Expression of HMGA2 Cooperates with Jak2V617F in the Development of Myelofibrosis

Abstract High Mobility Group AT Hook 2 (HMGA2) is a non-histone chromatin protein that regulates gene transcription and controls cell proliferation, survival and self-renewal of stem cells. HMGA2 is expressed at a low level in normal adult hematopoietic progenitors but is highly expressed in hematopoietic progenitors of patients with Myelofibrosis (MF). However, the contribution of HMGA2 to the pathogenesis of MF remains unknown. MF is the deadliest form of myeloprolifearative neoplasm (MPN) characterized by deposition of fibrous tissues in the bone marrow, increased megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. Median survival of patients with MF is less than 6 years. The JAK2V617F mutation has been found in 50-60% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, up-regulation of HMGA2 expression has been found in association with the JAK2V617F mutation in a significant percentage of patients with MF. To understand the role of JAK2V617F mutation in the pathogenesis of MPN, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mouse hematopoietic compartments is sufficient to induce a polycythemia vera (PV)-like MPN. Recently, we have shown that deletion of EZH2 promotes the development of MF in Jak2V617F knock-in mice and EZH2 deletion increases the expression of HMGA2 in hematopoietic progenitors of EZH2-deleted Jak2V617F mice. To directly assess the effects of concomitant expression of HMGA2 and heterozygous Jak2V617F in mice hematopoietic compartments, we expressed control vector or HMGA2 in wild type and heterozygous Jak2V617F knock-in mice BM by lentiviral transduction and performed bone marrow transplantation into lethally irradiated C57BL/6 recipient mice. Whereas recipients of vector-transduced Jak2V617F knock-in BM cells exhibited a PV-like MPN characterized by increased red blood cells (RBC), hemoglobin, hematocrit and platelets in their peripheral blood, recipients of HMGA2-transduced Jak2V617F knock-in BM showed reduced hemoglobin and hematocrit parameters compared with recipients of vector-expressing Jak2V617F BM cells. Interestingly, peripheral blood neutrophil and platelet counts were further increased in transplanted animals receiving HMGA2-transduced Jak2V617F BM cells. Expression of HMGA2 also resulted in significantly larger spleen size in the transplanted animals receiving HMGA2-expressing Jak2V617F BM cells. Flow cytometric analysis showed significant increase in megakaryocytic precursors (CD41+) but decrease in erythroid precursors (CD71+/Ter119+) in the BM and spleens of transplanted animals receiving HMGA2-expressing Jak2V617F BM compared with control vector-expressing Jak2V617F BM. Furthermore, the frequency of hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+) was significantly increased in recipients of HMGA2-transduced Jak2V617F knock-in BM compared with control vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM. Histopathologic analysis revealed extensive fibrosis in the BM and spleens from recipients of HMGA2-expressing Jak2V617F mice at 32 weeks after transplantation while BM and spleens from recipients of vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM showed very little or no fibrosis at this age. Together, these data suggest that expression of HMGA2 promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F. To gain insights into the mechanisms by which expression of HMGA2 accelerates the development of MF in Jak2V617F mice, we performed RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells. Gene set enrichment and pathway analyses revealed that the genes related to chemokine, TGF-β, MAP Kinase, PI3 kinase-Akt, mTOR and WNT signaling pathways were up-regulated in HMGA2-expressing Jak2V617F mice LSK compared with vector-expressing Jak2V617F LSK cells. We also found that HMGA2 directly binds to the promoter regions of some of these target genes and regulate their expression. Further studies will validate the targets of HMGA2 and determine their contribution in MF mediated by Jak2V617F. In conclusion, our studies show that expression of HMGA2 cooperates with Jak2V617F in the development of MF. Disclosures No relevant conflicts of interest to declare.

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Neutropenia Does Not Delay Lymphopoietic Regeneration in NODSCIDcγ−/− Mice

Blood ◽

10.1182/blood.v118.21.4831.4831 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4831-4831

Author(s):

Stefanie Bugl ◽

Stefan Wirths ◽

R Müller Martin ◽

Märklin Melanie ◽

Tina Wiesner ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Nk Cell ◽

Conflicts Of Interest ◽

Control Group ◽

Early Event ◽

Wild Type ◽

Hematopoietic Stem ◽

Lymphoid Progenitors ◽

Fate Decision

Abstract Abstract 4831 Introduction: Previously it was demonstrated that lymphopoiesis is rapidly established after transplantation of wild type stem cells into lymphopenic NODSCIDcγ−/− mice. These data were interpreted as evidence for an “empty” preformed lymphopoietic niche being replenished by lymphoid progenitors. We hypothesized that antibody-induced neutropenia might influence early post transplant fate decision to myeloid rather than lymphoid differentiation resulting in delayed lymphoid reconstitution. Materials and Methods: 25,000 flow sorted CD45.2-expressing wild type Lin-/Sca1+/c-Kit+ (LSK) cells from C57BL/6 mice were transplanted into sublethally irradiated B-/T-/NK-cell deficient NODSCIDcγ−/− mice (CD45.1). Three groups of n = 7 mice received anti-Gr1 or anti-1A8 i.p. every 48 h to induce continuous antibody-mediated neutropenia vs. PBS as control. Blood was harvested at regular intervals to monitor the engraftment. After 16, 22, and 34 days, animals were sacrificed and underwent blood and bone marrow analysis. Results: Hematopoietic regeneration started with the emergence of donor-derived monocytes in all groups as well as neutrophils in the control group as early as 9 days after transplantation. On day 14, B cells were to be detected for the first time, followed by T lymphocytes approximately 20 days after transplantation. Besides the fact that neutrophils were undetectable in the antibody treated groups, the peripheral blood revealed no significant changes between the neutropenic mice and the control group at any point of time. At the bone marrow level, an increase of LSK and granulocyte-macrophage progenitors (GMPs) at the expense of megakaryocyte erythrocyte progenitor cells (MEPs) was found in neutropenic mice. Common lymphoid progenitors (CLPs), however, were not significantly different. Conclusions: The engraftment of wild type donor cells after hematopoietic stem cell transplantation into NODSCIDcγ−/− mice started with the production of monocytes and neutrophils. B-lymphocytes were detectable by day 14 after transplantation. The production of T-cells started around day 20. Continuous antibody-mediated neutropenia did not significantly delay lymphoid regeneration. Although the marrow of neutropenic mice displayed increased proliferation of granulocyte progenitors, CLPs were unchanged. We conclude that the detection of donor-derived lymphocytes in the host peripheral blood is a relatively early event after LSK transplantation. Moreover, antibody induced neutropenia is not sufficient to induce sustainable changes in early hematopoietic fate decisions on the bone marrow level. Disclosures: No relevant conflicts of interest to declare.

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Erythroid Lineage Cells Are Found In Close Association With Bone In The Marrow Microenvironment

Blood ◽

10.1182/blood.v122.21.945.945 ◽

2013 ◽

Vol 122 (21) ◽

pp. 945-945

Author(s):

Rialnat Adebisi Lawal ◽

Kathleen E. McGrath ◽

Laura M. Calvi

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Erythroid Differentiation ◽

Enzymatic Digestion ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Osteolineage Cells

Abstract Osteolineage cells within the bone marrow microenvironment have been implicated in support and regulation of hematopoietic stem cells (HSCs). Recently, augmented hypoxia-inducible factor (HIF) signaling in osteoprogenitors has been shown to expand the HSC niche, and surprisingly these cells have also been demonstrated to express erythropoietin, the critical cytokine stimulating erythropoiesis. We therefore hypothesize that endosteal cells may represent an additional regulatory site for erythropoiesis. To further delineate the role of the osteolineage cells in the support of erythropoiesis, we isolated bone associated cells (BACs) with enzymatic digestion of adult C57bl/6 mice hind limbs after bone marrow flushing and depleted the BACs of CD45+ cells to enrich for osteogenic cells. We suspected some contribution of erythroid cells to CD45- BACs, however we were surprised to find that ter119+ cells represented a large percentage of BACs after enzymatic digestion. After CD45 depletion, ter119+ cells constituted about 30% percent compared to approximately 0.85% of CD45+ cells (33 ± 4.4vs. 0.85 ± 0.26, p= 0.0018) by flow cytometric analysis. Additionally, CD45 depleted BACs had approximately 46 fold higher osteocalcin expression than CD45+ cells (1300 ± 120 vs. 28 ± 9.5, p < 0.0001), while CD45/Ter119/CD31 depleted BACs had approximately 2000 fold higher osteocalcin expression than CD45/Ter119/CD31 (+) cells (2000 ± 520 vs. 0.98 ± 0.02, p= 0.0044) by qRT-PCR, confirming enrichment of the osteoblastic lineage by this immunophenotypic panel. These data suggest that there are a large number of erythroid lineage cells associated with the BACs along the endosteum. In the bone marrow of adult mice, ter119 + cells represented approximately 85% in the CD45- pool as compared to 5% in the CD45+ cell pool. To determine if the endosteum is an active site of erythropoiesis, we quantified erythroid progenitors and precursors in the BAC pool compared to whole bone marrow (wbm) and peripheral blood (pb) by both flow cytometric analysis and colony forming assays. Flow cytometric analysis demonstrated the presence of every phase of erythroid differentiation in the BAC pool, including the presence of phenotypic MEPs (wbm vs bac vs pb: 250 ± 30 vs 84 ± 22 vs 0), BFU-E (wbm vs bac vs pb: 300 ± 14 vs 110 ± 36 vs 0 ), CFU-E (wbm vs bac vs pb: 2900 ± 2 vs 430 ± 23 vs 1 ± 0.8) and proerythroblasts (wbm vs bac vs pb: 11000 ± 2500 vs 7600 ± 1600 vs 2300 ± 920) per million cells. The phenotypic frequency of CFU-E was particularly remarkable in the BACs (430 ± 23) as compared to peripheral blood (1 ± 0.8) , demonstrating that all stages of erythroid differentiation are found in tight association with the endosteum and are not due to contamination from circulating erythroid progenitors. Colony assays were performed for CFU-E (wbm vs. bac 108 ± 16 vs 6.3 ± 2 colonies per 20,000cells plated), BFU-E (wbm vs. bac 55 ±1.0 vs 2 ±1.0; colonies per 40,000 cells plated) and myeloid progenitors (wbm vs. bac 66 ± 28 vs 11 ± 2.5 ; colonies per 10,000 cells plated) also confirmed the presence of erythroid progenitors at endosteal sites. Together these results identify the endosteal surface as a site for erythroid differentiation. The presence of all phases of erythroid lineage differentiation in the BACs suggests a potential role for osteolineage cells for maintenance and regulation of erythropoiesis. Whether osteolineage cells contribute to erythroid lineage homeostasis and/or stress response, and whether activation or damage to osteolineage cells alters local erythroid differentiation remains to be demonstrated. However our data suggest further study of the endosteum and osteolineage cells as a potential and unexpected site of erythroid regulation, which could potentially be targeted to accelerate erythropoiesis and treat anemia. Disclosures: No relevant conflicts of interest to declare.

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Loss of De Novo DNA Methylation Causes Expansion of the Mouse Hematopoietic Stem Cell Pool

Blood ◽

10.1182/blood.v116.21.835.835 ◽

2010 ◽

Vol 116 (21) ◽

pp. 835-835 ◽

Cited By ~ 2

Author(s):

Grant A. Challen ◽

Jonathan S Berg ◽

Margaret A. Goodell

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

De Novo ◽

Conflicts Of Interest ◽

Cell Activity ◽

Aberrant Methylation ◽

Wild Type ◽

Hematopoietic Stem

Abstract Abstract 835 DNA methylation is one of the major epigenetic modifications in the vertebrate genome and is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a and Dnmt3b. We observed a dynamic expression profile of Dnmt3a and Dnmt3b in the hematopoietic system with both enzymes expressed at exponentially higher levels in hematopoietic stem cells (HSCs) compared to progenitors and differentiated cells and hypothesized that some of the unique characteristics of HSCs were epigenetically regulated by Dnmt3a and Dnmt3b. To study this, we crossed Dnmt3a and -3b conditional knock-out (KO) mice to Mx1-cre mice to generate inducible single- and double-KO (dKO) mice. We performed competitive transplantation of HSCs (side-population+c-Kit+Lineage-Sca-1+ = SPKLS) from these mice along with wild-type whole bone marrow competitor and induced deletion of the Dnmt3's in the donor cells by sequential pIpC injections in the wild-type recipients. No dramatic differences were observed in primary recipients in the absence of other hematopoietic perturbation, however when we re-transplanted Dnmt3a- and Dnmt3b-KO HSCs into secondary recipients, they exhibited surprisingly high peripheral blood reconstitution compared to control HSCs (>4-fold increase in engraftment). This was reflected in the bone marrow of these mice with a corresponding >4-fold expansion of the HSC pool (phenotypically defined by any of SPKLS; CD34-Flk2-KLS; CD150+CD48-KLS) with virtually all of these cells being derived from the Dnmt3a- and Dnmt3b-KO donor HSCs. Consistent with a previous study, we observed a decline in functional output of Dnmt3a/3b-dKO HSCs in secondary transplants in terms of peripheral blood chimerism, but surprisingly these mice also exhibited a modest expansion of the HSC pool (∼2-fold), the majority of which were derived from donor Dnmt3a/3b-dKO HSCs. In subsequent tertiary and quaternary transplantation, Dnmt3 single-KO HSCs remained highly superior in peripheral blood engraftment capacity relative to control HSCs and Dnmt3a/3b-dKO HSCs (Figure 1), although the expansion of the HSC pool in all Dnmt3-KOs continued to varying degrees. This enhanced HSC activity appears to be a cell autonomous mechanism as purified Dnmt3-KO SPKLS cells from transplanted mice have much greater hematopoietic colony forming potential in vitro compared to control HSCs on a per cell basis. However the observed HSC expansion does not appear attributable to either enhanced proliferation of Dnmt3-KO HSCs or more resistance to apoptosis. The serially-transplanted Dnmt3-KO HSCs are not overtly transformed, in that the levels of differentiated blood cells are still normal and the mice appear to be healthy. This may be akin to a pre-malignant state seen in human myelodysplastic syndrome. We have performed microarray expression profiling of serially-transplanted Dnmt3-KO HSCs and identified several candidate genes which are currently being investigated as the mechanism for HSC expansion. Our data suggest ablation of de novo DNA methylation in HSCs uncouples normal self-renewal and differentiation. These studies present further evidence for the contribution of epigenetic regulation to stem cell activity and provide a tantalizing link between potential aberrant methylation in HSCs contributing to leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

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Loss of EZH2 Inhibits Erythropoiesis and Accelerates the Development of Myelofibrosis in Jak2V617F Knock-in Mice

Blood ◽

10.1182/blood.v124.21.159.159 ◽

2014 ◽

Vol 124 (21) ◽

pp. 159-159

Author(s):

Yue Yang ◽

Hajime Akada ◽

Dipmoy Nath ◽

Robert E Hutchison ◽

Golam Mohi

Keyword(s):

Peripheral Blood ◽

Blood Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Flow Cytometric Analysis ◽

Gene Set Enrichment Analysis ◽

Jak2v617f Mutation ◽

Interferon Response ◽

Hematopoietic Stem ◽

Erythroid Precursors

Abstract EZH2, a component of the polycomb repressive complex 2 (PRC2), catalyzes the trimethylation of histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Inactivating mutations of EZH2 have been found in myelodysplastic syndromes and myeloproliferative neoplasms (MPNs) including myelofibrosis (MF). EZH2 mutations are associated with poor prognosis in patients with MF. However, the contribution of EZH2 mutations in the pathogenesis of MF remains unknown. The JAK2V617F mutation has been found in a majority of cases of MPNs including ~50% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, inactivating EZH2 mutations co-exist with JAK2V617F mutation in significant cases of MF. To understand the role of JAK2V617F in MPNs, we previously generated a conditional Jak2V617F knock-in mouse, which exhibits all the features of human PV. To determine if EZH2 mutations cooperate with JAK2V617F mutation in MF, we crossed the conditional EZH2 knock-out mice with conditional Jak2V617F knock-in mice and assessed the effects of concomitant deletion of EZH2 and expression of heterozygous Jak2V617F in mice hematopoietic compartments. Whereas Jak2V617F expression resulted in significant increase in red blood cells (RBC), hemoglobin, hematocrit, white blood cells and platelets in the peripheral blood of the Jak2V617F knock-in mice, deletion of EZH2 significantly reduced the RBC, hemoglobin, and hematocrit parameters in Jak2V617F knock-in mice. Interestingly, platelet counts were further increased in EZH2-deleted Jak2V617F-expressing mice. Flow cytometric analysis showed significant increase in CD71+Ter119neg/lo early erythroid precursors and decrease in CD71+Ter119high late erythroid precursors in the bone marrow (BM) and spleens of EZH2-deleted Jak2V617F mice suggesting a defect in erythroid differentiation upon EZH2 deletion in Jak2V617F mice. Notably, megakaryocytic precursors (CD41+CD61+) were significantly increased in the BM and spleens of EZH2-deleted Jak2V617F mice consistent with increased number of platelets in the peripheral blood of these mice. Similar to human PV, Jak2V617F expression resulted in cytokine-independent CFU-E colonies in the BM and spleens of Jak2V617F knock-in mice. However, deletion of EZH2 markedly inhibited cytokine-independent CFU-E colonies in the BM and spleens of Jak2V617F knock-in mice. Histopathologic analysis revealed extensive fibrosis in the BM and spleens of EZH2-deleted Jak2V617F mice at 24 weeks after induction while heterozygous Jak2V617F knock-in mice BM and spleens showed very mild fibrosis at this age. Control and EZH2-deficient mice did not exhibit any fibrosis in their BM or spleens. In order to determine whether the effects of EZH2 deletion in Jak2V617F mice were cell autonomous, BM cells from pIpC induced control, EZH2-deficient, Jak2V617F knock-in and EZH2-deleted Jak2V617F-expressing mice were transplanted into lethally irradiated syngeneic recipient mice. Transplanted animals receiving EZH2-deleted Jak2V617F BM developed severe fibrosis in their BM and spleens within 8 weeks after transplantation. Furthermore, recipients of EZH2-deleted Jak2V617F BM exhibited severe anemia and became moribund by 8 weeks after transplantation. In contrast, transplanted animals receiving control, EZH2-deficient or Jak2V617F BM did not exhibit fibrosis at 8 weeks after transplantation. Thus, the phenotypes observed in EZH2-deficient Jak2V617F mice are hematopoietic cell-autonomous. Together, these data suggest that loss of EZH2 inhibits erythropoiesis, promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F. To gain insights into the mechanisms by which EZH2 deficiency accelerates the development of MF in Jak2V617F mice, we performed microarray gene expression analysis on purified long-term hematopoietic stem cells (LT-HSC; Lin-c-kit+Sca-1+CD34-Flk2-). Gene set enrichment analysis revealed that interferon response-related genes and the genes related to TNF signaling pathway were up-regulated in LT-HSC of EZH2-deficient Jak2V617F mice compared with Jak2V617F LT-HSC. Further studies will validate the targets of EZH2 that are de-repressed upon EZH2 deletion in MF induced by Jak2V617F. In conclusion, our studies show that loss of EZH2 cooperates with Jak2V617F mutation in the development of MF. Disclosures No relevant conflicts of interest to declare.

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Sf3b1 K700E Mutation Impairs Pre-mRNA Splicing and Definitive Hematopoiesis in a Conditional Knock-in Mouse Model

Blood ◽

10.1182/blood.v126.23.140.140 ◽

2015 ◽

Vol 126 (23) ◽

pp. 140-140 ◽

Cited By ~ 3

Author(s):

Annalisa Mupo ◽

Vijitha Sathiaseelan ◽

Michael Seiler ◽

David Kent ◽

Shouyong Peng ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Wild Type ◽

Aberrant Splicing ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Stem And Progenitor Cells

Abstract Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by dysplastic hematopoiesis and peripheral blood cytopenias. Recently, somatic mutations affecting components of the spliceosomal machinery have been discovered in the majority of MDS patients. SF3B1 mutations are most frequent and strongly correlate with the presence of bone marrow ring sideroblasts and a favorable prognosis. SF3B1 mutations, including the K700E substitution which accounts for more than 50% of all mutations, are missense, heterozygous and cluster in a hotspot within the heat domain of the protein suggesting that they are gain-of-function variants. The molecular effects of SF3B1 mutations and the mechanisms through which they drive clonal expansion and dyserythropoiesis remain obscure. Therefore, to assess their molecular and phenotypic consequences, we generated a mouse model carrying a conditional floxed knock-in allele (Sf3b1flox-K700E/+) by homologous recombination of JM8 murine embryonic stem cells. To induce expression of Sf3b1 K700E in adult hematopoietic stem and progenitor cells, Sf3b1flox-K700E/+/Mx1-Cre+ were injected with pIpC from 4-8 weeks of age. Here we report the initial characterization of these animals. Monthly peripheral blood counts from mutants and wild-type (WT) littermates starting one month post-pIpC injection showed a reduction in hemoglobin levels (at 8 weeks WT=17g/dl mut=14.5g/dl, p<0.03). Additionally, flow cytometric analysis of bone marrow samples demonstrated a modest but consistent decrease in late erythroid progenitor cells (Ter119+ and CD71-/low). The myeloid compartment showed relative expansion of Gr1+/Mac1+ and Mac1+ cells whereas analysis of hematopoietic stem and progenitor cells (HSPCs) revealed a decrease in HSCs (% of total events WT=0.04%; Sf3b1flox-K700E/+=0.01%) in mutant mice. In competitive transplantation experiments into sub-lethally irradiated syngeneic recipients we observed a lower engraftment potential of Sf3b1flox-K700E Lin-ve HSPCs (CD45.2) compared to wild-type cells (CD45.1). Flow cytometric analysis of peripheral blood of recipient animals showed that Sf3b1flox-K700E cells contributed more to the myeloid lineage than wild-type cells (Sf3b1flox-K700E Mac1+/Gr1+ 8.95%; Mac1+ 15% vs WT Mac1+/Gr1+ 4.08%; Mac1+ 5.57%). At a median follow-up of 56 weeks, mutant animals did not show decreased survival or signs of illness as compared to WT controls. Finally, as Sfb31 mutations are predicted to affect splicing of pre-mRNA and consequently alter the gene expression, we performed RNAseq analysis in unselected and Lin-ve bone-marrow cells from mutant and controls animals. Comparison between wt and mutant samples showed deregulated expression of genes implicated in human MDS (Mmp9, Puma, Bcl2l1). We then looked at the pattern of aberrant splicing promoted by Sf3b1flox-K700E, and found that mutant animals have an increased use of cryptic 3'' splice sites (ss) throughout their genome. We showed that the majority of these alternative 3' ss are novel and we characterized them as being located 15 to 24 nucleotides upstream from the canonical 3' ss and associated with sequence features including a shorter polypyrimidine tract and an enrichment of adenines -8 to -18 bases upstream of the cryptic 3' ss. Interestingly, similar features have been reported in human cancers with SF3B1 hotspot mutations. We predict that ~33% of the mRNAs affected by aberrant splicing will include an aberrant premature termination codon, promoting RNA degradation through nonsense-mediated decay. In conclusion, our conditional Sf3b1K700E knock-in mouse is a faithful molecular model of the consequences of these mutations in the mouse hematopoietic system. The mild phenotype we observe in comparison to SF3B1-mutant human MDS may be explained by the requirement for additional mutations to progress to overt MDS and is more reminiscent of SF3B1-associated clonal hemopoiesis, relatively common phenomenon in elderly humans without overt hematological abnormalities. Additionally, our initial characterization of novel splice sites preferentially recognised by the mutant Sf3b1 protein suggests that transcriptional consequences of the mutation may differ between species, dependant on the degree of conservation of the relevant intronic regions. Disclosures Seiler: H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Campbell:14M genomics: Other: Co-founder and consultant.

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ALL Cells Mobilized by AMD3100 Are More Proliferative, and Remain In the Circulation Longer Than Normal HSC, Enhancing the Action of Vincristine

Blood ◽

10.1182/blood.v116.21.2137.2137 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2137-2137 ◽

Cited By ~ 1

Author(s):

Linda J. Bendall ◽

Robert Welschinger ◽

Florian Liedtke ◽

Carole Ford ◽

Aileen Dela Pena ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Bone Marrow Microenvironment ◽

Hematopoietic Progenitors ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Cell Mobilization ◽

Cycle Dependent

Abstract Abstract 2137 The chemokine CXCL12, and its receptor CXCR4, play an essential role in homing and engraftment of normal hematopoietic cells in the bone marrow, with the CXCR4 antagonist AMD3100 inducing the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. We have previously demonstrated that AMD3100 similarly induces the mobilization of acute lymphoblastic leukemia (ALL) cells into the peripheral blood. The bone marrow microenvironment is thought to provide a protective niche for ALL cells, contributing to chemo-resistance. As a result, compounds that disrupt leukemic cell interactions with the bone marrow microenvironment are of interest as chemo-sensitizing agents. However, the mobilization of normal hematopoietic stem and progenitor cells may also increase bone marrow toxicity. To better evaluate how such mobilizing agents affect normal hematopoietic progenitors and ALL cells, the temporal response of ALL cells to the CXCR4 antagonist AMD3100 was compared to that of normal hematopoietic progenitor cells using a NOD/SCID xenograft model of ALL and BALB/c mice respectively. ALL cells from all 7 pre-B ALL xenografts were mobilized into the peripheral blood by AMD3100. Mobilization was apparent 1 hour and maximal 3 hours after drug administration, similar to that observed for normal hematopoietic progenitors. However, ALL cells remained in the circulation for longer than normal hematopoietic progenitors. The number of ALL cells in the circulation remained significantly elevated in 6 of 7 xenografts examined, 6 hours post AMD3100 administration, a time point by which circulating normal hematopoietic progenitor levels had returned to baseline. No correlation between the expression of the chemokine receptor CXCR4 or the adhesion molecules VLA-4, VLA-5 or CD44, and the extent or duration of ALL cell mobilization was detected. In contrast, the overall motility of the ALL cells in chemotaxis assays was predictive of the extent of ALL cell mobilization. This was not due to CXCL12-specific chemotaxis because the association was lost when correction for background motility was undertaken. In addition, AMD3100 increased the proportion of actively cells ALL cells in the peripheral blood. This did not appear to be due to selective mobilization of cycling cells but reflected the more proliferative nature of bone marrow as compared to peripheral blood ALL cells. This is in contrast to the selective mobilization of quiescent normal hematopoietic stem and progenitor cells by AMD3100. Consistent with these findings, the addition of AMD3100 to the cell cycle dependent drug vincristine, increased the efficacy of this agent in NOD/SCID mice engrafted with ALL. Overall, this suggests that ALL cells will be more sensitive to effects of agents that disrupt interactions with the bone marrow microenvironment than normal progenitors, and that combining agents that disrupt ALL retention in the bone marrow may increase the therapeutic effect of cell cycle dependent chemotherapeutic agents. Disclosures: Bendall: Genzyme: Honoraria.

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RhoA Is An Essential Regulator of Mitosis and Survival During Hematopoietic Stem Cell Differentiation to Multipotent Progenitors

Blood ◽

10.1182/blood.v118.21.1273.1273 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1273-1273

Author(s):

Xuan Zhou ◽

Jaime Meléndez ◽

Yuxin Feng ◽

Richard Lang ◽

Yi Zheng

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Intracellular Signaling ◽

Conflicts Of Interest ◽

Stem Cell Differentiation ◽

Conditional Knockout ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Differentiated Cells ◽

Multipotent Progenitors

Abstract Abstract 1273 The maintenance and differentiation of hematopoietic stem cells (HSC) are critical for blood cell homeostasis, which is tightly regulated by a variety of factors. In spite of extensive investigation of HSC biology, however, the mechanism of regulation of HSC and progenitor cell division, particularly the unique molecular events controlling the mitosis process during HSC differentiation, remains unclear. RhoA GTPase is a critical intracellular signaling nodal that has been implicated in signal transduction from cytokines, chemokines, wnt/notch/shh, and adhesion molecules to impact on cell adhesion, migration, cell cycle progression, survival and gene expression. Recent mouse genetic studies in keratinocytes and embryonic fibroblast cells showed that RhoA is a key regulator of mitosis. By using an interferon-inducible RhoA conditional knockout mouse model (Mx-cre;RhoAlox/lox), we have made the discovery that RhoA plays an indispensible role in primitive hematopoietic progenitor differentiation through the regulation of mitosis and survival. RhoA deficient mice die at ∼10 days because of hematopoietic failure, as evidenced by a loss of bone marrow, splenocyte and PB blood cells. Syngenic as well as reverse transplant experiments demonstrate that these effects are intrinsic to the hematopoietic compartment. RhoA loss results in pancytopenia associated with a rapid exhaustion of the lin−c-kit+ (LK) phenotypic progenitor population (within 4 days after two polyI:C injections). Meanwhile, the lin−c-kit+sca1+ (LSK) primitive cell compartment is transiently increased in BM after RhoA deletion due to a compensatory loss of quiescence and increased cell cycle. Interestingly, we find that within the LSK population, there is a significant accumulation of LSKCD34+Flt2− short-term HSCs (ST-HSC) and a corresponding decrease in frequency of LSKCD34+Flt2+ multipotent progenitors (MPPs). Consistent with these phenotypes, the LK and more differentiated hematopoietic cell populations of RhoA knockout mice show an increased apoptosis while the survival activities of LSK and more primitive compartments of WT and RhoA KO mice remain comparable. These data suggest that RhoA plays an indispensible role in the step of ST-HSCs differentiation to MPP cells, possibly through the regulation of MPP cell survival. This hypothesis is further supported by a competitive transplantation experiment. Deletion of RhoA in a competitive transplantation model causes an extinction of donor derived (CD45.2+) differentiated cells (myeloid, erythroid, T and B cells) in the peripheral blood. Interestingly, bone marrow CD45.2+ LSK cells are only marginally affected by deletion of RhoA and RhoA−/− LSK cells are able to engraft into 2nd recipient, whereas CD45.2+ LK and more differentiated cells are mostly eliminated after RhoA deletion. This effect is associated with a decrease in the survival of CD45.2+ RhoA−/− LK, but not LSK cells. Further in vitro culture of isolated lin− progenitors demonstrates that RhoA deficiency results in a failure of cytokinesis, causing an accumulation of multinucleated cells, further suggesting that RhoA is essential for the cytokinesis of hematopoietic progenitors. Surprisingly, the well-defined Rho downstream target, actomyosin machinery, does not appear to be affected by RhoA knockout. We are further exploring the mechanism of RhoA contribution to the differentiation of HSCs by dissecting the signaling and functional relationship of RhoA regulated survival activity and cell cycle mitosis in early hematopoietic progenitors. Disclosures: No relevant conflicts of interest to declare.

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Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Yan Xiu ◽

John M Ashton ◽

Lianping Xing ◽

Yoshikazu Morita ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Double Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Physiological Processes ◽

Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽

10.1182/blood.v120.21.1224.1224 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1224-1224

Author(s):

Junke Zheng ◽

Chengcheng Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Actin Binding ◽

Wild Type ◽

Cell Fates ◽

Hematopoietic Stem ◽

Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Sparc Is Dispensable for Murine Hematopoiesis, Despite Its Suspected Role in 5q- Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v118.21.4822.4822 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4822-4822

Author(s):

Kavitha Siva ◽

Pekka Jaako ◽

Kenichi Miharada ◽

Emma Rörby ◽

Mats Ehinger ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Knockout Mice ◽

Peripheral Blood ◽

Blood Cells ◽

Lethal Dose ◽

Wild Type ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Expression Levels

Abstract Abstract 4822 Hematopoiesis is a complex process where a limited number of stem cells give rise to all mature blood cells. It involves interplay of several factors, many of which are yet to be identified. In a search for novel regulators of hematopoiesis, we chose to study SPARC (Secreted Protein Acidic and Rich in Cysteine, also known as Osteonection and BM40) because it is downregulated upon hematopoietic differentiation (Bruno et al., Mol Cell Biol, 2004) and might therefore play a role in the regulation of hematopoietic stem cells (HSC). SPARC is a matricellular protein that forms a major component of bone and is ubiquitously expressed in a variety of tissues. It is the founding member of a family of SPARC-like proteins. Several publications have indicated an important role for SPARC in hematopoiesis. In particular – knockdown of SPARC in zebrafish embryos resulted in an altered number of circulating blood cells, and a knockout mouse model showed thrombocytopenia and reduced erythroid colony formation. We carried out an in depth phenotypic and functional analysis of the hematopoietic system of SPARC knockout mice; using it as a model to gain insight into the role of SPARC in hematopoiesis. These mice are viable and fertile but show severe osteopenia and age-onset cataract at about six months of age. They also show an altered response to tumour growth and wound healing. We used mice (129SVJ background) (Gilmour et al. EMBO, 1998) that were less than six months old. These mice had normal peripheral blood counts and the bone marrow and spleen showed no alterations in morphology or cellularity. A detailed phenotypic analysis of precursors within the bone marrow showed no significant differences in myelo-erythroid precursors as compared to wild types (n=6). Though in vitro, the precursors showed lower ability to form BFU-E (n=5, p=0.048). In transplantations of lethally irradiated recipient mice, SPARC knockout cells gave rise to multi-lineage long-term reconstitution. Also, when competed with wild type cells, they provided reconstitution as well as their wild type counterparts. When SPARC knockout mice (n=8) were transplanted with wild type cells, there was normal reconstitution, indicating that a SPARC deficient niche can fully support normal hematopoiesis. We also tested if SPARC deficient mice respond differently to hematopoietic stress. We subjected mice (n=7) to sub lethal dose of irradiation and to experimentally induced anemia (n=7) and followed recovery by analyzing peripheral blood counts. In both SPARC knockouts and wild type mice, the blood counts recovered in a similar fashion. In conclusion, we find that SPARC is dispensable for murine hematopoiesis. It is possible that there are compensatory mechanisms involving other members of the SPARC family that ultimately lead to normal hematopoiesis in the murine model. In humans, SPARC maps to the deleted region in 5q MDS and has been reported to be 71 % down regulated in patient samples (Lehmann et al. Leukemia, 2007). It is the most prominent gene that is up regulated in response to lenalidomide, a drug that inhibits the malignant clone (Pellagatti et al. PNAS, 2007). SPARC is thus increasingly speculated to be involved in the pathophysiology of this hematopoetic disease. We analysed the expression levels of SPARC mRNA in the hematopoietic stem/progenitor cell compartment and found high expression levels in the CD34+ fraction of human cord blood cells. In contrast, there is very low level of SPARC expression in all compartments of murine HSCs. Therefore SPARC function may play a more important role in human hematopoiesis than in murine blood cell regulation. Disclosures: No relevant conflicts of interest to declare.

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