Growth Of Hematopoietic Progenitor Cells Research Articles

Abstract 1117: Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a player in hematopoiesis and AML

Abstract Background: Over 15 genome-wide association studies (GWASs) have identified a narrow region in chromosome 6q23.3 as a major hematopoietic quantitative trait locus (QTL) for many hematologic traits. The associated phenotypes have yet to be fully explained by genes in the region, including MYB. We noted a newly annotated microRNA within the QTL, miR-3662, and sought to characterize its role in the QTL by exploring its function in normal and malignant hematopoiesis, specifically acute myeloid leukemia (AML). Results: To assess the association between the QTL-defining SNPs and miR-3662 abundance, we genotyped 10 GWAS SNPs in 200 blood samples from non-leukemic individuals. The risk alleles of all 10 SNPs associated with higher miR-3662 abundance. Homozygosity for rs66650371, a three base pair (bp) deletion, showed the strongest association (P<.0001). Reinserting the three bp in cells homozygous for rs66650371 with CRISPR/Cas9 technology lowered miR-3662 abundance by 20%, suggesting rs66650371 directly affects miR-3662 transcription. MiR-3662 was nearly absent in primitive hematopoietic progenitor cells (HPCs) cells, whereafter its abundance increased throughout differentiation. MiR-3662 overexpression increased cell growth and colony formation of HPCs, particularly of the erythroid lineage. To explore the role of miR-3662 in malignant hematopoiesis, we overexpressed miR-3662 in AML cell lines (Kasumi1, KG1a, MV4-11) and primary AML patient blasts (n = 12). Overexpression of miR-3662 reduced cell growth and viability in vitro, and significantly reduced tumor size in a murine xenograft model (scramble vs. miR-3662; n = 9 mice/group). We used targeted RNA sequencing to identify differentially expressed genes between forced miR-3662-expresssing- and control AML patient blasts using a panel of 361 candidate genes (TruSeq RNA platform, Illumina). IKBKB was identified as a promising target, and direct regulation of IKBKB's 3′-UTR by miR-3662 was confirmed via luciferase assay. MiR-3662 overexpression lowered IKBKB mRNA and protein expression in both HPCs and AML patient blasts. As IKBKB promotes activation and nuclear localization of NF-κB, overexpression of miR-3662 led to decreased NF-κB transactivation potential via luciferase assay. Confocal microscopy of HPCs and AML cells, as well as subcellular fraction immunoblots, revealed decreased NF-κB nuclear localization upon miR-3662 overexpression. Re-introduction of IKBKB partly rescued miR-3662's phenotype by increasing nuclear localization of NF-κB, decreasing growth of HPCs, and increasing viability of AML cells. Conclusion: The risk alleles of the 6q23.3 region associated with higher miR-3662 abundance, and miR-3662 abundance was functionally linked to rs66650371. We characterized miR-3662 as an agent in normal and malignant hematopoiesis, and demonstrated that miR-3662 reduces NF-κB activity by targeting IKBKB. Citation Format: Sophia E. Maharry, Christopher J. Walker, Sandya Liyanarachchi, Sujay Mehta, James S. Blachly, Denis C. Guttridge, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a player in hematopoiesis and AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1117.

Enhanced Ex Vivo Expansion of Human Hematopoietic Progenitors on Native and Spin Coated Acellular Matrices Prepared from Bone Marrow Stromal Cells.

The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion.

The Contribution of Bone Marrow Stromal Cells to Leukemia Evolution: Differential Support of Leukemia Cell Growth at Diagnosis and Remission

Introduction: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of the hematopoietic progenitor cells characterized by excessive proliferation of early progenitor cells in the bone marrow (BM) and is hence associated with a variable prognosis. BM niches are specialized types of microenvironment that actively support the growth, maturation and maintenance of hematopoietic stem and progenitor cells. They include a complex network of extracellular matrix proteins, soluble growth factors, and cytokines. Little is known about the leukemic microenvironment or its clinical relevance to AML initiation and propagation. The aim of the study was to define the role of stromal cells in support of leukemic cell growth in active disease as compared to the remission (Rm) state and normal stoma. Thus, we have investigated the expression of various cytokines secreted by stromal cells and involved in hematopoietic progenitor maintenance and proliferation at disease diagnosis (Dx) and Rm. Additionally, we have characterized the growth pattern of leukemic cells on various stroma cells from different sources and disease stages.Methods: Leukemic and stromal cells from BM of 4 patients with AML were collected at Dx. Stromal cells were also collected in Rm. Leukemic cells were enriched for CD34+ using magnetic beads. Patients' own stroma (POS) obtained both at Dx and Rm and the HS-5 cell line (control) were seeded on gelatin-treated plates and were grown with MesenCult medium for 42 days. Morphologic and growth rate changes were monitored every week using inverted microscopy X20. Upon adherence of 40-50% of the stromal cells, CD34+ enriched leukemic cells derived at Dx were seeded on top of POS from both Dx and Rm and were grown together for additional 2 weeks. Co-cultures were harvested and RNA was extracted followed by RT-PCR to amplify the following hematopoietic regulating genes: hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and CXCL12. The results were expressed as arbitrary units normalized to GAPDH gene expression.Results: Expression of CXCL12 and HGF was elevated in a co-culture of leukemic cells and POS obtained at Dx compared to POS from Rm in all 4 patients. Additionally, the co-culture of leukemic cells and POS from Rm showed a higher expression of CXCL12 and HGF as compared to POS alone derived in Rm (Fig. 1). Mean values of cytokine expression were 37.33, 26.44 and 38.62 at Dx and 29.83, 25.55 and 24.55 in Rm for HGF, FGF and CXCL12, respectively. Additionally, a preferential growth advantage of colonies derived from single leukemic cells was observed when these cells were seeded on POS from Dx and relapse (Rx) compared to Rm (Fig. 2A). Moreover, colonies grew mainly in a co-culture with POS while no growth was noted on healthy stromal cells or stroma derived from a patient with myeloma (Fig. 2A) or another AML patient (Fig. 2B).Conclusions: Co-culture of AML cells and POS derived at Dx show a higher expression of CXCL12 and HGF compared to POS obtained in Rm, suggesting favorable microenvironment conditions supporting AML initiation and maintenance. A synergistic effect on expression of CXCL12 and HGF was observed in a co-culture of POS with leukemic cells as compared to POS or cell-line alone. Leukemic cells show preferential growth on POS and no growth on various malignant and normal stromal cells. Leukemic cell growth was diminished in Rm compared to Dx and Rx. Studying a larger number of patients as well as investigating the specific interaction of AML cells with stromal cells derived from various disease stages is needed to confirm the above results. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Novel Small Molecule FLT3 Inhibitors for the Treatment of FLT3-ITD AML

FMS-like Tyrosine Kinase 3 (FLT3) is a receptor tyrosine kinase that promotes growth and survival of hematopoietic stem and progenitor cells. Mutations in the gene encoding FLT3 are found in 20-30% of acute myeloid leukemias (AML), with approximately 25% of all AMLs containing an internal tandem duplication (ITD) in the juxtamembranal region. FLT3-ITD AML is associated with poor prognosis, with a 5 year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. As such, FLT3 inhibitors have been developed to treat FLT-ITD AML. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming. A Phase II study of quizartinib (AC220) showed few complete remissions in AML patients treated with AC220 as a single agent. Additionally, patients who relapse after FLT3 inhibitor treatment can develop FLT3-inhibitor resistant clones, many of which acquire a D835Y substitution. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse for FLT3-ITD AML patients.Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. After initial biochemical and functional analysis, NCGC-2327 emerged as our lead compound, and further optimized with improved solubility, stability, and permeability properties suitable for in vivo utility (NCGC-1481). Both compounds have excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 <5.08 x 10-10 M). AC220, NCGC-2327, and NCGC-1481 treatment of primary human FLT3-ITD-mutant AML cells show EC50 at subnanomolar concentrations (0.5 nM, 0.4 nM, and 0.1 nM respectively) as determined by CellTiter Glo proliferation assays. To ensure that our compounds were selectively effective against FLT3-ITD-mutant AML, primary human NRas-mutant AML cells treated with the inhibitors revealed an EC50 outside of the tested range (>30 µM). To assess the ability of the compounds to induce apoptosis, we treated FLT3-ITD-mutant AML cells with AC220, NCGC-2327, NCGC-1481, or Crenolanib (a FLT3 inhibitor that can inhibit FLT3-ITD-D835Y) for 72 hours. NCGC-148-treated cells showed the greatest levels of apoptosis (AnnexinV+) as compared to Crenolanib, AC220, and NCGC-2327 (P< 0.01). Consistent with the viability assays, NCGC-1481 treatment showed the greatest inhibition of leukemic progenitor function in methylcellulose (Vehicle: 189 ± 39, Crenolanib: 151 ± 32, AC220: 36 ± 3, NCGC-2327: 22 ± 3, and NCGC-1481: 3 ± 2) (P<0.01).Relapse and resistance is a primary concern for patients treated with AC220, therefore we investigated leukemic subclonal resistance in vitro after treatment with Crenolanib, AC220, NCGC-2327, or NCGC-1481. FLT3-ITD-mutant AML cells were treated for 72 hours, washed and then allowed to recover in the absence of the compounds for one week. Subclonal recovery was assessed by measuring cell viability (AnnexinV+) and leukemic progenitor function (methylcellulose) for up to 1 week post treatment. NCGC-2327 and NCGC-1481 delayed, and in some instances prevented, subclonal recovery as compared to AC220 or Crenolanib treatment.NCGC-2327 and NCGC-1481 show comparable potency to current FLT3 inhibitors (i.e., AC220 and Crenolanib) in regards to inhibition of FLT3 signaling, proliferation, and induction of apoptosis in FLT3-ITD-mutant AML. However, NCGC-2327 and NCGC-1481 are exquisitely effective at preventing subclonal recovery of FLT3-ITD-mutant AML as compared to both AC220 and Crenolanib. Taken together, these findings suggest our novel FLT3 inhibitors show 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.

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

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

Immunomodulatory potential of acemannan (polysaccharide from Aloe vera) against radiation induced mortality in Swiss albino mice

Plant polysaccharides have been reported to stimulate growth, differentiation and proliferation of hematopoietic progenitor and stem cells to protect against the deleterious effects of radiations. This study evaluated the radioprotective potential of acemannan, a major polysaccharide component of aloe vera gel. Treatment of mice with 50 mg/kg body weight of acemannan by oral gavage for 7 days was able to protect against the radiation-induced mortality. Seven-day pretreatment or post-treatment of mice with acemannan resulted in the increase in median survival by 60 and 20%, respectively. The decrease in mortality can be attributed to the induction of hematopoiesis (peripheral lymphocytes counts, spleen cellularity, spleen index) and the upregulation of cytokines like TNF-α and IL-1 by acemannan in irradiated mice. Data indicate that acemannan has the ability to protect mice against radiation-induced mortality by immunomodulation and can be developed as a radiation damage mitigation agent.

The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis

Growth differentiation factor 15 (GDF15) is a pleiotropic cytokine that belongs to the transforming growth factor-β superfamily. Elevated serum concentrations of this cytokine have been reported in patients with various malignancies. To assess the potential roles of GDF15 in hematologic malignancies, we measured its serum levels in patients with these diseases. We found that serum GDF15 levels were elevated in almost all these patients, particularly in patients with primary myelofibrosis (PMF). Immunohistochemical staining of bone marrow (BM) specimens revealed that GDF15 was strongly expressed by megakaryocytes, which may be sources of increased serum GDF15 in PMF patients. Therefore, we further assessed the contribution of GDF15 to the pathogenesis of PMF. Recombinant human (rh) GDF15 enhanced the growth of human BM mesenchymal stromal cells (BM-MSCs), and it enhanced the potential of these cells to support human hematopoietic progenitor cell growth in a co-culture system. rhGDF15 enhanced the growth of human primary fibroblasts, but it did not affect their expression of profibrotic genes. rhGDF15 induced osteoblastic differentiation of BM-MSCs in vitro, and pretreatment of BM-MSCs with rGDF15 enhanced the induction of bone formation in a xenograft mouse model. These results suggest that serum levels of GDF15 in PMF are elevated, that megakaryocytes are sources of this cytokine in BM, and that GDF15 may modulate the pathogenesis of PMF by enhancing proliferation and promoting osteogenic differentiation of BM-MSCs.

Chronic restraint stress after injury and shock is associated with persistent anemia despite prolonged elevation in erythropoietin levels

Following severe traumatic injury, critically ill patients have a prolonged hypercatacholamine state that is associated with bone marrow (BM) dysfunction and persistent anemia. However, current animal models of injury and shock result in a transient anemia. Daily restraint stress (chronic stress [CS]) has been shown to increase catecholamines. We hypothesize that adding CS following injury or injury and shock in rats will prolong the hypercatecholaminemia and prolong the initial anemia, despite elevated erythropoietin (EPO) levels. Male Sprague-Dawley rats (n = 6-8 per group) underwent lung contusion (LC) or combined LC/hemorrhagic shock (LCHS) followed by 6 days of CS. CS consisted of a 2-hour restraint period interrupted with repositioning and alarms every 30 minutes. At 7 days, urine was assessed for norepinephrine (NE) levels, blood for EPO and hemoglobin (Hgb), and BM for erythroid progenitor growth. Animals undergoing LC or combined LCHS predictably recovered by Day 7; urine NE, EPO, and Hgb levels were normal. The addition of CS to LC and LCHS models was associated with a significant elevation in NE on Day 6. The addition of CS to LC led to a persistent 20% to 25% decrease in the growth of BM hematopoietic progenitor cells. These findings were further exaggerated when CS was added following LCHS, resulting in a 20%q to 40% reduction in BM erythroid progenitor colony growth and a 20% decrease in Hgb when compared with LCHS alone. Exposing injured animals to CS results in prolonged elevation of NE and EPO, which is associated with worsening BM erythroid function and persistent anemia. Chronic restraint stress following injury and shock provides a clinically relevant model to further evaluate persistent injury-associated anemia seen in critically ill trauma patients. Furthermore, alleviating CS after severe injury is a potential therapeutic target to improve BM dysfunction and anemia.

MYST2 acetyltransferase expression and Histone H4 Lysine acetylation are suppressed in AML

Chromatin-modifying enzymes are frequently altered in acute myeloid leukemia (AML). In the current study, we identified MYST2, a core histone acetyltransferase, to be suppressed in blast cells from AML patients compared with nonmalignant hematopoietic progenitor cells. Functionally, loss of MYST2 accelerated leukemic growth and colony formation, while forced expression of MYST2 induced H4K5 acetylation (H4K5Ac) and suppressed hematopoietic progenitor cell growth. Consistently, global H4K5Ac levels were frequently decreased in AML blasts. Low levels of H4K5Ac were most prominent in patients with complex karyotype AML and were associated with inferior overall survival in univariate but not multivariate analysis. ChIP-seq experiments in primary AML patients' blasts revealed widespread H4K5Ac deregulation, most prominent at gene promoters. Taken together, MYST2 is a repressed growth suppressor in AML mediating reduced acetylation of histone 4 at residue 5 and is associated with inferior AML patient survival.

Neutrophils Promote Hematopoiesis Via a Novel Mechanism Involving Leucine Rich α-2 Glycoprotein (LRG)

Leucine-rich α2 glycoprotein (LRG), the founding member of the leucine-rich repeat superfamily of proteins, was initially identified in serum more than 30 years ago, but its biologic function has remained elusive. A role for LRG has been implicated in inflammation and angiogenesis. Our laboratory previously identified cDNA and genomic clones for human and murine LRG, and showed that ectopically expressed LRG localizes to the granule compartment in transfected myeloid cell lines and promotes their granulocytic differentiation. We also demonstrated that expression of LRG is transcriptionally regulated during neutrophil granulocyte differentiation in a manner similar to that reported for genes encoding the different subsets of neutrophil granule proteins. The presence of LRG in primary neutrophils and a role for LRG in hematopoiesis, however, have not been previously described. Based on our prior studies in transfected myeloid cell lines, we considered the tantalizing possibility that LRG is a novel neutrophil granule protein that is secreted extracellularly upon neutrophil activation to modulate hematopoiesis. To investigate this, we examined LRG in primary human neutrophils isolated from healthy volunteers. Immunoblot analysis of whole cell lysates from neutrophils (97% purity) identified a higher molecular weight LRG species in neutrophils (62 kDa) compared to serum (50 kDa); our data demonstrate the difference in apparent molecular weight is due to differential glycosylation. Immunofluorescence microscopy using antibodies to human LRG and antibodies to the neutrophil granule proteins myeloperoxidase (MPO), lactoferrin (LF), and matrix metalloproteinase 9 (MMP-9, also known as gelatinase), along with fluorescently-labeled secondary antibodies, demonstrated the presence of LRG in the cytoplasm of neutrophils in a compartment corresponding to LF. ELISA and immunoblot analyses of subcellular fractions from isolated neutrophils prepared by nitrogen cavitation demonstrated the presence of LRG in LF-containing fractions as well as some MMP-9-containing fractions, consistent with localization of LRG to the secondary/tertiary granule compartment. Neutrophil exocytosis assays using ionomycin, phorbol-12-myristate 13-acetate, and f-Met-Leu-Phe as stimulants also indicated that LRG is co-released with LF and MMP9, but not with MPO. Notably, LRG secreted from activated neutrophils could bind cytochrome c as reported for LRG purified from serum. Recent reports that LRG can also bind to the TGFβR1 receptor on endothelial cells prompted us to investigate the effects of LRG on TGFβ signaling in hematopoietic cells. LRG significantly antagonized the inhibitory effect of TGFβ on HL-60 cell proliferation (n=3; p<0.05) and also on colony growth of human hematopoietic progenitor cells. When LRG was added to hematopoietic progenitor cells cultured in TGFβ-containing Methocult (SF H4436, serum free), a 50% increase in CFU-GMs was observed. Collectively, these data suggest a novel mechanism whereby neutrophils modulate hematopoiesis in the microenvironment via extracellular release of LRG, and invoke an additional role for neutrophils in innate immunity that has not previously been reported DisclosuresNo relevant conflicts of interest to declare.

Abstract 5235: Identification of microRNA-3662 as a novel tumor suppressor miR

Abstract Background: Despite all progress, the prognosis of many acute myeloid leukemia (AML) patients remains poor. Identification of candidate genes for targeted treatment strategies may be beneficial for patient survival. Using online prediction programs, we identified miR-3662 as a novel tumor suppressor candidate in AML. Its predicted targets include numerous genes involved in hematopoietic differentiation and leukemogenesis, such as the oncogene BAALC. So far, miR-3662 has not been implicated in cancer biology. Aims: First, we strived to elucidate the role of miR-3662 in hematopoietic differentiation and leukemogenesis. Second, we aimed to experimentally validate BAALC as a miR-3662 target to explore the downstream biology of the tumor suppressor candidate. Methods &amp; Results: As tumor suppressor genes are often downregulated in cancer, we determined the expression of miR-3662 in peripheral blood blasts from cytogenetically normal (CN-) AML patients (n=10) and peripheral blood samples from non-cancer controls (n=20). Indeed, the non-cancer controls had an average 97-fold (P≤.001) higher miR-3662 expression compared to CN-AML patients, suggesting a downregulation of miR-3662 in leukemic cells. Next, we assessed the effects of forced miR-3662 expression and knock-down of miR-3662 on the growth and colony formation of hematopoietic progenitor cells (CD34+) from three healthy donors. While miR-3662 increased cell growth and colony formation, antagomiR-3662 reduced both parameters (miR-3662 vs. scramble: P≤.001; antagomiR-3662 vs. scramble: P≤.001). In contrast, colony forming assays with leukemia cell lines (OCI-AML3, KG1a) after stable infection with miR-3662 versus scramble reduced the cell growth and colony formation (miR-3662 vs. scramble: both P≤.001). To assess the effect of miR-3662 on leukemia cell survival, we performed caspase-3/7 assays with the leukemic blasts of five CN-AML patients infected with miR-3662 or scramble. miR-3662 increased caspase-3/7 activity (miR-3662 vs. scramble, P=.004), indicating miR-3662-mediated increased apoptosis. Thus, miR-3662 has tumor suppressive potential. Next we tested the effects of miR-3662 on the expression of its predicted target gene BAALC by overexpressing miR-3662 in two AML cell lines (KG1a, MV4-11) and blasts of CN-AML patients (n=10). miR-3662 reduced BAALC expression in both AML cell lines (KG1a: 70% reduction, MV4-11: 75% reduction, both P≤.001) and in the CN-AML patients (average reduction: 38.6%, P=.007). Finally, we performed luciferase assays with a BAALC 3′-UTR construct. Co-transfection with miR-3662 led to a 38% reduction of luciferase activity (miR-3662 vs. scramble, P≤.001), indicating that miR-3662 directly targets BAALC. Conclusion: miR-3662 is a novel tumor suppressor miR that has potential implications for leukemogenesis, as it increased colony formation and differentiation in CD34+ cells and decreased growth of leukemic cells. Citation Format: Sophia Maharry, Sujay Mehta, Sandya Liyanarachchi, Guido Marcucci, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. Identification of microRNA-3662 as a novel tumor suppressor miR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5235. doi:10.1158/1538-7445.AM2014-5235

Effects of adipogenic conditions on the in vitro growth of human hematopoietic stem and progenitor cells from umbilical cord blood

Effects of adipogenic conditions on the in vitro growth of human hematopoietic stem and progenitor cells from umbilical cord blood

Do all β-blockers attenuate the excess hematopoietic progenitor cell mobilization from the bone marrow following trauma/hemorrhagic shock?

Severe injury results in increased mobilization of hematopoietic progenitor cells (HPC) from the bone marrow (BM) to sites of injury, which may contribute to persistent BM dysfunction after trauma. Norepinephrine is a known inducer of HPC mobilization, and nonselective β-blockade with propranolol has been shown to decrease mobilization after trauma and hemorrhagic shock (HS). This study will determine the role of selective β-adrenergic receptor blockade in HPC mobilization in a combined model of lung contusion (LC) and HS. Male Sprague-Dawley rats were subjected to LC, followed by 45 minutes of HS. Animals were then randomized to receive atenolol (LCHS + β1B), butoxamine (LCHS + β2B), or SR59230A (LCHS + β3B) immediately after resuscitation and daily for 6 days. Control groups were composed of naive animals. BM cellularity, %HPCs in peripheral blood, and plasma granulocyte-colony stimulating factor levels were assessed at 3 hours and 7 days. Systemic plasma-mediated effects were evaluated in vitro by assessment of BM HPC growth. Injured lung tissue was graded histologically by a blinded reader. The use of β2B or β3B following LCHS restored BM cellularity and significantly decreased HPC mobilization. In contrast, β1B had no effect on HPC mobilization. Only β3B significantly reduced plasma G-CSF levels. When evaluating the plasma systemic effects, both β2B and β3B significantly improved BM HPC growth as compared with LCHS alone. The use of β2 and β3 blockade did not affect lung injury scores. Both β2 and β3 blockade can prevent excess HPC mobilization and BM dysfunction when given after trauma and HS, and the effects seem to be mediated systemically, without adverse effects on subsequent healing. Only treatment with β3 blockade reduced plasma G-CSF levels, suggesting different mechanisms for adrenergic-induced G-CSF release and mobilization of HPCs. This study adds to the evidence that therapeutic strategies that reduce the exaggerated sympathetic stimulation after severe injury are beneficial and reduce BM dysfunction.

The Role Of p53 In JAK2V617F-Induced Myeloproliferative Neoplasms

Ph- myeloproliferative neoplasms (MPNs) are hematopoietic malignancies in which one or more myeloid lineages are abnormally amplified. These diseases represent a group of chronic conditions including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. MPNs mainly affect older people and have an average onset age of 55 years. Complications associated with MPNs include development of acute leukemia as well as thrombosis, hemorrhage, and myeloid metaplasia. JAK2V617F is found in the majority of patients with MPNs, and an overwhelming number of studies have demonstrated its pathogenicity. However, the precise action of JAK2V617F is not well defined, and other molecular defects involved in MPNs remain elusive. In earlier studies, we have generated transgenic mice expressing JAK2V617F in the hematopoietic system and demonstrated that JAK2V617F can cause MPN-like phenotypes in an age- and transgene dose-dependent manner. In this study, we address the involvement of tumor suppressor p53 in the progression of JAK2V617F-induced MPN phenotypes. We crossed our JAK2V617F transgenic mice with p53 knockout mice. Under the p53+/+ background, our JAK2V617F transgenic mice developed a mild MPN-like phenotypes in 15 weeks. However, under the heterozygous knockout p53-/+ condition, the mice developed a strong MPN-like phenotype in 8 weeks, manifested in higher blood cell counts, more severe splenomegaly, and earlier onset of myelofibrosis. This suggests that p53 affects the progression of MPNs, and reduced levels of p53 activity may be responsible for the heterogeneous phenotypes observed in MPN patients. Furthermore, when p53 was deleted from both chromosomes, JAK2V617F mice developed acute erythroleukemia, megakaryoblastic leukemia, or myeloid leukemia and died in 20 weeks with greatly enlarged spleen (>10 times the normal size) and liver (twice the normal size) infiltrated with leukemic cells. This indicates that loss of p53 in addition to JAK2V617F causes leukemic transformation. This is consistent with the earlier findings that p53 mutations exist in JAK2V617F-positive leukemia cell lines and JAK2V617F-positive MPNs patients who developed acute myeloid leukemia. Our study also suggests that targeting JAK2V617F and p53 simultaneously may provide effective treatment for MPNs. We employed nutlin-3 that disrupts the MDM2-p53 interaction thereby reducing degradation of p53. In vitro cell culture studies demonstrated that JAK2 inhibitors and nutlin-3 synergistically inhibited the growth of hematopoietic progenitor cells from JAK2V617F transgenic mice and MPN patients. Finally, from one of the p53-/- JAK2V617F transgenic mice, we derived an erythroleukemia cell line designated J53Z1. J53Z1 cells are CD71high and Ter119low and are dependent on erythropoietin for survival. They were effectively inhibited by known JAK2 inhibitors and should be useful in cell-based assays for screening of JAK2 inhibitors. Altogether, our study demonstrates that the level of p53 dictates the progression of JAK2V617F-induced MPNs and targeting p53 and JAK2V617F simultaneously may provide effective treatment for JAK2V617F-positive MNPs. Disclosures:No relevant conflicts of interest to declare.

Sustained Alterations In Bone Marrow Stromal Cells From Patients With Myeloproliferative Neoplasms (MPN) Contribute To Remodelling Of The Bone Marrow Microenvironment Prior To The Manifestation Of Myelofibrosis

Myeloproliferative neoplasms (MPN) are characterized by the loss of normal hematopoiesis and the excessive production and accumulation of non-lymphoid cells and platelets in the BM. Clonal hematopoiesis in MPN is assumed to generate factors which induce profound changes in the non-clonal BM microenvironment. Alterations include massive deposition of extracellular matrix proteins (ECM), progression to bone marrow (BM)-fibrosis and osteosclerosis. We hypothesize that particularly in MPN, alterations in the cross talk between hematopoietic and bone marrow stromal cells (BMSC) play a critical role for an impaired bone marrow microenvironment long before overt signs of myelofibrosis can be detected by conventional methods.To dissect the hematopoiesis supporting capacity and extracellular matrix remodelling of BMSC from patients with MPN, we isolated BMSC from BM of patients with essential thrombocytemia (ET, n=5), polycythemia vera (PV, n=5), chronic myeloid leukemia (CML, n=5) and control BM (n=6). BMSC isolates were taken only from pre-fibrotic MPN patients (bone marrow trephine biopsy reticulin staining graded 0 or 1) and the resulting expansion cultures fullfilled MSC criteria according to the common consensus (Dominici et al., Cytotherapy, 2006; 8(4):315-317). When subjected to myeloid colony forming unit assays, MPN-BMSC conditioned supernatants showed a significantly reduced capacity to stimulate a CFU-GM/G/M growth of non-malignant hematopoietic stem and progenitor cells as compared to control BMSC (control (n=6) vs CML (n=5): p= 0.0032; control vs PV (n=5): p=0.016, control vs ET (n=4) not significant; student«s T- test ). BMSC-dependent matrix remodelling was analysed in a previously established robust matrix remodelling assay in vitro. MPN-BMSC displayed a pronounced increased matrix remodelling capacity compared to control BMSC. Interestingly, among the different MPN subtypes, this effect was highly significant in BMSC derived from patients with ET (Control (n=6) vs. ET (n=5): p<0.001). Furthermore, in vitro ECM production by MPN-BMSC was paralleled by ECM changes observed in matched bone marrow punches as shown by fibronectin immunohistochemistry. Co-expression of the stroma marker CD271 and fibronectin -as shown by confocal microscopy- points towards stroma-mediated ECM production in vivo. As upregulation of fibronectin expression was also detected in reticulin 0 graded BM punches, we hypothesized that fibronectin staining might be a potential marker for pre-fibrotic ECM changes in –so far- reticulin-negative MPN biopsies. To validate this hypothesis, we stained fibronectin in a tissue microarray (TMA), containing primary BM biopsies from patients with ET (n=14), PV (n=14), CML (n=14), MF (n=11) and controls (Non-Hodgkin's lymphoma without bone marrow involvement, n=17). Interestingly, within the reticulin-negative subcohort of pre-fibrotic MPN (n=34), fibronectin stained positive (grade 1 or higher) in 5/7 cases with ET (71%), 6/9 cases with PV (66%) and in 14/14 cases with CML (100%) as well as in all cases with PMF (100%). Furthermore, fibronectin staining correlated significantly with patients' decreased haemoglobin levels as shown by ANOVA analysis of routine clinical parameters (F=5.71; Prob> F 0.0037).We conclude that prior to the manifestation of fibrosis in MPN, BMSC loose their capacity to support non-malignant hematopoiesis and increase their matrix remodelling potential. As to our surprise, these effects are stably maintained in the absence of the malignant hematopoietic clone and since BMSC cultures develop over numerous population doublings in vitro from proliferating mesenchymal precursors, we hypothesize that cell intrinsic effects in BMSC from patients with MPN contribute to the development of myelofibrosis in MPN. [Display omitted] Disclosures:Bruemmendorf: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria.

Beta Blockade Protection of Bone Marrow Following Injury: A Critical Link between Heart Rate and Immunomodulation.

IntroductionSevere trauma induces a profound elevation of catecholamines that is associated with bone marrow (BM) hematopoietic progenitor cell (HPC) colony growth suppression, excessive BM HPC mobilization, and a persistent anemia. Previously, propranolol (BB) use after injury and shock has been shown to prevent this BM dysfunction and improve hemoglobin levels. This study seeks to further investigate the optimal therapeutic dose and timing of BB administration following injury and shock.MethodsMale Sprague-Dawley rats were subjected to a combined lung contusion (LC), hemorrhagic shock (HS) model ± BB. In our dose response experiments, animals received BB at 1, 2.5, 5, or 10 mg/kg immediately following resuscitation. In our therapeutic window experiments, following LCHS rats were given BB immediately, 1 hour, or 3 hours following resuscitation. BM and peripheral blood (PB) were collected in all animals to measure cellularity, BM HPC growth, circulating HPCs, and plasma G-CSF levels.ResultsPropranolol at 5 and 10 mg/kg significantly reduced HPC mobilization, restored BM cellularity and BM HPC growth, and decreased plasma G-CSF levels. Propranolol at 5 and 10 mg/kg also significantly decreased heart rate. When BB was administered beyond 1 hour after LCHS, its protective effects on cellularity, BM HPC growth, HPC mobilization, and plasma G-CSF levels were greatly diminished.ConclusionEarly Buse following injury and shock at a dose of at least 5mg/kg is required to maintain BM cellularity and HPC growth, prevent HPC mobilization, and reduce plasma G-CSF levels. This suggests that propranolol exerts its BM protective effect in a dose and time dependent fashion in a rodent model. Finally, heart rate may be a valuable clinical marker to assess effective dosing of propranolol.

Imatinib and Nilotinib Inhibit Hematopoietic Progenitor Cell Growth, but Do Not Prevent Adhesion, Migration and Engraftment of Human Cord Blood CD34+ Cells

BackgroundThe availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34+ cells.Design and MethodsAfter a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null) mice.ResultsTKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34+ cell cycle entry. Adhesion of CD34+ cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT.ConclusionsThese data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe.

Does selective beta-1 blockade provide bone marrow protection after trauma/hemorrhagic shock?

Previously, nonselective beta-blockade (BB) with propranolol demonstrated protection of the bone marrow (BM) after trauma and hemorrhagic shock (HS). Because selective beta-1 blockers are used commonly for their cardiac protection, the aim of this study was to more clearly define the role of specific beta adrenergic receptors in BM protection after trauma and HS. Male Sprague-Dawley rats underwent unilateral lung contusion (LC) followed by HS for 45minutes. After resuscitation, animals were injected with a selective beta-blocker, atenolol (B1B), butoxamine (B2B), or SR59230A (B3B). Animals were killed at 3 hours or 7 days. Heart rate and blood pressure were measured throughout the study period. BM cellularity, growth of hematopoietic progenitor cells (HPCs) in BM, and hemoglobin levels (Hb) were assessed. Treatment with a B2B or B3B after LCHS restored both BM cellularity and BM HPC colony growth at 3 hours and 7 days. In contrast, treatment with a B1B had no effect on BM cellularity or HPC growth but did decrease heart effectively rate throughout the study. Treatment with a B3B after LCHS increased Hb as compared with LCHS alone. After trauma and HS, protection of BM for 7 days was seen with use of either a selective beta-2 or beta-3 blocker. Use of a selective beta-1 blocker was ineffective in protecting the BM despite a physiologic decrease in heart rate. Therefore, the protection of BM is via the beta-2 and beta-3 receptors and it is not via a direct cardiovascular effect.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells

The concept that mesenchymal stromal cells (MSCs), a component of the hematopoietic microenvironment, can be a target for alloreactive effector cells in the context of graft-vs-host disease has not been investigated in detail. Mixed lymphocyte reaction (MLR) supernatant was used to mimic the inflammatory milieu induced by an allogeneic immune response in vitro. In addition to phenotype and proliferation, we monitored MSC differentiation, gene expression, and support of CD34(+) hematopoietic stem and progenitor cells after priming with MLR supernatant. Priming of MSCs with MLR supernatant led to an 11-fold decrease in cobblestone area-forming cells in the 4-week coculture (p < 0.05) and a threefold decrease of colony-forming unit macrophage in the colony-forming cell assay (p < 0.05). MSC proliferation over 8 days was increased 2.5-fold (p < 0.05). Osteogenic differentiation was enhanced, while adipogenesis was concurrently suppressed. In addition, the surface expression of HLA-DR and intercellular adhesion molecule-1 was increased 20-fold (p = 0.06) and 45-fold (p < 0.05), respectively. This was associated with increased adhesion of hematopoietic stem and progenitor cells to MLR-treated MSCs. In summary, our data shed light on the dysfunction of the stromal environment during graft-vs-host disease, possibly aggravating cytopenia and leading to an enhanced immunogenicity of MSCs.

The HDAC inhibitor Givinostat modulates the hematopoietic transcription factors NFE2 and C-MYB in JAK2V617F myeloproliferative neoplasm cells

We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)(V617F) myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2(V617F) MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2(V617F) cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2(V617F) cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34(+) cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription.