, normal CD34+CD38- HSCs completely lacked ASCT1 expression, whereas CD34+CD38-LSCs expressed at a high level, indicating the possibility that the high expression of ASCT1 should be a LSC specific machinery for enhanced GSH synthesis.Thus, human AML cells predominantly utilize aerobic respiration that is supported by a high level of GSH, and AML specific ASCT1 expression presumably contributes to the high level of GSH. These data suggest that ASCT1 should be a promising molecule to specifically target AML stem/progenitor cells. DisclosuresAkashi:Celgene: Research Funding; Astellas Pharma: Research Funding; Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol Meyers Squibb: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Sunitomo Dainippon Pharma: Consultancy.

Megakaryocyte Expansion and Platelet CD34 Expression Observed in GFI1BQ287*-Related Bleeding and Platelet Disorder Is Caused By Quenching of the Lysine Specific Demethylase LSD1/KDM1A

The heterozygous Q287* mutation in Growth Factor Independence 1B (GFI1B) causes an autosomal-dominant bleeding disorder characterized by gray platelets as a result of reduced α-granule content.Affected individuals also exhibited macro-thrombocytopenia, increased megakaryocyte numbers and platelet CD34 expression. GFI1B functions as transcriptional repressor by recruiting the histone modifying enzyme LSD1/KDM1A. The C-terminally truncated GFI1B-Q287* mutant has lost its repressive function and inhibits the function of wild type GFI1B in a dominant-negative manner.To study how mutant GFI1B affects megakaryopoiesis, we expressed it in megakaryoblastic MEG01 cells by retroviral transduction. Compared to empty vector transduced cells, expression of GFI1B-Q287* caused a significant growth advantage, which is in line with the strong increase in megakaryocytes in the bone marrow of affected individuals. In contrast to GFI1B-Q287*, expression of GFI1B-WT significantly impaired MEG01 expansion (Figure 1).GFI1B recruits LSD1 through its proline and lysine amino acids at positions 2 and 8, respectively. Individual alanine mutations at these positions disturb the LSD1 interaction. We tested whether the LSD1 interaction was required for the growth phenotypes induced by GFI1B-WT and GFI1B-Q287* by separately introducing alanine mutations (P2A and K8A). This showed that both mutations nullified the growth advantage and disadvantage of GFI1B-Q287* and GFI1B-WT, respectively (Figure 1). Thus, GFI1B-WT may limit megakaryoblast expansion through LSD1 recruitment. The mutant protein may inhibit wild type GFI1B by quenching LSD1. Indeed, preliminary results show that co-expression of LSD1 and GFI1B-Q287* resulted in loss of the proliferative advantage as seen for GFI1B-Q287* alone.Figure 1: P2A and K8A mutations in GFI1B nullified the growth advantage of GFI1B-Q287* and the and growth disadvantage GFI1B-WT. GFP% of MEG01 cells was followed for 26 days. Data is normalized to day 5.To address this finding in more detail, we treated normal human CD34+ bone marrow cells with 4 µM LSD1 inhibitor GSK2879552 and induced megakaryocyte differentiation. This showed an 1.4-fold increased CD34+/CD41+ megakaryoblast expansion after 2 days of treatment. In addition, we observed that CD34 expression was retained and elevated. Thus, LSD1 inhibition of primary CD34+ cells during in vitro megakaryocyte differentiation phenocopies disease characteristics observed in individuals with the GFI1B-Q287* mutation.To further study megakaryopoiesis, we generated induced pluripotent stem (IPS) cells from two individuals with GFI1B-Q287* plus a healthy control. Hematopoietic differentiation of IPS colonies was induced using Stemline II, VEGF and BMP4 for the first 6 days and from day 6 onward in CellQuin, VEGF, BMP4, SCF, IL-1β, IL-3, IL-6 and TPO. Non-adherent hematopoietic cells where harvested and differentiated towards megakaryocytes using CellQuin, IL-1β and TPO. Upon megakaryocyte differentiation a 10-fold increase in expansion of megakaryocytes, sustained CD34 expression and increased hypolobulation was observed compared to GFI1B-WT differentiated IPS cells. Thus, GFI1B-Q287* IPS originated megakaryocytes phenocopy disease characteristics observed in individuals with the GFI1B-Q287* mutation.Our data indicate that the LSD1-GFI1B interaction is key to controlling megakaryoblast expansion and that it is important for proper megakaryocyte maturation as assessed by CD34 expression. These data also suggest that mutant GFI1B-Q287* inhibits the function of wild type GFI1B by quenching the demethylase LSD1. Induced LSD1 expression in the megakaryocyte lineage may be of therapeutic relevance for GFI1B related bleeding and platelet disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Generation of a new de novo model of human AML from C-MYC-transduced normal human CD34+ cord blood cells transplanted into NRG-3/GM-CSF/S mice

Generation of a new de novo model of human AML from C-MYC-transduced normal human CD34+ cord blood cells transplanted into NRG-3/GM-CSF/S mice

1. A Direct Comparison of Foamy and Lentiviral Vector Genotoxicity in SCID-Repopulating Cells Shows Foamy Vectors Are Less Prone to Clonal Dominance

Hematopoietic stem cell (HSC) gene therapy has immense potential for numerous diseases including primary immunodeficiencies. However, in some SCID-X1 patients treated with gammaretroviral vectors, leukemia occurred as a result of vector mediated genotoxicity. Lentiviral vectors are less genotoxic than gammaretroviral vectors and have become the retroviral vector of choice in HSC clinical trials. Foamy retroviral vectors have a promising integration profile and are less prone to read-through transcription when directly compared to gammaretroviral or lentiviral vectors. Here we directly compared the safety and efficacy of foamy vectors to lentiviral vectors in human cord blood CD34+ repopulating cells in immunodeficient mice (NSG). To increase their genotoxic potential, foamy and lentiviral vectors with identical transgene cassettes that utilized a known genotoxic spleen focus forming virus promoter were used. Our rationale was that in order to compare the relative safety of these two vector systems in normal human CD34+ cells, within the relatively short time span of repopulating cells in mice, a highly genotoxic promoter would be the best way to elicit differences in genotoxicity. Both foamy and lentiviral vectors resulted in efficient marking in vivo with 12.2% of peripheral blood human CD45+ cells expressing EGFP for foamy vectors, and 9.6% for lentiviral vectors at 19 weeks after transplantation. A total of 837 foamy vector and 423 lentiviral vector unique integration sites were recovered in human SCID repopulating cells 19 weeks after transplantation. Foamy vector proviruses were observed less often within genes (40%) than lentiviral vectors (74%), and were also observed less often within proto-oncogenes (6.6%) than lentiviral vectors (9.7%). Analysis of clonality in repopulating cells within individual mice showed that the foamy vector group was more polyclonal with fewer dominant clones (2/6 mice) than in the lentiviral vector group (8/8 mice) (Figure 1Figure 1). Dominant clones were defined as a single integration site having over 20% of captures. Five of the thirteen dominant clones from the lentiviral group had integrations near known proto-oncogenes (Table 1) but in the foamy vector group, none of the three dominant clones had a known proto-oncogene near the integration site. In summary, in a direct comparison in human CD34+ SCID repopulating cells, foamy vectors resulted in fewer dominant clones and more polyclonal repopulation than lentiviral vectors. Our data further supports the relative safety of foamy vectors for HSC gene therapy.View Large Image | Download PowerPoint Slide

Helicase-like transcription factor is a RUNX1 target whose downregulation promotes genomic instability and correlates with complex cytogenetic features in acute myeloid leukemia.

Helicase-like transcription factor is a SWI/SNF chromatin remodeling factor involved in various biological processes. However, little is known about its role in hematopoiesis. In this study, we measured helicase-like transcription factor mRNA expression in the bone marrow of 204 adult patients with de novo acute myeloid leukemia. Patients were dichotomized into low and high expression groups at the median level for clinicopathological correlations. Helicase-like transcription factor levels were dramatically reduced in the low expression patient group compared to those in the normal controls (n=40) (P<0.0001). Low helicase-like transcription factor expression correlated positively with French-American-British M4/M5 subtypes (P<0.0001) and complex cytogenetic abnormalities (P=0.02 for ≥3 abnormalities;P=0.004 for ≥5 abnormalities) but negatively with CEBPA double mutations (P=0.012). Also, low expression correlated with poorer overall (P=0.005) and event-free (P=0.006) survival in the intermediate-risk cytogenetic subgroup. Consistent with the more aggressive disease associated with low expression, helicase-like transcription factor knockdown in leukemic cells promoted proliferation and chromosomal instability that was accompanied by downregulation of mitotic regulators and impaired DNA damage response. The significance of helicase-like transcription factor in genome maintenance was further indicated by its markedly elevated expression in normal human CD34(+)hematopoietic stem cells. We further demonstrated that helicase-like transcription factor was a RUNX1 target and transcriptionally repressed by RUNX1-ETO and site-specific DNA methylation through a duplicated RUNX1 binding site in its promoter. Taken together, our findings provide new mechanistic insights on genomic instability linked to helicase-like transcription factor deregulation, and strongly suggest a tumor suppressor function of the SWI/SNF protein in acute myeloid leukemia.

Cord Blood-Derived Quiescent CD34+ Cells Are More Transcriptionally Matched to AML Blasts Than Cytokine-Induced Normal Human Hematopoietic CD34+ Cells.

Acute myeloid leukemia (AML) is characterized by developmental arrest, which is thought to arise from transcriptional dysregulation of myeloid development programs. Hematopoietic stem and progenitor cells (HSPCs) isolated from human blood are frequently used as a normal comparator in AML studies. Previous studies have reported changes in the transcriptional program of genes involved in proliferation, differentiation, apoptosis, and homing when HSPCs were expanded ex vivo. The intrinsic functional differences between quiescent and dividing CD34+ HSPCs prompted us to determine whether fresh or cytokine-induced cord blood-derived CD34+ HSPCs are a more appropriate normal control compared to AML blasts. Based on principal component analysis and gene expression profiling we demonstrate that CD34+ HSPCs that do not undergo ex vivo expansion are transcriptionally similar to minimally differentiated AML blasts. This was confirmed by comparing the cell cycle status of the AML blasts and the HSPCs. We suggest that freshly isolated CD34+ HSPCs that do not undergo ex vivo expansion would serve as a better control to identify novel transcriptional targets in the AML blast population.

Deletion of Pleiotrophin (PTN), a Vascular Niche Paracrine Factor, Abrogates Chronic Myeloid Leukemia Pathogenesis In Vivo

Tyrosine kinase inhibitor (TKI) therapy revolutionized the treatment of patients with chronic myeloid leukemia (CML), but TKI therapy alone is not curative. Leukemic stem cells (LSCs) are considered to be resistant to TKI therapy in CML, although the mechanisms which confer LSC resistance to TKI-mediated eradication remain poorly understood. Our laboratory has previously described the role of PTN, a paracrine factor which is secreted by bone marrow endothelial cells (BM ECs) and is necessary for the self-renewal and regeneration of normal hematopoietic stem cells (HSCs) in vivo. We hypothesized that PTN signaling in the BM vascular niche might also be important in CML pathogenesis and perpetuation of LSCs. In order to test our hypothesis, we crossed SCL-tTA/BCR-ABL mice, which develop CML over time under the control of doxycycline-treatment, with either PTN-/- or PTN+/+ mice, and compared CML pathogenesis in vivo. SCLtTA/BCR-ABL;PTN+/+ mice displayed evolution of chronic phase CML (splenomegaly, increased WBCs, left-shifted myelopoiesis, decreased survival) within 90-120 days. In contrast, SCLtTA/BCR-ABL;PTN-/- mice displayed normal range WBCs, decreased splenomegaly and significantly increased survival over the same time period. At 300 days post-induction of BCR-ABL, 33% percent of SCLtTA/BCR-ABL;PTN-/- mice remain alive compared to only 7% of SCLtTA/BCR-ABL;PTN+/+ mice (p<0.0001). Importantly, treatment of human CML cells with a specific anti-PTN antibody (BD Biosciences) significantly decreased CML colony formation in methylcellulose (P=0.02), whereas normal human CD34+ cells were unaffected. This result suggested the novel possibility that human CML cells may produce PTN in an autocrine manner as a means to propagate disease. These studies suggest that genetic or pharmacologic antagonism of PTN signaling is a potent strategy to inhibit CML pathogenesis. Ongoing studies will determine if PTN-targeted therapy, with or without TKIs, can mediate the eradication of LSCs which are responsible for perpetuation of this disease. DisclosuresHimburg:Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

Nuclear Export Inhibitor KPT-8602 Is Highly Active Against Leukemic Blasts and Leukemia-Initiating Cells in Patient-Derived Xenograft Models of AML

Currently available combination chemotherapy for AML induces complete remission in about 75% of younger and 45% of older patients, but often fails to induce long-term disease-free survival and is toxic to normal hematopoietic cells. The frequent disease relapse observed in chemotherapy treated AML patients is thought to occur through the inability of the existing drugs to specifically target the self-renewing leukemia-initiating cells (LICs). These cells have the functional capacity to replenish AML blasts. An attractive target for AML therapy is the nuclear export protein exportin 1 (XPO1), which is functionally required for continuous nuclear export of a subset of proteins and mRNAs. Small molecule Selective Inhibitor of Nuclear Export (SINE) compounds covalently bind to Cysteine528 in the cargo-binding groove of XPO1 and inhibit nuclear export. The orally bioavailable clinical SINE compound, selinexor (KPT-330), is currently in Phase 1 and 2 clinical trials in adult patients with AML (NCT01607892, NCT02088541, NCT02249091, NCT02403310, NCT02093403, NCT02485535, NCT02299518, NCT02416908, NCT02212561, and NCT01607892) and in a Phase 1 trial for relapsed childhood ALL and AML (NCT02091245). The results of these trials are encouraging, as they have demonstrated that selinexor alone or in combination is active in inducing remission in patients with relapsed or refractory AML. Here, we explore the anti-leukemia activity of a next generation SINE compound, KPT-8602.In preclinical toxicology studies as well as clinical trials, noted adverse events for selinexor are anorexia with weight loss, fatigue and thrombocytopenia, which limit the frequency of dosing to no more than every other day, three times a week. KPT-8602 is orally bioavailable and has similar pharmacokinetic properties to selinexor, with reduced brain penetration. Preliminary toxicology studies in rats and monkeys suggest that KPT-8602 has a substantially better tolerability profile, with reduced fatigue, thrombocytopenia, and anorexia compared to selinexor. Thus, KPT-8602 can be dosed orally on a daily basis for 5 days each week. The increased drug exposure with daily dosing and better tolerability of KPT-8602 leads directly to superior efficacy in pre-clinical models compared with selinexor.To define the anti-leukemia activity of KPT-8602 against primary AML blasts and LICs in a relevant pre-clinical setting, we established patient-derived xenograft (PDX) models, in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Mice engrafted with leukemic blasts were treated with vehicle, selinexor (20 mg/kg three times per week), or KPT-8602 (15 mg/kg daily for 4 weeks). KPT-8602 was highly active against blast cells from three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN), AML with complex karyotype (AML-CK), and MDS-derived AML (MDS/AML)), as evidenced by a reduction in leukemic engraftment in primary mice after treatment (Fig. 1A-C). At these doses, KPT-8602 demonstrated higher anti-leukemic activity than selinexor, with no leukemic cells detected in the bone marrow of two of the eight AML-CN PDX treated mice (Fig. 1A-C). In secondary transplantation assays, KPT-8602 greatly reduced the frequency of LICs in the PDX model derived from AML-CN cells, indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs (Fig. 1D). In mice engrafted with normal cord blood human CD34+ cells, KPT-8602 reduced bone marrow human CD45+ cell numbers (Fig. 1E), but the impact on normal hematopoietic stem and progenitor cells, as determined by secondary transplantation assays (Fig. 1E), was modest when compared to the LIC reduction after KPT-8602 treatment. These findings demonstrate that KPT-8602 has better tolerability, perhaps through decreased CNS penetration, and is better at eliminating both AML LIC and blast cells when compared to selinexor. Therefore, KPT-8602 should be tested for efficacy and tolerability in patients with relapsed and refractory AML, with the goal to overcome a difficult obstacle in curing AML; destroying the LIC compartment while sparing normal hematopoietic cells. [Display omitted] DisclosuresEtchin:Karyopharm: Research Funding. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Ellis:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutics, Inc: Employment. Stone:Agios: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Karyopharm: Consultancy; Celator: Consultancy; Novartis: Research Funding; Merck: Consultancy; Juno: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Roche/Genetech: Consultancy; Celgene: Consultancy. DeAngelo:Bristol Myers Squibb: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Ariad: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership. Look:Karyopharm Therapeutics, Inc: Research Funding.

Knockdown of HCK Reduces Cell Death and Erythroid Differentiation in Human CD34+ Hematopoietic Progenitor Cells

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The identification of genes and cellular pathways active in leukemia cells but not in normal hematopoietic stem/progenitor cells (HSC) may help to understand the key steps in the MDS and AML pathogenesis and lead to new approaches to further enhance the treatment of both diseases, considered incurable with non-transplantation therapy. Src kinase family (SFK) is a central mediator in multiple oncogenic signaling pathways and some SFK members (Hck, Lyn, Fgr, Fyn) had previously been described as overexpressed or activated in leukemic cells. However, to this moment, the role of hematopoietic cell kinase (HCK), the unique SFK member restricted expressed in hematopoietic cells, had not been characterized in MDS and AML pathogenesis as well as in HSC. In order to better understand the HCK importance in hematopoiesis, we used lentiviral shRNA vectors to knockdown the HCK expression in primary human CD34+ HSC. The HCK levels were reduced in approximately 70-80% (shHCK) compared to the control lentiviral shRNA (shControl-GFP). To promote erythroid differentiation, human CD34+ transduced cells were grown in methylcellulose for 7 days and in liquid media for another 6 days. During this experiment, shHCK cells showed decreased cell viability (fold change compared to shControl-GFP = 0.55, P<.0001, n=3) combined with an increase in CD71+ expression (fold change compared to shControl-GFP = 3, P<.01, n=3), indicating a delay in erythroid differentiation. As expected, shControl-GFP cells showed a decreased GATA1 expression during erythroid differentiation. Meanwhile, shHCK cells did not modulate GATA1 expression. Interestingly, without any stimulus, HCK knockdown in CD34+ cells significantly decreased apoptosis (AnnexinV+ cells) compared to shControl-GFP (fold change = 0.52, P<.01, n=4). Attempts have been made to overexpress HCK in CD34+ HSC, however more than 80% cells were apoptotic and further assays were not possible. Thus, in HSC, HCK participates of erythroid differentiation and apoptosis signaling. According to the HCK importance on HSC and that SFK inhibitors are undergoing early phase clinical testing, a specific inhibitory activity compound for HCK, named iHCK-37, had been developed by Dr Maurizzio Botta. We tested this compound on primary normal human CD34+ cells originated from healthy donors bone marrow samples and also from cord blood units. The iHCK-37 treatment did not change proliferation, survival and death of these normal CD34+ cells. Conversely, MDS and AML CD34+ cells treated with the same drug exhibited a dose-dependent growth inhibition. Likewise, following iHCK-37 treatment of MDS and AML total bone marrow mononuclear cells, the BFU-E and CFUs colony numbers were significantly decreased compared to untreated cells (vehicle). We also observed a potent in vitro antiproliferative activity of iHCK-37 against a panel of leukemia cell lines, with uM IC50 values in AML (5.0 - 5.8uM) and chronic myeloid leukemia (9.1 - 19.2uM). In addition, the combinatory in vitro treatment of iHCK-37 and 5-Azacitidine (Aza) also demonstrated additive effects relative to either drug alone. Interestingly, iHCK-37 or iHCK-37 plus Aza treatments of dysplastic and leukemia cells enhanced apoptosis and resulted in increased BAX and reduced BCL-XL protein levels. This result could be clinically relevant for MDS, as Aza is the only treatment available for higher-risk MDS, but with low response rates and frequent induced resistance and refractoriness over time. In summary, we herein have shown that HCK mRNA knockdown of normal CD34+ cells resulted in growth inhibition, decreased cell death and reduced erythroid differentiation, suggesting that HCK is essential for normal hematopoiesis. We presume that the deregulation of HCK pathway in leukemic cells might be crucial for MDS and AML pathogenesis. On the other hand, the inhibition of HCK protein activity with a specific inhibitor was able to restore the apoptotic pathways of leukemic cells, acting on cancer cells without alter any signaling of normal cells. Moreover, the specific inhibitor may have antineoplastic effect that can even be additive to current available drugs. Our study adds new insights to the role of HCK in MDS and AML as well as into potential new anticancer treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition

AbstractCancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34+ progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GACK320A allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.

Abstract 4445: Selective inhibitor of nuclear exporter CRM1/XPO1, Selinexor (KPT-330), exhibits remarkable activity against AML leukemia-initiating cells while sparing normal hematopoietic cells

Abstract Current treatments for acute myeloid leukemia (AML) often fail to induce long-term remissions and are toxic to normal tissues, prompting the need to develop new targeted therapies. The frequent disease relapse that is observed in patients with AML is thought to occur because of the inability of the existing drugs to target the self-renewing leukemia-initiating cells (LICs). An attractive new strategy for AML therapy is inhibition of the nuclear export protein exporter 1 (XPO1), or CRM1. XPO1 regulates export of proteins that contain leucine-rich nuclear export signals (NES), including protein adaptors that mediate transport of RNA. XPO1 cargo encompass tumor suppressor proteins, cell cycle regulators, and apoptotic proteins. Recently, small molecule inhibitors of nuclear export (SINE) that inhibit the export function of XPO1 by targeting Cys528 in its NES-binding groove, were developed using an in silico molecular modeling. Selinexor (KPT-330), the orally bioavailable SINE compound, is in Phase 1 and 2 studies in adult patients with AML (NCT01607892 and NCT02088541) and in a Phase 1 study for relapsed childhood ALL and AML initiated in March 2014 (NCT02091245). To define the anti-leukemic activity of selinexor against primary AML blasts and LICs in a clinically relevant setting, we established mouse models of primary human leukemia, or patient-derived xenografts (PDX), in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcγnull (NSG) mice. Mice engrafted with leukemic blasts were treated with either vehicle or selinexor. Selinexor was highly active against blast cells from two of the three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN) and complex karyotype AML (AML-CK1 and AML-CK2)), as evidenced by a reduction in leukemic engraftment in primary mice after 4 weeks of treatment. Secondary transplantation assays indicated that selinexor greatly reduced the frequency of LICs in PDX models derived from all three patients (6- to 430- fold reduction compared to controls), indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs. These findings show that selinexor has potent activity against LICs, even when it has only moderate activity against the bulk AML cell population. Furthermore, preliminary results of combination studies of selinexor with Ara-C, a standard chemotherapeutic agent, demonstrate synergistic effect of the two drugs against LICs in a PDX model of AML-CN. Importantly, 4 weeks of selinexor treatment demonstrated minimal toxicity in mice engrafted with normal human CD34+ hematopoietic cells. These findings demonstrate that inhibition of nuclear export with selinexor overcomes an important obstacle to cure of AML, which is to destroy the very critical LIC compartment while sparing normal hematopoietic cells. Citation Format: Julia Etchin, Bonnie Thi Le, Alla Berezovskaya, Amy S. Conway, Weihsu C. Chen, Alex Kentsis, Marc R. Mansour, Richard M. Stone, Ilene A. Galinsky, Daniel J. DeAngelo, Dilara McCauley, Michael Kauffman, Sharon Shacham, Jean CY Wang, Andrew L. Kung, Thomas Look. Selective inhibitor of nuclear exporter CRM1/XPO1, Selinexor (KPT-330), exhibits remarkable activity against AML leukemia-initiating cells while sparing normal hematopoietic cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4445. doi:10.1158/1538-7445.AM2015-4445

27. Aberrant Expression of the Stem Cell microRNA-126 Induces B Cell Malignancy

MicroRNAs are essential regulators of normal and malignant hematopoiesis. miRNAs are relevant for gene therapy, since they can be exploited to fine-tune the expression profile of vector constructs or to alter viral tropism (GentnerN Chiriaco et al, 2014; Escobar et al, 2014) and described the function of miR-126 in HSC where it regulates the balance between quiescence and self-renewal (Lechman et al, 2012). We here report a novel role for miR-126 in the induction and maintenance of high-grade B cell malignancies. By ectopically expressing miR-126 in transplanted BM cells, we observed that up to 60% of mice (n=71) developed B cell malignancies. LV insertion site (IS) analysis revealed that all tumors were monoclonal. We then tracked back leukemic clone to different hematopoietic lineages prospectively purified from the mice 2-6 months before disease onset. IS sharing between normal lineages and leukemic clone suggests stem or multipotent progenitor cell as origin for most tumors. Importantly, we show that miR-126 is the direct cause of genesis and maintenance of leukemia, since leukemogenesis is abolished when miRNA expression is inhibited by doxycycline (doxy) using a tetracycline-repressible miR-126 cassette, and established symptomatic leukemia completely regresses when miR-126 is switched off by doxy through induction of apoptosis. Transcriptional profiling indicated that miR-126 regulates multiple genes in p53 pathway both in murine blasts and in normal human CD34+ cells. Previous work suggested expression of miR-126 in acute lymphoblastic leukemia (ALL) and germinal center lymphoma. To further establish the relevance of miR-126 in human disease, we measured miR-126 expression in blasts from 16 adult patients with ALL. miR-126 was highly expressed in most studied ALL cases (Phil+: n=11, Phil-: n=5), at similar levels as CD34+ cells. We then down-regulated miR-126 in primary blasts from human B-ALL patients (n=5), and we observed increased apoptosis and impaired engraftment in xenograft models after primary and secondary transplantation (miR-126/KD: n=32 mice; Ctrl: n=37 mice), demonstrating the relevance of miR-126 in human B-ALL. In conclusion, we present a novel spontaneous mouse model for high grade B cell malignancies which are addicted to miR-126 expression, provide insight into the dynamic process of leukemogenesis by clonal IS tracking and unveil key tumor signaling pathways controlled by miR-126. Down-regulation of miR-126 could be exploited as therapeutic strategy in ALL, since it would deplete leukemic cells while expanding normal HSC, two ways to restore normal hematopoieis.

Autocrine TNF-α Signaling in Hematopoietic Stem Cells Promotes Myeloproliferative Disease Progression through Activation of TNFR2

The JAK2V617F mutation is a unifying feature in the majority of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN. In particular, expression of tumor necrosis factor alpha (TNF-α) is increased by constitutive JAK2 kinase activity and imparts a competitive advantage on JAK2V617Fexpressing cells.To identify cell types responsible for increased levels of TNF-α we performed intracellular cytokine staining in leukocytes from myelofibrosis (MF) patients and normal controls. Using a panel of markers to define cellular subsets we found expression of TNF-α was uniformly low in unstimulated cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p<0.005), while expression in mature populations was not different. In a murine model of JAK2V617F driven MPN, where JAK2V617F cells are identified by GFP, TNF-α expression was 3-fold higher in JAK2V617F-expressing HSCs (GFP+) compared to GFP-controls (p<0.005), irrespective of LPS stimulation.To evaluate anti-TNF therapy in MPNs, we treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α. Etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of TNF-α activity in plasma. As TNF-α may activate pro-apoptotic (predominantly through TNF receptor 1, TNFR1) and pro-survival pathways (predominantly through TNF receptor 2, TNFR2), we reasoned that global blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Additionally we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2. Colony assays performed after 3 days in liquid culture with TNFR-blocking antibodies confirmed that interrupting TNFR2-mediated survival signaling resulted in decreased colony formation (61% reduction) while blocking TNFR1 increased colony formation (5% increase). In addition we tested CD34+ cells from MF patient samples with TNFR-blocking antibodies. As seen with the murine MPN cells, colony formation was decreased with the TNFR2 blocking antibody (30% reduction) and increased with the TNFR1 blocking antibody (48% increase) consistent with a differential activation of survival signals by TNF-α in MPN cells. These studies were performed without addition of exogenous TNF-α, which confirms the ability and requirement for these primitive JAK2V617Fcells to utilize TNF-α production to actively support survival.Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, while TNFR1 activation is suppressive. If selective inhibition of TNFR2 shifts the equilibrium toward normal hematopoietic cells, this would support the use of TNFR2 blockade to treat MF and other myeloproliferative neoplasms. Genotyping of human MF and murine MPN colonies cultured with TNF receptor blocking antibodies and experiments in normal human CD34+ cells are ongoing and will be presented. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

Design, Optimization, and Pre-Clinical Evaluation of Direct, Mechanism-Based STAT3 Inhibitors for Treating Myeloid Disorders

We have identified STAT3 as a convergence point for oncogenic signaling in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) lacking BCR-ABL1 kinase domain mutations. In addition, we found that STAT3 activity contributes to disease in other myeloid disorders, including acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). Utilizing TKI-resistant CML as a model system, we identified BP-5-087 as a small molecule inhibitor of STAT3 that reduces STAT3 phosphorylation and nuclear transactivation (Eiring et al. Leukemia, 2014).Binding of BP-5-087 to the STAT3 SH2 domain was initially assessed using fluorescence polarization (FP) assays and high-resolution computational docking simulations. To further validate the binding motif of BP-5-087, we conducted time-resolved electrospray ionization mass spectrometry/hydrogen-deuterium exchange experiments. Fold-change in deuterium uptake was analyzed for 68 STAT3 peptides representing 71% sequence coverage, and mapped onto the crystal structure of STAT3. This analysis precisely defined the binding epitope for BP-5-087 within the STAT3 SH2 domain.We next tested the effects of BP-5-087 in several myeloid malignancies using relevant disease models. (i) CML stem and progenitor cells from TKI-resistant patients without kinase domain mutations were treated with BP-5-087 ex vivo, using short-term liquid culture, clonogenic and LTC-IC assays. BP-5-087 treatment significantly reduced colony formation by CML stem and progenitor cells (p<0.01), with no effect on normal human CD34+ cord blood (CB) cells. (ii) Similarly, BP-5-087 also increased apoptosis and reduced viability (p<0.05) of primary AML blasts treated ex vivo with BP-5-087 for 72 hours in liquid culture. (iii) CD34+ cells from patients with myelofibrosis were also treated with BP-5-087 in clonogenic assays, and similar to CML, BP-5-087 reduced myeloid colony formation, although to a lesser extent.The in vivo activity of BP-5-087 was next evaluated in a murine model of JAK2 V617F-induced MPN. Briefly, Balb/c bone marrow was transduced with JAK2 V617F-GFP, followed by injection into lethally irradiated recipients. After disease induction, mice were treated with BP-5-087 (25 mg/kg) by once-daily oral gavage. No toxicities were observed after 40 days of treatment in BP-5-087-treated mice. While BP-5-087 did not significantly reduce the percentage of GFP+ cells, there was a 41% reduction of spleen weight in BP-5-087-treated mice compared to vehicle-treated controls (p<0.05). Post study analysis revealed BP-5-087 plasma concentrations <1 μM, suggesting that insufficient bioavailability contributed to the modest in vivo effects.To advance the lead optimization of our STAT3 inhibitor series, we instituted a comprehensive screening cascade. We first developed a computational model (quantitative structure-activity relationship, QSAR) to guide and prioritize selection of new inhibitor candidates for synthesis. Compounds are initially ranked using a methanethiosulfonate (MTS)-based cell viability assay in a TKI-resistant, STAT3-dependent CML cell line (AR230R). Inhibition of STAT3 is confirmed using a cell-based STAT3 reporter assay and an in vitro FP-based binding assay. Optimization of potency is balanced by the goals of reducing molecular weight (MW) and calculated LogP (cLogP) compared to BP-5-087 (MW: 694.8; cLogP: 7.3). Compounds with improvements in these categories are then subject to toxicity testing utilizing clonogenic assays with CD34+ CB cells. Non-toxic compounds are evaluated for their pharmacokinetic profile in Balb/c mice and tested for activity in primary samples from CML, AML and MPN patients. These activities have directed us to a lead compound, AM-1-124, which displays significant improvements in potency, MW, cLogP, and in vivo half-life compared to BP-5-087. AM-1-124 had minimal effects in the CB toxicity assay and induced apoptosis in primary AML patient samples at 2-fold lower concentrations than BP-5-087. With AM-1-124 as our current lead compound, we are continuing our iterative evaluation of novel STAT3 inhibitors utilizing our screening cascade. Design and testing of optimized, orally active inhibitors will enable further evaluation of STAT3 as a target in animal models of myeloid leukemia and will justify the clinical development of these compounds for patients in need of new targeted therapies. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.