Growth Of Hematopoietic Progenitors Research Articles

A synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34+ cells

Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type-dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)-mediated very rapid apoptosis occurs within 1 hour in CD34(+) cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34(+) cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34(+) cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34(+) cells, neither exogenous IFN-α nor IFN-β induces rapid apoptosis in CD34(+) cells and neutralization or blocking of type-I IFN receptor does not rescue CD34(+) cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TLR3, signaling triggers poly I:C-induced rapid apoptosis of human CD34(+) cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders.

New Insights on Assessing Intra-Family Selectivity for Jak2 Inhibitors

Abstract 5150Essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) are myeloproliferative disorders (MPDs) characterized by a chronic over-production of cells of one or more blood cell lineages and/or bone marrow fibrosis which may, on occasion, progress to acute myeloid leukemia. The V617F gain of function mutation in the pseudokinase domain of Jak2, which results in constitutive activation of Jak2, is the most frequent mutation associated with MPD. Constitutively activated Jak2 can lead to dysregulated downstream signaling pathways (STAT, MAP kinase, and PI3 kinase) which in turn trigger abnormal growth, survival and differentiation of hematopoietic progenitors. Therefore, inhibition of constitutively activated Jak2 may offer therapeutic potential. Designing a Jak2V617F specific inhibitor encounters challenges due to the lack of enzymatic activity of the pseudokinase domain of Jak2. In lieu of a Jak2V617F mutant selective inhibitor, a highly selective inhibitor of Jak2 is likely an attainable goal.Jak2 is a member of the Jak family of kinases including Jak1, Jak3, and Tyk2. Highly selective Jak2 inhibitors may provide a better safety margin in chronic dosing settings in ET and PV patients since inhibiting other Jak family members could cause side-effects such as immunosuppression. Attaining the desired selectivity of Jak2 inhibition versus the other family members has been challenging and few compounds have been reported to date that have the desired Jak2 selectivity. This can be attributed to the high homology of the ATP binding pocket among Jak family members, but is also hampered by a lack of assays capable of distinguishing the Jak-selectivity profile in a physiologically relevant setting.We compared the potency and selectivity of compounds tested in a pSTAT5 AlphaScreen® assay panel consisting of isogenic Ba/F3 cell lines individually expressing translocated ETS leukemia (TEL) fusions of each Jak-family member (Ba/F3-TEL-Jak) with data from corresponding Jak enzyme assays. Here we report that the selectivity of inhibitor compounds illustrated in enzyme assays did not correlate with the selectivity profile in cell lines due to different shifts in potency for each family member between enzyme and cells (Figure 1). As a consequence the selectivity of compounds for Jak2 against Jak1 observed in enzyme assays may be reduced or reversed in cellular assays. On the other hand, Jak2 selectivity over Jak3 seen in the enzyme assays was conserved in the cellular assay. Thus, we propose that compounds that exhibit greater potency on Jak2 compared to Jak1 in the enzyme assays are needed and should be the main focus of medicinal chemistry efforts in order to attain Jak2 selectivity over Jak1 in a cellular context. [Display omitted] We also compared the potency and selectivity of compounds in the isogenic Ba/F3-TEL-Jak cell lines with data obtained with cytokine stimulated peripheral blood mononuclear cells (PBMCs). The potency and selectivity of compounds in PBMCs are determined by measuring the inhibition of phosphorylation of STAT5 in TPO or GM-CSF stimulated platelets or monocytes (mediated by Jak2) and in IL-2 stimulated lymphocytes (mediated by Jak1 and Jak3). We found that potency correlated well between PBMCs and Ba/F3-TEL-Jak2 cells, and the rank order of compounds based on IC50 values obtained with Ba/F3-TEL-Jak cell lines were conserved well in PBMCs; the compound selectivity profiles derived from the Ba/F3-TEL-Jak cell assays were predictive of Jak2 selectivity profiles obtained in the PBMC assays. Therefore, inclusion of Ba/F3-TEL-Jak pSTAT5 cellular assays may be useful for Jak family inhibitor development. Our results also suggest that relying solely on enzyme potency and selectivity data can be misleading, and that evaluating cellular selectivity in a biologically relevant context may provide a more meaningful understanding of selectivity and lead to the development of more selective Jak2 compounds. Disclosures:Liu:Amgen, Inc: Employment. Yu:Amgen: Employment. Pistillo:Amgen: Employment. Lee:Amgen: Employment. Schenkel:Amgen: Employment. Geuns-Meyer:Amgen: Employment. Archibeque:Amgen: Employment. Sinclair:Amgen: Employment. Emkey:Amgen: Employment. Molineux:Amgen: Employment.

Phase 1 Dose-Ranging Study of Oral Ezatiostat Hydrochloride (Telintra®, TLK199) in Combination with Lenalidomide (Revlimid®) in Patients with Non-Deletion(5q) Low to Intermediate-1 Risk Myelodysplastic Syndrome (MDS)

Abstract 2778 Introduction:Lenalidomide is approved for the treatment of del(5q) MDS in US and Japan. In Low to Intermediate-1 (Int-1) risk non-del(5q) MDS, lenalidomide treatment is less effective with a lower response rate (25%) and shorter response duration [Raza A. et al, Blood, 2008.111,1]. Ezatiostat, a glutathione S-transferase P1-1 (GST P1-1) inhibitor, activates Jun kinase, promoting the growth and maturation of hematopoietic progenitors while inducing apoptosis in malignant cells. Based on the novel mechanism of action, response rates, non-overlapping toxicities, and tolerability observed in a single agent ezatiostat Phase 2 study in MDS, a study of the combination of ezatiostat and lenalidomide was conducted to determine the safety and efficacy of ezatiostat with lenalidomide in non-del(5q) Low to Int-1 risk MDS. Methods: In this multicenter Phase 1 dose-ranging study, ezatiostat was given at a starting dose of 2000 mg in combination with lenalidomide at 10 mg, days 1–21 of a 28-day cycle. In stage 1, 3–6 patients in a standard 3+3 design were treated before escalation to the ezatiostat/lenalidomide 2500/10 mg dose level. Treatment was given until lack of MDS response or unacceptable toxicity. Hematologic improvement-erythroid (HI-E) rates were determined by the MDS International Working Group (IWG; 2006) criteria. Results: Eighteen pts (median age 73 yrs; range 57–82; 72% male), with World Health Organization classifications: 4 refractory anemia (RA), 2 RA with excess blasts-1, 4 refractory cytopenia with multilineage dysplasia (RCMD), 5 RCMD with ring sideroblasts, 2 MDS-unclassified, 1 MDS/myeloproliferative disorder-U were enrolled. Thirteen pts (72%) were Int-1 risk, 5 (28%) Low risk; 4 pts (22%) had abnormal cytogenetics. Twelve RBC transfusion-dependent pts (67%) required a median of 6 units (range 4–10)/8-weeks. Two pts (11%) were platelet transfusion dependent. A total of 67 treatment cycles were given (median 3.5 cycles/pt [range 1–11]) and only 6 cycles (9%) required dose reductions and 8 (12%) dose delays. Two of 6 pts reported DLTs (Grade 3 diarrhea and Grade 3 rash) at 2500/10 mg, with 9 additional pts receiving the recommended combination dose of 2000/10 mg. Eleven of 18 pts were evaluable (4 at 2500/10 mg and 7 at 2000/10 mg), and 3 pts are still on therapy with insufficient treatment duration to be evaluable. The HI-E rate was 43% (3/7; 95% CI, 10%–82%) for pts at the recommended 2000/10 mg dose and 6 pts are continuing therapy at the time of analysis. Three of 8 (38%; 95% CI, 9%–76%) RBC transfusion-dependent evaluable pts achieved transfusion independence including 1 responder who did not respond to prior lenalidomide. In responders, the median increase in hemoglobin level was 3.4 g/dL (from 7.9 g/dL). In 2 of 4 thrombocytopenic pts, a HI-platelet (HI-P) response was observed. A bilineage (HI-E and HI-P) response in 2 of 4 pts with anemia and thrombocytopenia was reported. One RBC and platelet transfusion-dependent pt who had a poor response to prior anti-thymocyte globulin treatment achieved complete RBC and platelet transfusion independence. The combination was generally well tolerated with no unexpected toxicities. Most common treatment-related non-hematologic adverse events (AEs) were Grades 1 and 2 including: fatigue (6%, 28%), swelling (0%, 11%), anorexia (11%, 6%), rash (0%, 6%), skin odor (11%, 6%), nausea (39%, 11%), diarrhea (22%,17%), vomiting (28%,17%), upper abdominal pain (5.6%, 5.6%), and constipation (11%, 0%). Grade 3 events were rash (11%), nausea (6%), diarrhea (17%), and vomiting (6%). Most common hematologic-related AEs were Grades 1 and 2 thrombocytopenia (11%, 6%) and neutropenia (0%, 11%). Grade 3–4 AEs were thrombocytopenia (11%, 17%), neutropenia (17%, 11%), anemia (6%, 6%), and febrile neutropenia (11%, 0%). Conclusions: Ezatiostat is the first GST P1-1 inhibitor to cause clinically significant reductions in RBC and platelet transfusions, including RBC and platelet transfusion independence. Since ezatiostat is non-myelosuppressive, it is a good candidate for combination with lenalidomide and in this study, the combination was well tolerated. Interestingly, ezatiostat may also have the potential to enhance lenalidomide’s efficacy. The recommended doses of this combination regimen for future studies is the ezatiostat/lenalidomide 2000/10 mg. Disclosures:Off Label Use: Lenalidomide was used off-label in patients with non-del5q MDS. Mulford:Celgene: Speakers Bureau. Brown:Telik, Inc.: Employment, Equity Ownership. Meng:Telik, Inc.: Employment, Equity Ownership. Lyons:Incyte: Research Funding; Telik: Research Funding; Alexion: Consultancy, Honoraria; Novartis: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Sekeres:Celgene: Consultancy, Honoraria, Speakers Bureau. Mesa:NS Pharma: Research Funding; Astra Zeneca: Research Funding; SBio: Research Funding; Lilly: Research Funding; Incyte: Research Funding; Celgene: Research Funding.

Cytopenias in Large Granular Lymphocyte Leukemia Are Mediated by Pro-Fibrotic Mesenchymal Stem Cells in the Bone Marrow Niche with Functional Restoration by FGFb

Abstract 932Large Granular Lymphocyte Leukemia (LGLL) is a chronic lymphoproliferative syndrome of clonal mature T or NK cells frequently associated with peripheral cytopenias including neutropenia and anemia. The ability of LGLL cells to lyse pro-erythrocytes, the association with secondary autoimmune disorders, and the chronically activated state of LGLL clones suggest that the disease is secondary to a systemic reactive process. Yet, the mechanism causing severe and chronic cytopenias remains unresolved. Because LGLL cells heavily reside in the bone marrow and the bone marrow microenvironment plays such a large role in supporting hematopoiesis, we hypothesized that constituents of the bone marrow microenvironment may be dysfunctional in LGLL patients, and contribute toward the pathogenesis of LGLL and the devolvement of cytopenias.To address this hypothesis, bone marrow core biopsies, aspirates, and peripheral smears taken from 24 patients diagnosed with LGLL were analyzed retrospectively by three independent pathology reviews. Reticulin and trichrome stains revealed clinically relevant myelofibrosis in 21 patients at the time of diagnosis. Of these, 15 had severe myelofibrosis (MF2-3, semi-quantitative grading). The severity of myelofibrosis correlated with neutropenia, anemia, splenomegaly, secondary autoimmune disorders, and the degree of LGLL bone marrow infiltration. The severity of fibrosis also correlated with novel disease aspects reported here for the first time, including increased pseudo-Pelger-Huet and immature neutrophils in peripheral smears, and increased monocytes in the bone marrow and periphery.Because myelofibrosis is so strongly associated with disease severity, we sought to understand the pathogenesis of medullary fibrosis using primary mesenchymal stem cell (MSC) cultures isolated from bone marrow aspirates of LGLL patients or healthy controls. Patient MSC cultures displayed abnormal morphologies, impaired growth kinetics, and reduced growth potential compared to controls, suggesting that patient MSCs may be prematurely senescent. Microarray analysis showed global gene expression changes as healthy MSCs expand in culture including increased expression of pro-hematopoietic cytokines and autocrine growth factors vital to MSC self-renewal such as basic fibroblast growth factor (FGFb) and leukemia inhibitory factor (LIF). Patient MSC cultures appeared incapable of these gene expression changes, instead maintaining low levels of pro-hematopoietic cytokines, FGFb, and LIF expression, while maintaining heightened collagen expression. Cytokine secretion was confirmed using ELISA, and immunofluorescent staining of collagen on MSC cultures verified abnormally elevated collagen deposition from patient MSCs. In particular, collagen I and collagen III matrices were heavily deposited over patient MSCs reiterating the increased trichrome and reticulin staining seen in patient bone marrow biopsies. In an effort to rescue senescence, exogenous FGFb and LIF were added to patient MSCs. FGFb, but not LIF, completely restored the growth kinetics, growth potential, and morphology of these cells. Moreover, collagen deposition of patient MSCs was comparable to healthy donors after exposure to FGFb.We also investigated the ability of MSC cultures to support the growth of hematopoietic progenitors. Healthy bone marrow mononuclear cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and placed in co-culture with patient or control MSCs. Four days later, cellular division of CD34+ cells was analyzed by CFSE dilution. Control MSCs were able to support high levels of CD34+ cell division after expansion in culture. Conversely, patient MSCs supported only minimal growth of CD34+ cells. This suggests that gene expression changes seen in healthy MSC cultures during expansion may be required to support hematopoiesis, and that these changes are blocked in senescent MSCs in LGLL patients. Collectively, this data implicates aberrant MSCs and collagen production in the pathogenesis of LGLL, and suggests that autocrine growth factors such as FGFb, may represent a novel therapeutic option to rescue pro-hematopoietic behavior and reduce excessive buildup of collagen fibers in the bone marrow microenvironment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Role of MicroRNA-486-5p Overexpression In CML CD34+ Cells In Modulating BCR-ABL Mediated Hematopoietic Stem/Progenitor Cell Transformation and Imatinib Sensitivity

Abstract 1667MicroRNAs are key regulators of gene expression that regulate normal differentiation and contribute to malignant transformation of hematopoietic cells. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. In both normal and CML cells, miR-486 expression level was significantly higher in MEP compared to HSC, GMP and CMP populations. Treatment with Imatinib resulted in reduced expression of miR-486-5p in CML CD34+ cells, suggesting that upregulation of miR-486-5p expression was at least in part BCR-ABL kinase dependent. Consistent with this ectopic expression of BCR-ABL in cord blood CD34+ cells using retroviral vectors resulted in 4.2 fold increase in miR-486-5p expression. miR-486-5p is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is enriched in muscle cells. However, the role of miR-486-5p in normal and leukemic hematopoiesis has not been evaluated. To explore the role of miR-486-5p in growth and differentiation of hematopoietic progenitor cells (HSPC), we first overexpressed hsa-miR-486-5p pre-microRNA in normal CD34+ cells using lentiviral vectors. CB CD34+ cells overexpressing miRNA-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days compared to cells expressing control vectors, with increased numbers of erythroid cells and reduced numbers of myeloid cells. We further investigated the role of miR-486-5p on growth and differentiation of normal and leukemic HSPC by inhibiting miR-486-5p expression using a modified pmiRZip lentivirus vector expressing an anti-miR-486-5p sequence and comparing to cells expressing a control scrambled anti-miRNA sequence. Expression of anti-miR-486-5p resulted in reduced proliferation of normal CD34+ cells (32±10% inhibition) and BCR-ABL transformed CD34+ cells (38±7 % inhibition) with significantly greater inhibition of erythroid compared to myeloid cells. Anti-miR486-5p expression resulted in significantly increased apoptosis of BCR-ABL-transformed CD34+ cells but not normal CD34+ cells (CML CD34+ cells: scramble 11.1±2.4%, anti-miR-486-5p 14.7±1.7 % p=0.02; Normal CD34+ cells: scramble 9.7±5.4%, anti-486-5p 13.4±7.9% p=0.15). Importantly, anti-miR-486-5p significantly enhanced the sensitivity of BCR-ABL transformed CD34+ cells to imatinib-mediated apoptosis [combination of scramble with IM: 17.7±8.1%; anti-miR-486-5p with IM: 26.4±13%]. A search for conserved miR-486-5p target genes in the TargetScan database identified the important hematopoietic negative regulatory factors Foxo1 and Pten amongst the highest ranking targets. Using pMIR-REPORT constructs containing miR-486-5p seed sites within the Foxo1 and Pten 3’-UTR we showed that Foxo1 and Pten are direct targets of miR-486-5p. Expression of anti-miR-486-5p increased Foxo1 and Pten protein expression and decreased active Akt in normal and CML CD34+ cells. Knockdown of Foxo1 using shRNA partly blocked the suppressive effects of anti-miR486-5p on the growth of CD34+ cells. In summary, we have shown that miR-486-5p expression is modulated during hematopoietic differentiation and plays an important role in regulating hematopoietic progenitor growth and differentiation towards the erythroid lineage. We further show that miR-486-5p expression is enhanced in CML CD34+ cells, related at least in part to BCR-ABL kinase activity, and contributes to enhanced progenitor growth and survival. Inhibition of miR-486-5p results in enhanced sensitivity of CML CD34+ cells to IM-induced apoptosis. miR-486-5p effects are mediated at least in part through inhibition of Foxo1 and Pten expression. We conclude that miR-486-5p represents a novel regulatory mechanism that promotes erythroid differentiation in normal hematopoiesis and modulates Bcr-Abl-mediated transformation and tyrosine kinase inhibitor sensitivity in CML progenitors. Disclosures:No relevant conflicts of interest to declare.

α1-adrenergic drugs modulate differentiation and cell death of human erythroleukemia cells through non adrenergic mechanism

Preliminary data showed that α1-adrenergic antagonists induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells. To test the hypothesis whether survival and differentiation of erythroleukemia cells are under control of α1-adrenergic signalling, we examined α1-adrenoceptor expression of erythroleukemia cells and compared the in vitro effects of α-adrenergic antagonists with those of agonists. We discovered that α1-adrenergic agonists suppress both erythroid differentiation and growth of erythroleukemia cells concomitant with lipofuscin accumulation, autophagy and necrotic cell death. α1-adrenergic agonists also inhibit the in vitro growth of physiologic hematopoietic progenitors obtained from umbilical cord blood with high selectivity for the erythroid lineage. Interestingly, the observed effects could not be related to α1-adrenoceptors, even though agonists and antagonists displayed opposing effects regarding cellular growth and differentiation of erythroleukemia cells. Our data suggest that the effects of α1-adrenergic drugs are related to a non-adrenoceptor binding site, controlling the fate of erythroid progenitor cells towards differentiation and cell death. Since the observed effects are not mediated through adrenoceptors, the physiologic relevance of our data remains unclear, so far. Nevertheless, the identification of the still unknown binding site(s) might disclose new insights into regulation of erythroid differentiation and cell death.

Modulation of hematopoiesis through histamine receptor signaling

Histamine is one of the most versatile biogenic amines targeting a variety of cells through extra- and intracellular binding sites and specific receptors, which trigger different signal transduction pathways. It has been associated with cell growth ever since G. Kahlson demonstrated that its synthesis was increased in rapidly growing tissues of plants and animals. He proposed that the newly formed amine, as opposed to its stored counterpart, might play a major role in growth processes. Later on, a number of investigators provided evidence for the contribution of histamine to the expansion of normal and malignant cells, whether of hematopoietic origin or not. These studies have generated conflicting results, revealing growth-promoting as well as inhibitory effects, most likely because the final outcome of exposure to histamine depends on the signaling pathways triggered by distinct receptors and their differential distribution among the target population. The purpose of the present review is to outline our current understanding of the regulatory functions of histamine during growth and differentiation of hematopoietic progenitors, focusing on those mediated through its H4 receptor.

Selective Expansion of CD34+ Cells from Mouse Bone Marrow Cultured on LH/P MP-Coated Plates with Adequate Cytokines

Low-molecular-weight heparin/protamine microparticles (LH/P MPs) serve as carriers for controlled release of heparin-binding cytokines. LH/P MPs were stably coated onto plastic surfaces by drying. The purpose of this study is to evaluate a culture method for selective expansion of CD34+ cells using LH/P MPs as cytokine-binding matrix. Ficoll-purified mouse bone marrow cells (mouse FP-BMCs) containing CD34+ cells were cultured on LH/P MP-coated plates in the presence of stem cell factor (SCF), thrombopoietin (Tpo), and Flt-3 ligand (Flt-3) in hematopoietic progenitor growth medium (HPGM) supplemented with 4% heat-inactivated fetal bovine serum (FBS). After 8 days of culture, the total cell count increased 4.6-fold, and flow cytometry analyses revealed that 23.8% of the initial cells and 57.4% of the expanded cells were CD34 positive. Therefore, CD34+ cells were estimated to have increased 11.0-fold. In contrast, cultured CD34+ cells on uncoated tissue culture plates increased 5.8-fold in an identical medium.

Phase 2 Randomized Multicenter Study of Extended Dosing Schedules of Oral Ezatiostat HCl (Telintra), a Glutathione Analog Prodrug GSTP1-1 Inhibitor, In Low to Intermediate-1 Risk Myelodysplastic Syndrome (MDS)

AbstractAbstract 2910 Introduction:MDS is characterized by defects in hematopoietic cell growth, differentiation and apoptosis. Ezatiostat, a glutathione S-transferase inhibitor, activates Jun kinase, promoting the growth and maturation of hematopoietic progenitors while inducing apoptosis in leukemic blasts. The purpose of this Phase 2 study was to determine the efficacy and safety of ezatiostat on extended dose schedules in MDS. Methods:Patients (pts) with International Prognostic Scoring System Low or Int-1 risk MDS were enrolled. After initial dose-ranging in 14 pts, subsequent pts were randomized to 2 selected dose schedules: 37 pts at 3 gm daily for 2 weeks followed by a 1-week rest period and 36 pts at 2 gm daily for 3 weeks followed by a 1-week rest period. Treatment was given until lack of MDS response or unacceptable toxicity. A logistic regression analysis was conducted to identify significant MDS disease factors associated with hematologic improvement-erythroid (HI-E) rates as determined by the MDS International Working Group (2006) criteria. Results:Seventy-three pts were median age 73 years (range 48–89) 70% male, and with World Health Organization classification as follows: 11 refractory anemia (RA), 15 RA with ring sideroblasts, 5 RA with excess blasts-1, 24 refractory cytopenia with multilineage dysplasia, 9 MDS-unclassified, 3 MDS/Myeloproliferative disorder-U, 4 MDS-del 5q, and 2 Unknown. Fifty pts (68%) were INT-1 risk, 23 (32%) were Low risk; 27 pts (37%) had abnormal karyotypes with 23 (85%) complex/poor prognostic types. RBC transfusion-dependent pts required a median of 6 (range 4–19) U/8 weeks prior to study entry. Prior treatments included: 34 (47%) DNA methyltransferase (DMTI), 28 (38%) lenalidomide, 56 (77%) erythropoietin, and 32 (44%) growth factors. The HI-E response rate was similar for evaluable pts on the 2 schedules, therefore the data were pooled. The overall HI-E rate was 22% (13/60) [95% CI, 12.1%–34.2%]. The median duration of response was 34 weeks. Eleven of 38 (29%) RBC transfusion-dependent pts had transfusion reductions (reduction of 4U/8weeks) with 4 pts (11%) achieving transfusion independence. A 40% HI-E rate (6/15 pts) [95% CI, 16.3%–67.7%], was observed in pts who had prior lenalidomide, but no prior DMTI, with 5/11 pts (45%) achieving significant RBC transfusion reduction and 3/11 pts (27%) achieving transfusion independence. A 28% HI-E rate (5/18 pts) [95% CI, 9.7%–53.5%] was observed in pts who were lenalidomide and DMTI naive, with 4/8 pts (50%) achieving clinically significant RBC transfusion reduction. Sixteen pts had received prior DMTI but no prior lenalidomide treatment, a 0% HI-E rate (95%CI: 0%-20.6%) was reported. Prior DMTI treatment predicts a 6-fold decrease in the odds for HI-E response to ezatiostat (p=0.027). A 19% HI-Neutrophil (HI-N) rate was observed in 4/21 pts with neutropenia (95% CI, 5.4%–41.9%) and a bilineage (HI-E and HI-N) rate of 20% (95% CI, 5.7%–43.7%) in 4/20 pts with anemia and neutropenia. A 3.7% (95% CI, 0.1%–19%) HI-P rate was observed in 1/27 thrombocytopenic pts and a trilineage (HI-E, HI-N, HI-P) rate of 9.1% (95% CI, 0.2%–41.3%) in 1/11 pts with trilineage cytopenia. There were 2 cytogenetic complete responses, 1 with 45X,-Y[4], 46, XY [16] and 1 with del5q. A total of 431 ezatiostat cycles were given, median 4.0 (range 1–14). Only 15 cycles (3.5%) required dose reduction and 37 cycles (8.6%) dose delays. Most common ezatiostat-related adverse events (AEs) were non-hematologic Grade 1 and 2 gastrointestinal (GI) including: nausea (45%, 17%), diarrhea (25%, 8%), and vomiting (30%, 12%). Grade 3 events were: nausea (1%), diarrhea (3%), and vomiting (2%). Prior DMTI treatment was associated with an increased incidence of GI AEs. Pts with prior DMTI use had GI AEs (Grades 1, 2, 3): nausea (51%, 20%, 2%), vomiting (34%, 7%, 2%), diarrhea (22%, 10%, 5%). DMTI-naive pts had fewer GI AEs (Grades 1, 2, 3): nausea (39%, 15%, 0%), vomiting (26%, 15%, 2%), diarrhea (28%, 7%, 2%). Conclusions:Ezatiostat is the first GSTP1-1 inhibitor shown to cause clinically significant reduction in RBC transfusions, including transfusion independence, as well as HI-N and HI-P. Ezatiostat has shown clinically significant efficacy in lenalidomide naive and lenaliomide treated pts. The tolerability and activity profile of ezatiostat may offer an important treatment option for pts with low to INT-1 risk MDS. Disclosures:Raza:Telik, Inc.: Research Funding. Galili:Telik, Inc.: Research Funding. Godwin:Scripps Health: Honoraria. Boccia:Telik, Inc.: Research Funding. Rarick:Telik, Inc.: Research Funding. Meng:Telik, Inc.: Employment, Equity Ownership. Jones:Telik, Inc.: Employment, Equity Ownership. Brown:Telik, Inc.: Employment, Equity Ownership, Patents & Royalties. Young:Telik, Inc.: Employment, Equity Ownership. Sekeres:Celgene Corp: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

In vitro growth of hematopoietic progenitors and stromal bone marrow cells from patients with multiple myeloma

In the present study we have determined the content of hematopoietic and stromal progenitors in multiple myeloma (MM) bone marrow, and assessed their in vitro growth. Marrow cells were obtained from 17 MM patients at the time of diagnosis, and from 6 hematologically normal subjects. When mononuclear cells (MNC) from MM marrow were cultured, reduced numbers of hematopoietic progenitors were detected and their growth in long-term cultures was deficient, as compared to cultures of normal cells. When cell fractions enriched for CD34 + Lin − cells were obtained, the levels of hematopoietic progenitors from MM marrow were within the normal range, and so was their growth kinetics in liquid suspension cultures. The levels of fibroblast progenitors in MM were not statistically different from those in normal marrow; however, their proliferation potential was significantly reduced. Conditioned media from MM-derived MNC and stroma cells contained factors that inhibited normal progenitor cell growth. Our observations suggest that hematopoietic progenitors in MM marrow are intrinsically normal; however, their growth in LTMC may be hampered by the presence of abnormal accessory and stroma cells. These results suggest that besides its role in the generation of osteolytic lesions and the expansion of the myeloma clone, the marrow microenvironment in MM may have a negative effect on hematopoiesis.

The mTOR Inhibitor, RAD001, Inhibits the Growth of Cells From Patients with Myeloproliferative Neoplasms.

Abstract 2914Poster Board II-890The JAK2 and MPL somatic mutations in chronic myeloproliferative neoplasms (MPN) result in constitutive activation of JAK2/STAT pathway; in-vitro JAK2 inhibitors reduced the proliferation of JAK2V617F mutant cells while in early clinical trials in patients (pts) with myelofibrosis resulted in clinical improvement, although the effects on the burden of JAK2-mutant clone were less impressive than anticipated. On the other hand, little is known about other signalling pathways, such as PI3K/Akt and ERK, that also resulted activated in cells harboring the JAK2V617F mutation. In this study we focused on the mammalian target of rapamycin (mTOR), a downstream target of key signalling molecules including Akt, PI3K, GTPase Ras and Rheb; mTOR is often abnormally activated in cancer cells and represents an attractive target for anticancer therapy, as shown by the effects of mTOR inhibitors in solid tumors and lymphomas. First, we documented constitutive phosphorylation of mTOR and of the eukaryotic initiation factor 4E-binding protein 1 (4EBP1), a downstream target of mTOR, in Ba/F3 cells transduced with JAK2 V617F (kindly provided by R. Skoda); conversely, phosphorylation of both mTOR and 4EBP1 in wild-type (wt) Ba/F3 cells was largely dependent on exposure to IL3. Then, to assess the effects of mTOR inhibition on the growth of JAK2V617F mutant cells we used the specific mTOR inhibitor RAD001. We observed that day-14 clonogenic growth of JAK2V617F mutant cells HEL, UKE-1 (a gift of W. Fiedler) and SET2 was potently inhibited by RAD001 compared to BCR-ABL-pos K562 cells: IC50 values were 85±30 nM, 60±20 nM, 330±50nM and 12,000±340 nM, respectively (all P<.001). Also the IC50 of JAK2V617Fpos Ba/F3 cells (2.0±3.0 nM) was significantly lower than that of wt Ba/F3 cells (1,990±500 nM). RAD001 caused an accumulation of HEL cells in the G0 phase of the cell cycle from 60.5±11.5% to 71.8±10.3%, and reduced cells in the S-phase from 19.1±5.9% to 10.7±1.6%. The proportion of apoptotic HEL cells increased from 8.9±1.8% to 40.8±8.3% after 48 hr with 100 nM RAD001. We then evaluated whether inhibition of mTOR could affect the growth of hematopoietic progenitors from MPN pts by using serial dilutions of the drug added to cytokine-supplemented clonogenic cultures. Results shown in the Table demonstrated that RAD001 efficiently prevented the growth of clonogenic progenitors from Polycythemia Vera (PV) or Primary Myelofibrosis (PMF) pts compared to controls (Ctrl) (P<.01 for all). The proportion of wt and JAK2V617F-mutant colonies was measured in cytokine supplemented cultures of 5 pts by single-colony genotyping ; we found that JAK2V617Fpos colonies decreased of a median value of 30% (range 10-50%) in the presence of RAD001. Finally, we found that the sensitivity to RAD001 of erythropoietin-independent erythroid progenitors from PV pts was significantly greater for JAK2V617F homozygous (IC50= 3.0±2.0 nM) than heterozygous (33.0±5.0 nM; P<.01) progenitors. In HEL and UKE1 cells, and in JAK2V617Fpos Ba/F3, exposed to RAD001 the phosphorylation of mTOR and 4EBP1 was efficiently prevented, as expected; however, we also found that RAD001 caused a significant reduction of STAT5 phosphorylation (Western Blotting and FACS analysis). To confirm that reduction of pSTAT5 was actually dependent on mTOR inhibition, we transduced (Amaxa) HEL cells with siRNA against mTOR; we observed a parallel reduction of 4EBP1 and STAT5 phosphorylation at 48-72 hr that followed a >50% decrease of mTOR mRNA and protein content. In conclusion, we observed constitutive activation of mTOR pathway associated with V617F mutation and provided evidence of a significant sensitivity to RAD001 of JAK2V617F mutated cell lines (HEL, Ba/F3) and hematopoietic progenitors from MPN patients. These effects may be largely related to a previously uncharacterized effect on STAT5 phosphorylation by member(s) of the mTOR pathway, that is inhibited by RAD001. Overall, these data suggest an involvement of mTOR in MPN pathogenesis, and anticipate a possible relevance for treatment. A phase I/II trial with RAD001 in pts with myelofibrosis is currently ongoing in Italian Institutions (AM Vannucchi et al., ASH 2009, abstract submitted).RAD001 IC50 (nM)BFU-ECFU-GMCFU-MkCtrl (n=5)360 ± 10366 ± 7488 ± 8PV (n=5)4 ± 248 ± 230 ± 4PMF (n=5)4 ± 140 ± 730 ± 7 Disclosures:No relevant conflicts of interest to declare.

Tanespimycin Reverses Bortezomib-Induced Inhibition of Granulopoiesis.

Abstract 3846Poster Board III-782 INTRODUCTIONTanespimycin, an Hsp90 inhibitor, is in phase 3 clinical trials with bortezomib for the treatment of multiple myeloma (MM). Neutropenia and thrombocytopenia are commonly observed during bortezomib treatment in patients with MM. However, in a phase 1/2 study of tanespimycin + bortezomib in patients with MM, the incidence and severity of neutropenia was low (Richardson ASCO 2009). Here we present the in vitro effects of tanespimycin and bortezomib on hematopoiesis and granulopoiesis in cell culture systems using mononuclear cells from healthy subjects and cryopreserved hematopoietic stem cells. METHODS: Mononuclear cells were separated from fresh whole healthy human bone marrow cells and cultured for up to 14 days in suspension in the presence of tanespimycin (1 pM–300 μM) and bortezomib (1 nM–300 μM). Committed hematopoietic progenitor growth (CFC-GEMM [granulocyte/erythroid/macrophage/megakaryocyte], BFU-E [erythroid], GM-CFC [granulocyte/macrophage], MkCFC [megakaryocyte]) of cultured cells was assessed using intracellular ATP bioluminescence in the HemoGenix HALO® system, which couples specific hematopoietic lineage growth conditions with a surrogate for cell count. In addition, cryopreserved healthy human hematopoietic stem cells were cultured in semisolid methylcellulose and colonies (CFC-GEMM, CFU-E, BFU-E) were microscopically enumerated. RESULTS: When bortezomib and tanespimycin were given alone in suspension cultures, IC50 values for bortezomib-induced cytotoxicity and tanespimycin-induced cytotoxicity were approximately 1 nM to 2 nM and 100 nM, respectively, for all hematopoietic lineages. In semisolid methylcellulose cultures, 1 nM to 10 nM bortezomib induced a concentration-dependent inhibition of granulocyte-macrophage colony formation from 2% to 46% and of erythroid colony formation from 8% to 35%. While tanespimycin alone at a concentration of 10 nM had little or no effect on erythropoiesis or granulocytopoiesis, when bortezomib (1 nM or 3 nM) and tanespimycin (10 nM) were cocultured, tanespimycin completely reversed bortezomib-induced inhibition of erythropoiesis and granulopoiesis. More severe erythropoietic inhibition at 10 nM bortezomib was not reversed by tanespimycin, while granulopoietic inhibition was mitigated approximately 23%. At concentrations of melphalan (1 μM) and camptothecin (3 nM) that inhibit in vitro hematopoiesis approximately 50% and 90%, respectively, 10 nM tanespimycin had no effect. CONCLUSION: At clinically relevant drug concentrations in hematopoietic cultures, tanespimycin reverses bortezomib-induced inhibition of granulopoiesis, consistent with the low frequency of neutropenia observed in clinical trials of tanespimycin + bortezomib. This suggests tanespimycin may prevent bortezomib-induced apoptosis of granulocyte-macrophage progenitors. Disclosures:Flint:Bristol-Myers Squibb: Employment, Equity Ownership. Oberdoerster:Bristol-Myers Squibb: Employment. Price:Bristol-Myers Squibb: Employment. Foster:Bristol-Myers Squibb: Employment. Gemzik:Bristol-Myers Squibb: Employment. Berman:Bristol-Myers Squibb: Employment.

IL-3 and Oncogenic Abl Regulate the Myeloblast Transcriptome by Altering mRNA Stability

The growth factor interleukin-3 (IL-3) promotes the survival and growth of multipotent hematopoietic progenitors and stimulates myelopoiesis. It has also been reported to oppose terminal granulopoiesis and to support leukemic cell growth through autocrine or paracrine mechanisms. The degree to which IL-3 acts at the posttranscriptional level is largely unknown. We have conducted global mRNA decay profiling and bioinformatic analyses in 32Dcl3 myeloblasts indicating that IL-3 caused immediate early stabilization of hundreds of transcripts in pathways relevant to myeloblast function. Stabilized transcripts were enriched for AU-Response elements (AREs), and an ARE-containing domain from the interleukin-6 (IL-6) 3′-UTR rendered a heterologous gene responsive to IL-3-mediated transcript stabilization. Many IL-3-stabilized transcripts had been associated with leukemic transformation. Deregulated Abl kinase shared with IL-3 the ability to delay turnover of transcripts involved in proliferation or differentiation blockade, relying, in part, on signaling through the Mek/Erk pathway. These findings support a model of IL-3 action through mRNA stability control and suggest that aberrant stabilization of an mRNA network linked to IL-3 contributes to leukemic cell growth.

Direct Confirmation of Quiescent Property of AML Stem Cells by Showing Markedly Decreased Plating Efficiency in Single Leukemic Stem Cell Culture

Cancer stem cell, recently discovered to exist in colon cancer and brain tumor, is resistant to anti-cancer drugs and radiotherapy, demanding the development of new drugs and treatment strategies targeting tumor stem cells. Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). Various anti-cancer medicines, particularly anti-proliferative agents, have been ineffective in treating LSC due to its slower division process and longer interphase compared to normal stem cell and hematopoietic cell. This study comparatively examined growth and proliferation capacity (plating efficiency) of clonogenic hematopoietic progenitors and LSC from healthy donors and AML patients using single cell sorting and culture system (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA). A total of 384 normal hematopoietic stem cells (CD34+CD38+/CD38−) were obtained from peripheral bloods and cord bloods donated by four donors using single cell sorter, and individual single cells were cultured in 96-well plates with each well containing 100ul of serum media, 100ng/ml of stem cell factor, 100ng/ml of Flt-3, 100ng/ml of thrombopoietin and 50ng/ml of G-CSF for five days. 768 single LSC (CD34+CD38−) and 384 single CD34+CD38+ cells were obtained from three AML patients. Growth and proliferation capacities of normal hematopoietic stem cell and LSC were determined in terms of plating efficiency (number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Plating efficiency of individual normal single hematopoietic stem cells varied between samples. Eighty eight out of 192 single stem cells originated from cord blood grew into more than two cells, yielding plating efficiency of 45.8% and cells from the peripheral blood of two healthy donors 30.2% (58/192). In contrast, single LSC originated from the AML patients showed significantly lower plating efficiency with 14.6% (42/288), 3.6% (7/192) and 8.0% (23/288). These results directly confirmed quiescent and slowly dividing properties of LSC. In addition, plating efficiency of normal hematopoietic stem cells was shown to vary between their originating locations in healthy donors.

The Polyamide-Chlorambucil Conjugate 1R-Chl Effectively Inhibits Proliferation and Induces Apoptosis in CML Progenitor Cells

Imatinib mesylate (IM) is highly effective in the treatment of chronic myelogenous leukemia (CML). However, resistance of malignant progenitors to elimination by IM remains a clinical challenge and new strategies to improve elimination of residual primitive CML cells are required. The polyamide-chlorambucil conjugate 1R-Chl has been developed with the rationale that coupling to a polyamide will increase the DNA sequence specificity of the alkylating agent, and decrease unwanted side effects while retaining the ability to kill cancer cells. In previous studies 1R-Chl was shown to inhibit growth of K562 CML cells in vitro and in vivo (Mol Cancer Ther. 2008; 7:769). Histone H4c, which is highly expressed in a wide range of cancer cells, was identified as a gene target of 1R-Chl. H4c downregulation was associated with chromatin decondensation, possibly exposing otherwise hidden 1R-Chl binding sites. The purpose of the current studies was to investigate whether 1R-Chl could effectively induce apoptosis and inhibit growth of primary CML hematopoietic progenitors. CD34+ progenitors obtained from untreated CML patients and from healthy donors were cultured for 96 hours in growth factor supplemented medium in a range of concentrations of 1R-Chl (0–1μM), its inactive stereoisomer 1S-Chl (0–1μM), and with 5μM IM for comparison. Cells were labeled with CFSE prior to culture and with Annexin-V at culmination of culture to allow flow cytometry assessment of the effects of drug exposure on cell proliferation and apoptosis. In addition CD34+ cells incubated under the same conditions were plated in methylcellulose progenitor culture to assess effects on colony forming cell (CFC) growth. 1R-Chl treatment resulted in significantly increased apoptosis of CML CD34+ cells (from 5.0±1.2% [control] to 41.2±8.0% ([1μM 1R-Chl], n=6, p=001). In contrast, 1S-Chl (1μM) or IM (5μM) treatment did not significantly increase CML CD34+ cell apoptosis. Importantly 1R-Chl, 1S-Chl and IM treatment did not significantly increase apoptosis in normal CD34+ cells (n=3). Combined treatment with 1R-Chl and IM did not result in increased apoptosis of CML CD34+ cells compared to 1R-Chl alone (n=3). Analysis of CFSE fluorescence indicated that 1R-Chl treatment induced dose dependent reduction in proliferation of CML CD34+ progenitors (proliferation relative to untreated cells of 0.39±0.1 with 1μM 1R-Chl, n=4–6, p=.01) with an IC50 value of 0.4μM. The antiproliferative effects of 1R-Chl were significantly greater than those of 1S-Chl (0.6±0.2, n=4–6, p=.02) and IM (0.6±0.1, n=4–6, p=.03). Minor antiproliferative effects were observed in normal controls (0.8±0.1, n=3, p=.0005 [1μM 1R-Chl]; 0.9±0.1, n=3, p=.008 [1μM 1S-Chl] and 0.98±0.1, n=3, p=NS [5μM IM]). The combination of IM and 1R-Chl did not result in further reduction in proliferation of CML CD34+ cells compared to 1R-Chl alone (n=3). A trend towards an increased proportion of undivided cells was observed in response to 1R-Chl, 1S-Chl and IM treatment (from 1.5±0.5% [control] to 19.1±8.7%, 4.4±1.9%, and 11.3±6.7%, n=4–6, respectively). CML CFC growth was suppressed by 96.0±1.5% by 1μM 1R-Chl (n=4–5, p<.001) with an IC50 value of 0.25μM. In comparison, 1S-Chl did not significantly inhibit CML CFC growth and IM suppressed CFC CML growth by 76.1±8.9% (n=4–5, p<.001). 1R-Chl (1μM) suppressed normal CFC growth by 59.3±13.9% (n=3, p=.005, IC50=0.88μM), whereas 1S-Chl and IM did not significantly inhibit normal CFC growth. In summary we observed that the alkylating agent-polyamide conjugate 1R-Chl exerted significant and selective pro-apoptotic and antiproliferative effects on CML CD34+ progenitors. The pro-apoptotic effects of 1R-Chl were more potent than those observed with IM, although as with IM treatment, non-dividing CML progenitors were not eliminated. We conclude that 1R-Chl demonstrates considerable activity against primary CML progenitors and that further studies of its potential role in CML therapy are warranted.

IL-3 Coordination of the Myeloblast Transcriptome by Modulating mRNA Stability

The growth factor interleukin-3 (IL-3) promotes the survival and growth of multipotent hematopoietic progenitors and stimulates myelopoiesis. It has also been reported to oppose terminal granulopoiesis and to support leukemic cell growth through autocrine or paracrine mechanisms. While most investigations of the action of hematopoietic cytokines such as IL-3 have focused on cytokine signal transduction leading to transcriptional activation, IL3 has been reported to downregulate proapoptotic Bim in factor-dependent lymphocytes (Matsui et. al., Mol. Cell, 2007). We hypothesized that IL-3 could support the myeloblast phenotype and oppose differentiation by stabilizing transcripts involved in proliferation or differentiation blockade. Kinetic microarray analysis of actinomycin chase experiments in IL-3-dependent 32Dcl3 myeloblasts demonstrated that IL-3 caused stabilization of hundreds of transcripts in pathways relevant to myeloblast function. Control of transcript decay by IL-3 did not require new transcription. Among the most IL-3 stabilized transcripts, 8 out of the top 22 gene ontology categories at a corrected significance level <0.001 were related to cell cycling, and 5 were related to RNA processing. Northern Blotting validated stabilization of transcripts by IL-3, including transcripts associated with proliferation and leukemic transformation (pik3cd, myb, pim-1), hematopoietic development (cited2), differentiation control (cdkn1a) and RNA processing (BRF1, BRF2). A domain was localized in the 3′-utr of IL-6 that mediated IL-3 responsiveness. Proteins known to modulate mRNA stability bound to this domain and to other IL-3 controlled transcripts. The v-Abl oncoprotein (that can support cytokine-independent proliferation of 32Dcl3 myeloblasts) was also shown to stabilize several of the IL-3 targeted transcripts. These findings support a new model of IL-3 action through coordinated mRNA stability control and suggest that leukemic oncoproteins can hijack the ability of IL3 to regulate the decay rate of critical transcripts in myeloblasts.

Prolonged continuous in vitro human platelet production using three-dimensional scaffolds

Methods producing human platelets using growth on plastic, on feeder layers, or in suspension have been described. We hypothesized that growth of hematopoietic progenitors in a three-dimensional (3D) scaffold would enhance platelet production sans feeder layer. We grew CD34 positively selected human cord blood cells in surgical-grade woven polyester fabric or purpose-built hydrogel scaffolds using a cocktail of cytokines. We found production of functional platelets over 10 days with two-dimensional (2D), 24 days with 3D scaffolds in wells, and more than 32 days in a single-pass 3D perfusion bioreactor system. Platelet numbers produced daily were higher in 3D than 2D, and much higher in the 3D perfusion bioreactor system. Platelet output increased in hydrogel scaffolds coated with thrombopoietin and/or fibronectin, although this effect was largely obviated with markedly increased production caused by changes in added cytokines. In response to thrombin, the platelets produced aggregated and displayed increased surface CD62 and CD63. Use of 3D scaffolds, especially in a bioreactor-maintained milieu, may allow construction of devices for clinical platelet production without cellular feeder layers.

Cytokine-immobilized microparticle-coated plates for culturing hematopoietic progenitor cells

The purpose of this study was to provide a culture method for an effective expansion of human CD 34 positive hematopoietic progenitor cells (CD 34 (+) HCs) utilizing low molecular weight heparin/protamine microparticles (LH/P MPs) which can be stably coated onto plastic surfaces and cytokines. CD 34 (+) HCs optimally proliferated on LH/P MP-coated plates in the presence of stem cell factor (SCF; 5 ng/ml), thrombopoietin (Tpo; 10 ng/ml), and Flt-3 ligand (Flt-3; 10 ng/ml) in hematopoietic progenitor growth medium (HPGM). After 6 days, the total cells expanded 16.5-fold. Those cytokines were shown to be partially immobilized on the LH/P MP-coated plates, and the immobilized cytokines were gradually released into the medium with half releasing time of 3–4 days. Since flow cytometry analyses revealed that 90% of initial cells and 44.5% of expanded cells were CD 34 positive, CD 34 (+) HCs were estimated to have increased 8.0-fold after 6 days, and to have increased to over 31.9-fold after 12 days. In contrast, cultured CD 34 (+) HCs on non-coated tissue culture plates increased only 2.9-fold in the identical medium after 6 days, and only 5.2-fold after 12 days.

Inhibition of the TGF-β receptor I kinase promotes hematopoiesis in MDS

AbstractMDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of transforming growth factor–β (TGF-β) receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34+ cells, providing direct evidence of overactivation of TGF-β pathway in this disease. Suppression of the TGF-β signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-β–mediated gene activation in BM stromal cells, and reverses TGF-β–mediated cell-cycle arrest in BM CD34+ cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-β1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-β signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS.

Antiproliferative and Apoptotic Effects of Two New Pd(II) Methylsarcosinedithiocarbamate Derivatives on Human Acute Myeloid Leukemia Cells In Vitro

[Pd(MSDT)Cl]n palladium, chloro[methyl N-(dithiocarboxy-kS,kS')-N-methylglycinate], and [Pd(MSDT) Br]n palladium, bromo[methyl N-(dithiocarboxy-kS,kS')-N-methylglycinate], palladium (Pd)(II) derivatives are two newly synthesized Pd(II) derivatives of methylsarcosinedithiocarbamate (MSDT), containing a sulfur chelating ligand that is able to strongly bind the metal center, so preventing interactions with sulfur-containing enzymes. In fact, these reactions are believed to be responsible for the nephrotoxicity induced by platinum (II)-based drugs. Their activity has been evaluated in a panel of acute myeloid leukemia (AML) cell lines representing different French-American-British (FAB) subtypes and in the Philadelphia (Ph)-positive cell line K-562 and compared to cisplatin. Both compounds suppressed, in a dose-dependent manner, colony formation in methylcellulose with ID50 values comparable to those of the reference drug cisplatin, excluding the ML-3 cell line (ID50 10-fold lower than cisplatin). Exposure of HL-60, ML-3, NB-4, and THP-1 cell lines to a cytotoxic concentration of [Pd(MSDT)Br]n (5 microM) determined: downregulation of the antiapoptotic molecule Bcl-2, upregulation of the proapoptotic molecule Bax; apoptosis induction, as evaluated by APO2.7 and annexin V staining; mitochondrial membrane permeabilization; and DNA fragmentation. In ML-3 cells the Pd(II) complexes were more active than cisplatin in apoptosis induction. Finally, [Pd(MSDT)Br]n showed an inhibitory effect on clonogenic growth of hematopoietic progenitors (CFU-GM, CFU-GEMM, and BFU-E) with both ID50 and ID90 comparable to those of cisplatin. Remarkably, the Pd(II) complex was more potent in inhibiting the clonogenic growth of the less differentiated AML cell lines KG-1a, HL-60, NB-4, ML-3, and THP-1 (ID50 ranging from 0.02 +/- 0.001 to 0.52 +/- 0.04 microM), compared to normal hematopoietic progenitors (ID50 of 2.1 +/- 0.1, 3.8 +/- 0.4, and 2.5 +/- 0.2 microM) for CFU-GEMM, BFU-E, and CFU-GM, respectively). These data suggest that leukemic cells of myelomonoblast lineage might represent a preferential target for its cytotoxic activity compared to normal committed hemopoietic progenitor cells. Altogether, our results indicate that these new Pd(II) dithiocarbamate derivatives might represent novel potentially active drugs for the management of some selected myeloid leukemia strains, able to conjugate cytostatic and apoptotic activity with reduced toxicity.