Proliferation Of Hematopoietic Progenitor Cells Research Articles

Influence of Genetic Variants in EGF and Other Genes on Hematological Traits in Korean Populations by a Genome-Wide Approach.

Hematological traits are important health indicators and are used as diagnostic clinical parameters for human disorders. Recently, genome-wide association studies (GWAS) identified many genetic loci associated with hematological traits in diverse ethnic groups. However, additional GWAS are necessary to elucidate the breadth of genetic variation and the underlying genetic architecture represented by hematological metrics. To identify additional genetic loci influencing hematological traits (such as hematocrit, hemoglobin concentration, white blood cell count, red blood cell count, and platelet count), we conducted GWAS and meta-analyses on data from 12,509 Korean individuals grouped into population-based cohorts. Of interest is EGF, a factor plays a role in the proliferation and differentiation of hematopoietic progenitor cells. We identified a novel EGF variant, which associated with platelet count in our study (P combined = 2.44 × 10−15). Our study also replicated 16 genetic associations related to five hematological traits with genome-wide significance (P < 5 × 10−8) that were previously established in other ethnic groups. Of these, variants influencing platelet count are distributed across several genes and have pleiotropic effects in coronary artery disease and dyslipidemia. Our findings may aid in elucidating molecular mechanisms underlying not only hematopoiesis but also inflammatory and cardiovascular diseases.

Adjuvant granulocyte colony-stimulating factor therapy results in improved spatial learning and stimulates hippocampal neurogenesis in a mouse model of pneumococcal meningitis.

Despite the development of new antibiotic agents, mortality of pneumococcal meningitis remains high. In addition, meningitis results in severe long-term morbidity, most prominently cognitive deficits. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of hematopoietic progenitor cells and increases the number of circulating neutrophil granulocytes. This study investigated the effect of adjuvant G-CSF treatment on cognitive function after pneumococcal meningitis. C57BL/6 mice were infected by subarachnoid injection of Streptococcus pneumoniae serotype 3 and treated with ceftriaxone and G-CSF subcutaneously or ceftriaxone alone for 5 days. Clinical scores, motor performance, and mortality during bacterial meningitis were unaffected by adjuvant G-CSF treatment. No effect of G-CSF treatment on production of proinflammatory cytokines or activation of microglia or astrocytes was observed. The G-CSF treatment did, however, result in hippocampal neurogenesis and improved spatial learning performance 6 weeks after meningitis. These results suggest that G-CSF might offer a new adjuvant therapeutic approach in bacterial meningitis to reduce long-term cognitive deficits.

Thrombopoietin Affects the Behavior of Myelofibrosis Stem Cells

Recently the presence of myelofibrosis (MF) stem cells (MF-SC) in the spleens of MF patients which when transplanted into NOD/SCID/IL2R null (NSG) mice were capable of generating multiple hematopoietic lineages that belonged to the malignant clone has been observed. Although MF is characterized by marrow megakaryocyte (Mk) hyperplasia, limited numbers of human marrow Mks were, however, observed in these transplanted mice and evidence of both marrow and splenic fibrosis 9 months after the transplantation was lacking (Wang X, et al. J Clin Invest. 2012; 122:3888). Splenic MF CD34+ cells did retain the capacity to differentiate in vitro into CD41a+ and CD61+ Mks in the presence of thrombopoietin (TPO). We therefore investigated whether the administration of romiplostim, a novel human TPO peptide mimetic which lacks homology to endogenous TPO in MF humanized mice, might create appropriate environmental cues which affect the behavior of MF-SCs.Elevated levels of TPO (345±114ng/ml) were detected in MF plasmas (n=13) as compared to levels detected in normal plasma (10±4ng/ml, n=6, P=0.049), indicating the possibility that TPO affects MF-SCs and MF hematopoietic progenitor cells (HPC). Following the culture of CB (n=3) or PB MF CD34+ cells (n=4) for 1 and 2 wks in serum free expansion media (SFEM) supplemented with SCF (50ng/ml) + romiplostim (100ng/ml), the numbers of total cells, CD34+Lin- cells and assayable HPCs, including CFU-Mk, CFU-GM and BFU-E, CD41a+CD34-CD15- cells and CD15+CD34-CD41a- cells generated were greater (CB) or similar (MF) to the number generated in the cultures containing SCF + TPO (100ng/ml). Moreover, similar proportions of colonies (CFU-GM+BFU-E) generated in cultures supplemented with SCF+TPO or SCF+ romiplostim (100ng/ml, 1000ng/ml) were JAK2V617F-positive. These findings suggest that both romiplostim and TPO are capable of promoting MF-SC and HPC proliferation in vitro.To assess the effects of romiplostim on human (h) platelet production, CB CD34+ cells (5×105) were transplanted via the tail vein into eight- to nine-week-old sublethally irradiated (240 cGy) NSG mice. One week after transplantation, mice were treated with water or 10, 100 or 1000 µg/kg of romiplostim. Both the percentage and number of hCD41a+ platelets in the PB increased 4 days following the treatment with 10ug/kg romiplostim, and peaked at day 8 with a 2.58±0.29 fold increase in the percentage (P<0.05) and 3.51±1.32 fold increase in the absolute number (P<0.05) of hCD41a+ platelets being observed. The number of human platelets in the PB, were reduced to the levels detected in mice not receiving romiplostim by day 15.MF splenic CD34+ cells (5-10×105) were next transplanted into romiplostim (10ug/kg or 30ug/kg) treated or control NSG mice. Two months after the transplantation, a 1.83±0.62 fold increase in the number of hCD41a+ cells was observed in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not treated with romiplostim. Moreover, a 7.13±2.13 fold elevation in the percentage of hCD45+ cells was detected in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not receiving romiplostim. Moreover, enhanced hCD45+ cell chimerism was achieved in both the marrow (55.6%, 7.2%) and spleen (47.7%, 1.5%) of mice receiving splenic CD34+ cells from 2 MF patients and romiplostim as compared with mice not receiving romiplostim (marrow: 30.4%, 5.6%; spleen: 23.6%, 0.9%), respectively. The splenic MF CD34+ cells receiving or not receiving romiplostim had a similar differentiation patterns in the marrow and spleen, however, the numbers of hCD33+, hCD19+, hCD3+, hCD41a+ and hCD15+ cells detected in mice treated with romiplostim were dramatically greater than those detected in mice not receiving romiplostim. Greater numbers of hCD34+ cells were detected in the BM (7.1%; 3.5%) of recipient mice receiving romiplostim as that detected in mice not receiving romiplostim (6.6%; 0.6%). Furthermore, a similar degree of hCD45+ cell and hCD34+ cell chimerism was observed in both the marrow and spleen of mice receiving splenic MF CD34+ cells and romiplostim 4 months after the transplantation. These findings suggest that the administration of thrombopietin agonist has a profound effect on the behavior of MF-SCs and that the elevated levels of TPO documented in MF patients likely has important effects on the biology of MF-SCs. DisclosuresWang:The MPN Research Foundation (MPNRF) and the Leukemia & Lymphoma Society (LLS) : Research Funding.

Oxymetholone Therapy of Fanconi Anemia Induces Hematopoietic Stem Cell Cycling By Suppressing Osteopontin Transcription

Androgens are widely used for treating Fanconi anemia and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2-/- mice were used to assess the therapeutic efficacy of oxymetholone and its mechanism of action. 18-month old Fancd2-/- mice recapitulated key human Fanconi anemia phenotypes including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic oxymetholone treatment significantly improved these hematological parameters by stimulating the proliferation of hematopoietic stem and progenitor cells. RNAseq analysis implicated down-regulation of osteopontin as an important mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic oxymetholone therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Krüppel-like Factors KLF4 and KLF2 Regulate microRNA-150 Expression in Myeloid Leukemias

Background: Acute myeloid leukemia (AML) is characterized by increased self-renewal of leukemia stem/progenitor cells and failure of differentiation to mature myeloid cells. MicroRNAs (miRNAs) are small single stranded non-coding RNAs 19 to 24 nucleotides in length that regulate expression of tens to hundreds of genes via mRNA degradation or translational repression. MiRNA contributions to normal hematopoiesis have been described and deletion of key miRNA processing enzymes in murine and human cells suggests that miRNA loss contributes to the cancer phenotype and aberrant differentiation in leukemia.By combining observations of miRNA expression in normal hematopoietic progenitor cells and patient AML cells and high-throughput lentiviral expression library screening approaches in AML cell lines we have identified candidate miRNAs that contribute to altered proliferation and differentiation in AML cells. We have previously established 1) that miR-150 expression is decreased in a large subset of primary patient AML samples, in particular poor risk cytogenetic groups, 2) and that miR-150 re-expression induces myeloid differentiation and decreases cell proliferation of normal hematopoietic progenitor cells and AML cell lines and primary patient cells in part through downregulation of MYB expression. MiR-150 loss is relevant in other hematopoietic and solid tumor malignancies where re-expression inhibits cell proliferation, promotes apoptosis and induces reversal of endothelial to mesenchymal transition.Transcription factors are important regulators of myeloid differentiation and cell proliferation. Moreover, as highlighted by recent sequencing of the AML genome, alterations in myeloid transcription factors through mutation, gene rearrangement, and altered expression play a significant role in leukemogenesis. Consequently, we have focused on how myeloid transcription factors regulate miRNA expression, specifically for miR-150.Results: Using 5’RACE from healthy bone marrow RNA, we identified a major transcription start site at 214 basepairs upstream of the pre-miR-150 hairpin. We identified the minimal miR-150 promoter region as -266 to +259 basepairs from the major transcription start site using miR-150 promoter truncation luciferase constructs assayed in myeloid leukemia cell lines (THP-1, K562, and KG1a) and a lymphoid leukemia cell line (Jurkat). We identified DNA binding sites for the Krüppel-like factor (KLF) family of transcription factors that are necessary for miR-150 promoter activity using site-directed DNA mutagenesis of the luciferase reporters. KLFs regulate proliferation, differentiation, pluripotency, migration and inflammation. Depending on cell type and context, KLFs can function as tumor suppressors or oncogenes. To identify which KLF isoforms regulate miR-150 expression, we assayed the ability of KLFs 2, 3, 4, 5, 6, 7, 9, and 10 to induce miR-150 promoter activity using the luciferase reporters and endogenous miR-150 expression by quantitative PCR. KLF2 and KLF4 overexpression increased miR-150 promoter activity in luciferase assays 50-fold and 450-fold respectively in K562 cells. Furthermore, KLF2 and KLF4 induced endogenous miR-150 expression 20-fold and 100-fold respectively as detected by quantitative PCR in both THP-1 and K562 cells. Prior work has established that KLF2 and KLF4 regulate the differentiation of monocytes. We then confirmed that KLF2 and KLF4 overexpression promotes myeloid differentiation of THP-1 cells by flow cytometry and gene expression that was partially reversed by inhibition of miR-150 expression.Conclusions: Previous studies have determined that KLF2 and KLF4 expression are decreased or absent in a significant subset of AML cases. Our observations suggest that loss of KLF2 and KLF4 expression contributes to decreased miR-150 expression which in turn alters cell proliferation and differentiation. Other studies have implicated the cell cycle inhibitor p21WAF1/CIP1 and altered PPAR gamma signaling downstream of KLF4. Nonetheless, our mechanistic understanding is limited. Our work suggests that the loss of miR-150 and other miRNAs downstream of these transcription factors also contributes. Understanding the interactions between KLFs, miR-150 and other miRNAs has broader significance as KLF2 and KLF4 expression is altered in other hematopoietic and solid tumors. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Hematopoietic Stem and Progenitor Cell Fate Determination By the Dpy30 Subunit of Set1/Mll Complexes

Many hematological diseases result from aberrant chromatin and gene regulation in the maintenance, proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). As the major H3K4 methylation enzymes in mammals, the SET1/MLL family complexes occupy a crucial position in developmental biology, and are considered potential drug targets for epigenetic therapeutics due to the intimate connection of H3K4 methylation with gene expression as well as the extensive association of several subunits in these complexes with many diseases including multiple blood cancers. The SET1/MLL complexes comprise either SET1A, SET1B, MLL1, MLL2, MLL3, or MLL4 as the catalytic subunit, and WDR5, RBBP5, ASH2L, and DPY30 as integral core subunits necessary for the full methylation activity. However, it remains unclear how the enzymatic activity (H3K4 methylation) of these complexes regulates normal and abnormal hematopoiesis, potentially through regulating target genes critically involved in HSPC fate determination. Our previous finding of the direct and important activity of Dpy30 in facilitating genome-wide H3K4 methylation (Jiang et al., Cell 144:513-525, 2011) allows an effective interrogation of the functional role of H3K4 methylation through genetic studies of Dpy30.We have previously shown that Dpy30 is crucial for efficient differentiation of embryonic stem cells by facilitating the induction of many bivalently marked developmental genes (Jiang et al., Cell, 2011). We then demonstrated an important role of human DPY30 in ex vivo proliferation and differentiation of mobilized hematopoietic progenitors, as well as in zebrafish hematopoiesis (Yang et al., Blood, accepted). To further determine a role for Dpy30 and its associated H3K4 methylation in regulating HSPC maintenance and differentiation, we have generated a Dpy30 conditional knockout (KO) mouse model. Hematopoietic KO of Dpy30 in mice resulted in marked reduction of cellular H3K4 methylation and severe pancytopenia. Surprisingly, in contrast to the rapid HSC depletion upon hematopoietic loss of Mll, we detected a massive accumulation of phenotypic early HSPCs at the expense of more downstream hematopoietic cells in the Dpy30 KO mouse bone marrow (BM), despite little effects on cell proliferation and apoptosis. Competitive transplantation assays revealed a profound defect of the Dpy30 KO BM in multilineage hematopoietic reconstitution. These results are most consistent with a defect in hematopoietic differentiation. We have also started mixed BM chimera assays to further investigate the role of Dpy30 in HSPC fate determination. Our early time point results strongly support a defect in hematopoietic differentiation following Dpy30 loss, and demonstrate a role of Dpy30 in the efficient induction of many lineage regulatory genes during the transitions of the hematopoietic cell fate. Our data from later time points in the transplantation assays will allow us to discover if Dpy30 loss has a potential effect on HSC self-renewal, and will be presented and discussed. By revealing a previously unrecognized role of the H3K4 methylation activity of the Set1/Mll complexes in regulating HSPC fate determination, our studies may have important implications for developing therapeutic strategies against certain HSPC-based hematological diseases. DisclosuresNo relevant conflicts of interest to declare.

The Microanatomy of the Leukemic Stem Cell Niche in Murine Chronic Myelogenous Leukemia

Objectives and background: Constituents of the bone marrow microenvironment (BMM) influence the proliferation, differentiation and location of hematopoietic stem and progenitor cells (HSPC). Dependent on their maturation stage, different subsets of HSPC are localized at distinct sites in the BMM. This location depends on HSPC-intrinsic, as well as HSPC-extrinsic factors. The BMM protects leukemic stem cells (LSC) from treatment with tyrosine kinase inhibitors or chemotherapy. We, therefore, investigated the microanantomy of the LSC niche hypothesizing that it may differ from the normal HSPC niche.Methods: We used a combination of confocal and 2-photon intravital microscopy (IVM) of the murine calvarium and well-described retroviral models of BCR-ABL1+chronic myelogenous leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL).Results: We show here that BCR-ABL1+Lin–c-Kit+Sca-1+ (LKS) CD150+CD48– (LKS SLAM) cells, which harbor the LSC fraction in the CML model, homed to locations further away from the endosteum than their normal counterparts. Prior in-vitro treatment of BCR-ABL1+ LKS with imatinib mesylate, considered standard of care in CML, reversed this phenotype and the cells were found closer to the endosteum.Native BCR-ABL1, as well as the imatinib-resistant BCR-ABL1 point mutants BCR-ABL1Y253F, BCR-ABL1E255K, BCR-ABL1T315I and BCR-ABL1M351T had similar intrinsic catalytic activity, but the BCR-ABL1Y253F, BCR-ABL1E255K, and BCR-ABL1T315I mutants increased the IL-3-independent proliferative capacity of 32D cells relative to native BCR-ABL1. BCR-ABL1Y253F and BCR-ABL1M351T caused increased transformation of primary BM B-lymphoid progenitors in vitro and led to accelerated induction of B-ALL in mice. In the CML model, BCR-ABL1Y253F and BCR-ABL1T315Iinduced myeloproliferative neoplasia with shortened survival and features of accelerated phase disease compared to native BCR-ABL1, whereas BCR-ABL1T315I LKS cells homed closer to osteoblastic cells than LKS cells expressing native BCR-ABL1.Sequential in vivo tracking of leukemic progenitor growth by IVM showed a similar nadir in the number of cells per leukemic cell 'nest’ 11 days after irradiation and IV transplantation in recipients of DsRed+BCR-ABL1+ or empty vector control-transduced bone marrow. However, between days 18-25 after transplantation there was a significant increase in the number of cells per leukemic cell 'nest’ compared to the empty vector control group. Sequential immunohistochemistry and TUNEL assays of leukemic bone sections in imatinib- or vehicle-treated recipient mice with CML showed that initial BCR-ABL1+ growth tends to occur at locations further away from the endosteum, whereas erythroid islands were found closer to the endosteum and trabeculae. Apoptosis in response to imatinib appeared most prominent in the metaphysis. Lastly, we could demonstrate by IVM in the CML model that treatment of mice with a combination of imatinib plus granulocyte colony-stimulating factor led to 'emptying’ of the LSC niche and superior eradication of BCR-ABL1+ leukemic cells compared to treatment with imatinib alone.Conclusions: In summary, these data suggest that the microanatomy of the LSC niche in CML differs from the normal hematopoietic niche. BCR-ABL1 mutation status may affect the positioning of CML LSC in the microenvironment, and location in the niche may be altered pharmacologically, suggesting that niche location may influence clinical outcome. DisclosuresKrause:Glycomimetics. Inc.: Research Funding.

The Effects of Lipocalin (LCN2) on Hematopoiesis in Primary Myelofibrosis

Myelofibrosis (MF) is characterized by anemia, thrombocytopenia and marrow fibrosis. Malignant rather than normal hematopoietic progenitor cells (HPC) preferentially proliferate. A form of MF can also occur in patients with a prior history of PV or ET which is referred to as PV or ET related MF (PV-MF and ET-MF). In MF, excessive proliferation of marrow stromal cells occurs in response to cytokines elaborated by the malignant clone. Previously, we found that lipocalin-2 (LCN2) levels were elevated in patient plasmas with MPN, and a greatest degree of elevation was observed in the PMF plasma and PMF conditioned medium. We also found that LCN2 increased PMF HPC proliferation and increased numbers of JAK2V617F homozygous HPCs, but depressed normal BM HPC (Lu. et al, 2013 ASH meeting).In this study, a series of experiments were performed to further explore the effects of LCN2 on MF pathogenesis. ELISA data showed that plasma LCN2 levels not only were higher in all MF patients, plasma LCN2 levels in PV-MF as well as ET- MF were significantly greater than that in PV or ET plasma (p=0.0002 and p=0.0175, respectively). Importantly, in MPN patients, a non-parametric spearman correlation coefficient analysis suggested an inverse relationship between the patients’ hemoglobin levels and LCN2 levels (coefficient=-0.35, p<0.0001) and between platelet counts and LNC2 levels (coefficient=-0.26, p=0.0044). Immunofluorescence staining showed a greater proportion of PMF MNCs expressed LCN2. And immunohistochemical results revealed that LCN2 expressing cells were present in the PMF bone marrow and spleens samples and the LCN2 expression was restricted to cells belonging to the myeloid lineage rather than erythroid precursors or megakaryocytes. These data strongly confirmed that a higher level of LCN2 was expressed in MF patients. We then found that LCN2 significantly increased the generation of ROS (reactive oxygen species) in normal BM CD34+ cells but not PMF CD34+ cells (p<0.05). Furthermore, exposure of normal marrow to the LCN2 dramatically increased the CD34+/annexin V+ cell population in a dose dependent fashion. These apoptotic effects were blocked by addition of N-Acetyl-Cysteine (NAC), a ROS scavenger. By contrast, LCN2 did not increase the degree of apoptosis of PMF CD34+ cells when added at similar doses. These data indicate that LCN2 selectively promotes the survival of PMF CD34+ cells but leads to the apoptosis of normal BM CD34+ cells by increasing ROS. With 6 different individual BM samples, we demonstrated that PMF MNC cells that produce high levels of LCN2 as well as a recombinant form of LCN2 strongly promoted BM marrow adherent cells (MAC) proliferation. Similarly, LCN2 added to semisolid cultures of normal marrow cells resulted in the appearance of increased numbers of fibroblast-like cells. In addition, the ability of LCN2 to promote MAC proliferation was blocked by addition of NAC. These results suggest that LCN2 related generation of ROS induces MAC proliferation. The MAC in both control and LCN2 containing conditions expressed vimentin, Von Willebrand factor (VWF) and α-smooth muscle actin (a-SMA), while in LCN2 cultures expressed higher levels of CD105 (Endoglin) and CD106 (VCAM-1). These MAC therefore expressed features characteristic of mesenchymal stem cells, fibroblasts and endothelial cells. The MAC under control conditions expressed CD90+ on their cell surface, while incubation with LCN2 resulted in diminished cell surface CD90 expression, and its localization to the nucleus.We propose that LCN2 acts in an autocrine fashion to promote malignant PMF hematopoiesis as documented by its ability to preferentially favor the appearance of JAK2V617F homozygous hematopoietic colonies in vitro, but also acts in a paracrine fashion to suppress the proliferation of normal HPC and to affect the marrow microenvironment by promoting stromal cell proliferation. Our findings indicate that LCN2 affects these numerous biological effects by promoting the generation of ROS. We also propose that therapeutic strategies that can impair the actions of LCN2 or inactivate ROS are likely to be beneficial to MPN patients since they improve disease related cytopenias as well as impede the development of progressive marrow fibrosis. Such agents affecting this alternative therapeutic target might potentially be used alone or in combination with other presently available therapeutic modalities. DisclosuresNo relevant conflicts of interest to declare.

Oxymetholone Therapy of Fanconi Anemia Suppresses Osteopontin Transcription and Induces Hematopoietic Stem Cell Cycling

SummaryAndrogens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2−/− mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2−/− mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure.

Related-to-receptor tyrosine kinase receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice

Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a noncanonical Wnt ligand receptor termed 'related-to-receptor tyrosine kinase' (Ryk). Expression of Ryk on HSPCs invivo is associated with a lower rate of proliferation, and, following treatment with fluorouracil (5-FU), the percentage of Ryk(+/high) HSPCs increased and the percentage of Ryk(-/low) HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling invivo resulted in increased hematopoietic-stem-cell proliferation and decreased hematopoietic-stem-cell function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury.

Pten function in zebrafish: Anything but a fish story

Zebrafish is an excellent model system for the analysis of gene function. We and others use zebrafish to investigate the function of the tumor suppressor, Pten, in tumorigenesis and embryonic development. Zebrafish have two pten genes, ptena and ptenb. The recently identified N-terminal extension of human PTEN that may facilitate cell membrane transfer, appears not to be conserved in zebrafish Ptena or Ptenb. Mutants that retain a single wild type pten allele develop tumors, predominantly hemangiosarcomas. Homozygous double mutants are embryonic lethal. Zebrafish embryos lacking functional Pten display enhanced proliferation of endothelial cells, resulting in hyperbranching of blood vessels. In addition, ptena−/−ptenb−/− mutant embryos display enhanced proliferation of hematopoietic stem and progenitor cells and concomitant arrest of differentiation, although Pten-deficient cells commit to all blood cell lineages. Zebrafish is an ideal model for intravital imaging and future work using ptena−/−ptenb−/− mutants will enhance our understanding of the function of Pten in vivo.

Abstract 3058: Bone marrow hematopoietic stem cell niche activation and mobilization fosters the metastatic niche

Abstract Metastasis remains the main cause of mortality for patients with cancer. The process whereby a localized tumor becomes metastatic remains poorly understood. Metastasis was previously thought to be a late-stage, unidirectional migration of cells from the primary tumor to a distant site, due to acquired genetic mutations that conferred necessary metastatic attributes to the cell. However, current research demonstrates that dissemination is not a late phenomenon and although loss of metastasis suppressor genes or gain of acquired mutations can contribute to the metastatic phenotype, the cross-talk between the tumor and its microenvironment has proven to be a major driver of this process. We have found that recruited bone marrow-derived cells and changes in resident stromal cells form a microenvironment that is conducive to metastatic progression, termed the pre-metastatic niche. The recruited and resident cells that form this microenvironment are active areas of investigation. Because the bone marrow is the source for most of the recruited cell types in these distant tissue sites, we postulated that activation of the bone marrow's hematopoietic compartment plays a key role in metastatic progression. We sought to profile the changes that occur within the hematopoietic stem cell niche of the bone marrow and subsequent changes in hematopoiesis in the setting of tumor development. Within the bone marrow of tumor bearing mice we found increased proliferation of hematopoietic stem and progenitor cells. These cells are also increased in the circulation of both tumor bearing mice and patients with malignancy. Furthermore, we have determined that elevated levels of circulating hematopoietic progenitor cells can be predictive of metastatic risk, as those patients with the highest levels at diagnosis developed metastases. This biomarker may be useful in stratifying therapy for patients with localized disease who have the highest metastatic risk. Better understanding of the steps and individual factors required to establish a metastatic microenvironment can lead to novel adjuvant therapies to target this process. Citation Format: Amber J. Giles, Meera Murgai, Yorleny Vicioso, Steven Highfil, Crystal Mackall, Leonard Wexler, David Lyden, Rosandra Natasha Kaplan. Bone marrow hematopoietic stem cell niche activation and mobilization fosters the metastatic niche. [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 3058. doi:10.1158/1538-7445.AM2014-3058

The DPY30 subunit in SET1/MLL complexes regulates the proliferation and differentiation of hematopoietic progenitor cells

AbstractEpigenetic mechanisms, including histone modifications, have emerged as important factors influencing cell fate determination. The functional role of H3K4 methylation, however, remains largely unclear in the maintenance and differentiation of hematopoietic stem cells (HSCs)/hematopoietic progenitor cells (HPCs). Here we show that DPY30, a shared core subunit of the SET1/MLL family methyltransferase complexes and a facilitator of their H3K4 methylation activity, is important for ex vivo proliferation and differentiation of human CD34+ HPCs. DPY30 promotes HPC proliferation by directly regulating the expression of genes critical for cell proliferation. Interestingly, while DPY30 knockdown in HPCs impaired their differentiation into the myelomonocytic lineage, it potently promoted hemoglobin production and affected the kinetics of their differentiation into the erythroid lineage. In an in vivo model, we show that morpholino-mediated dpy30 knockdown resulted in severe defects in the development of the zebrafish hematopoietic system, which could be partially rescued by coinjection of dpy30 messenger RNA. Taken together, our results establish a critical role of DPY30 in the proliferation and appropriate differentiation of hematopoietic progenitor cells and in animal hematopoiesis. Finally, we also demonstrate a crucial role of DPY30 in the growth of several MLL1-fusion–mediated leukemia cell lines.

Cholesterol and lipoprotein metabolism: Early Career Committee contribution.

Cholesterol and lipoprotein metabolism: Early Career Committee contribution.

Гематологічні ефекти інгібітора протеїнкіназ похідного малеіміду за 1,2-диметилгідразиніндукованого канцерогенезу товстої кишки щурів

The effect of the protein kinase inhibitor maleimide derivative (MI-, 1, 1-(4-Cl-benzyl)-3-CI-4-(CF3-phenylamino)- 1H-pyrrole-2,5-dione) on blood cells of rats with 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis has been studied. Administration of MI-I1 at 2.7 mg/kg for 20 weeks on DMH-induced carcinogenesis prevents anemia, which is a consequence of cancer and complicates it. This is confirmed by a reduction in the numberofreticulocytes (0,19(0,15;0,21)x 10(12)/1)and restoration of mean corpuscular hemoglobin (18,02 (17,44;19,03) pg) and mean corpuscular hemoglobin concentration (309,42 (292,38;318,27) g/L) to a control value (0,17 (0,15;0,19), 18,31 (17,95;18,45), 310,78 (306,25;316,18), respectively) in contrast to the group DMH (0,28 (0,24;0,39); 17,50 (17,00;17,96); 288,10 (284,71;303,73), respectively). MI-I normalizes the number of monocytes (1,40 (0,95;2,50)x 10(9)/L) and platelets (646,32 (575,23;700,50)xl10(9)/L) in the blood after 26 weeks ofexperiment; in the DMH group, the values are significantly higher (1,97 (1,52;2,58), 783,90 (687,64;922,27), respectively) as compared to control group (1,23 (0,94; 1,68), 629,34 (590,19;711,48), respectively). MI- 1 reduces the involvement of these cells in the tumors progression and metastasis. Reduction of the monocytosis and thrombocytosis may be mediated by: 1) a decrease in the number and size of tumors and, consequently, the influence of their cytokines on hematopoietic tissue; 2) suppression of proliferation and differentiation of hematopoietic progenitor cells through inhibiting of receptor protein kinases of vascular endothelial and epidermal growth factors and non-receptor PDKI-, Src- and Syk- kinases, that are involved in hematopoiesis and carcinogenesis.

PRL2/PTP4A2 phosphatase is important for hematopoietic stem cell self-renewal.

Hematopoietic stem cell (HSC) self-renewal is tightly controlled by cytokines and other signals in the microenvironment. While stem cell factor (SCF) is an early acting cytokine that activates the receptor tyrosine kinase KIT and promotes HSC maintenance, how SCF/KIT signaling is regulated in HSCs is poorly understood. The protein tyrosine phosphatase 4A (PTP4A) family (aka PRL [phosphatase of regenerating liver] phosphatases), consisting of PTP4A1/PRL1, PTP4A2/PRL2, and PTP4A3/PRL3, represents an intriguing group of phosphatases implicated in cell proliferation and tumorigenesis. However, the role of PTP4A in hematopoiesis remains elusive. To define the role of PTP4A in hematopoiesis, we analyzed HSC behavior in Ptp4a2 (Prl2) deficient mice. We found that Ptp4a2 deficiency impairs HSC self-renewal as revealed by serial bone marrow transplantation assays. Moreover, we observed that Ptp4a2 null hematopoietic stem and progenitor cells (HSPCs) are more quiescent and show reduced activation of the AKT and ERK signaling. Importantly, we discovered that the ability of PTP4A2 to enhance HSPC proliferation and activation of AKT and ERK signaling depends on its phosphatase activity. Furthermore, we found that PTP4A2 is important for SCF-mediated HSPC proliferation and loss of Ptp4a2 decreased the ability of oncogenic KIT/D814V mutant in promoting hematopoietic progenitor cell proliferation. Thus, PTP4A2 plays critical roles in regulating HSC self-renewal and mediating SCF/KIT signaling.

Tumor suppressor DYRK1A effects on proliferation and chemoresistance of AML cells by downregulating c-Myc.

Acute myeloid leukemia (AML), caused by abnormal proliferation and accumulation of hematopoietic progenitor cells, is one of the most common malignancies in adults. We reported here DYRK1A expression level was reduced in the bone marrow of adult AML patients, comparing to normal controls. Overexpression of DYRK1A inhibited the proliferation of AML cell lines by increasing the proportion of cells undergoing G0/G1 phase. We reasoned that the proliferative inhibition was due to downregulation of c-Myc by DYRK1A, through mediating its degradation. Moreover, overexpression of c-Myc markedly reversed AML cell growth inhibition induced by DYRK1A. DYRK1A also had significantly lower expression in relapsed/refractory AML patients, comparing to newly-diagnosed AML patients, which indicated the role of DYRK1A in chemoresistance of AML. Our study provided functional evidences for DYRK1A as a potential tumor suppressor in AML.

MuLV-related endogenous retroviral elements and Flt3 participate in aberrant end-joining events that promote B-cell leukemogenesis.

During V(D)J recombination of immunoglobulin genes, p53 and nonhomologous end-joining (NHEJ) suppress aberrant rejoining of DNA double-strand breaks induced by recombinase-activating genes (Rags)-1/2, thus maintaining genomic stability and limiting malignant transformation during B-cell development. However, Rag deficiency does not prevent B-cell leukemogenesis in p53/NHEJ mutant mice, revealing that p53 and NHEJ also suppress Rag-independent mechanisms of B-cell leukemogenesis. Using several cytogenomic approaches, we identified a novel class of activating mutations in Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase important for normal hematopoiesis in Rag/p53/NHEJ triple-mutant (TM) B-cell leukemias. These mutant Flt3 alleles were created by complex genomic rearrangements with Moloney leukemia virus (MuLV)-related endogenous retroviral (ERV) elements, generating ERV-Flt3 fusion genes encoding an N-terminally truncated mutant form of Flt3 (trFlt3) that was transcribed from ERV long terminal repeats. trFlt3 protein lacked most of the Flt3 extracellular domain and induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, expression of trFlt3 in p53/NHEJ mutant hematopoietic progenitor cells promoted development of clinically aggressive B-cell leukemia. Thus, repetitive MuLV-related ERV sequences can participate in aberrant end-joining events that promote development of aggressive B-cell leukemia.

Anti-radiation effect and mechanism studies of ethanol extracts from Spatholobus suberectus and its active component catechin

To study the anti-radiation effect and mechanism of ethanol extracts from Spatholobus suberectus and its active component catechin, ICR mice were exposed to 6Gy irradiation and randomly divided into normal group, model group, positive control group (amifostine, 43.6 mg•kg⁻¹, iv 30 min before irradiation), SSD group (10, 20, 40 g•kg⁻¹) and catechin group (50, 100, 200 mg•kg⁻¹). The mice were administered the appropriate drugs once a day after irradiation for 28 consecutive days. Blood samples were collected from the tail end and the number of peripheral blood cells was counted before irradiation and on day 1, 3, 7, 14, 21 and 28 using a microcell counter. Changes of thymus and spleen index of mice on day 7 were observed. The serum SOD, GSH-Px activity and MDA level were detected by the colorimetric method. The colony forming ability of bone marrow hematopoietic progenitor cells on day 7 was detected by semi solid culture method. The HE staining was adopted to observe the pathological changes. The apoptosis of bone marrow cells was detected by flow cytometry. The expression of cleaved caspase-3 and Bax of bone marrow cells were measured separately by western-blotting and immunohistochemistry method. SSD and catechin can both significantly revert the irradiated-induced decline in hematological parameters (RBC, WBC, PLT, Hb), improve thymus and spleen index, significantly enhance serum SOD and GSH-Px activity and decrease the MDA level. The proliferation and differentiation of hematopoietic progenitor cells in bone marrow were promoted, the apoptosis of bone marrow cells was significantly up-regulated and the expression of cleaved caspase-3 and Bax was significantly reduced in SSD and catechin group. SSD and catechin have significant anti-radiation effect and its mechanism may be related to hematopoietic promoting, antioxidant and anti-apoptotic effects.

Two ellagic acids isolated from roots of Sanguisorba officinalis L. promote hematopoietic progenitor cell proliferation and megakaryocyte differentiation.

Using a bioassay-directed chromatographic separation, two ellagic acids were obtained from the ethyl acetate extract of the roots of Sanguisorba officinalis L. On the basis of chemical and spectroscopic methods, the two ellagic acids were identified as 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside and 3,3',4-tri-O-methylellagic acid. Stimulation of cell proliferation was assayed in hematopoietic progenitor cells using the Cell Counting kit-8 method. The megakaryocyte differentiation was determined in human erythroleukemia (HEL) cells using Giemsa staining and flow cytometry analysis. The ellagic acids significantly stimulated the proliferation of Baf3/Mpl cells. Morphology analysis and megakaryocyte specific-marker CD41 staining confirmed that the ellagic acids induced megakaryocyte differentiation in HEL cells. This is the first time that 3,3',4-tri-O-methylellagic acid or 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside are reported to induce megakaryopoiesis, suggesting a class of small molecules which differ from others non-peptidyl, and appears to have potential for clinical development as a therapeutic agent for patients with blood platelet disorders.