The Role Of tet2 DNA Methylcytosine Dioxygenase In Zebrafish Early Hematopoiesis

Abstract Loss-of-function mutations in Ten-Eleven-Translocation 2 (TET2) gene have been identified in various human myeloid and lymphoid malignancies. Recently, the TET gene family (TET1, TET2, and TET3) was found to function as DNA methylcytosine dioxygenase that is able to oxidize 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). In Tet2-deficient mouse models, Tet2 has been shown to play an important role in regulating self-renewal and differentiation of hematopoietic stem cells. These Tet2-deficient mice would gradually develop a chronic myeloid neoplasm resembling human chronic myelomonocytic leukemia suggesting that TET2 may function as a tumor suppressor. In the present study, we investigated the role of tet2 in zebrafish early hematopoiesis. During zebrafish early development, the expression of tet1, tet2, and tet3 by qRT-PCR can be detected mainly after the segmentation stage (26-somite), with fluctuated expression levels thereafter. Whole-mount in situ hybridization revealed that tet2 expression was strong over aorta-gonad-mesonephros region at 48 hours post-fertilization (hpf). Morpholino oligonucleotide (MO) knock-down of tet2 increased the expression of tet1, tet3, dnmt3aa, gata-1, alpha-Hb and fli1a (48 hpf) as well as rag2 and lck (4 days post-fertilization), and the expression of spi1b and mpo decreased (48 hpf). The expression of primitive hematopoietic stem cell markers scl and lmo2, as well as dnmt3ab, beta-Hb, l-plastin, and rag1 were unaffected. The levels of 5-mC and 5-hmC measured by ELISA were also decreased after MO knock-down of tet2. The number of gata-1 expressing red blood cells increased after tet2 MO knock-down as evaluated by flow-cytometry indicating that tet2 deficiency increased erythropoiesis. These preliminary results suggest that tet2 might play a role in the epigenetic regulation of zebrafish early hematopoiesis including erythropoiesis. Recently, transcription activator-like effector nuclease (TALEN) has been shown to generate targeted genomic editing in zebrafish. To validate our observation, we therefore utilized customized TALENs pair to generate tet2 knock-out zebrafish animal model. We designed a pair of TALENs targeting first exon of tet2 and our tet2 TALENs were able to generate insertion and/or deletion in targeted region of tet2 exon 1 in 25% to 44% zebrafish embryos. We obtained a total of fifteen different tet2 mutation genotypes F1 fish, and seven of them were predicted to cause early termination of transcription. The in-cross of these F1 genotypes matched the Mendelian inheritance. The tet2-/- knock-out F2 zebrafish is not embryonic lethal and can grow to sexually mature adult fish. The detailed analysis of tet2-/- knock-out zebrafish early hematopoiesis will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants

Blood ◽

10.1182/blood.v126.23.2373.2373 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2373-2373

Author(s):

Erica Bresciani ◽

Blake Carrington ◽

Erika Mijin Kwon ◽

Marypat Jones ◽

Stephen Wincovitch ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Time Lapse ◽

Loss Of Function ◽

Transcription Activator ◽

Zebrafish Model ◽

Hematopoietic Stem ◽

Definitive Hematopoiesis

Abstract Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models. In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain,, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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SETD2, a H3K36 Lysine Methyltransferase, Is Essential for Adult Normal Hematopoiesis and Leukemia Stem Cells

Blood ◽

10.1182/blood.v128.22.1055.1055 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1055-1055

Author(s):

Yile Zhou ◽

Yunzhu Dong ◽

Jiachen Bu ◽

Xiaomei Yan ◽

Yoshihiro Hayashi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

The Other ◽

Leukemia Stem Cells ◽

Transcriptional Networks ◽

Loss Of Function ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are characterized by their capability for self-renewal and multi-potency. Hematopoiesis is dynamically controlled by the interplay between epigenetic and transcriptional networks. Dysregulation of these networks can lead to unfitness of hematopoiesis, cell transformation, and hematological diseases. The human SETD2 gene was originally isolated from HSCs and progenitors. SETD2 is a histone methyltransferase, which specifically catalyzes tri-methylation of histone 3 lysine 36 (H3K36me3). SETD2 functions as a tumor suppressor, as loss-of-function mutations have been identified in many cancers. However, the role of SETD2 in hematopoiesis has not been fully understood. To assess the function of Setd2 in hematopoiesis, we generated three Setd2 mouse alleles with Crispr/CAS9 technology; Setd2F2478/WT knock-in, Setd2Exon6-Δ/WT, and Setd2-Exon6flox/flox/Mx1-Cre conditional knockout alleles, as homozygous Setd2 mutation showed embryonic lethality. Setd2-F2478 point mutation, which is located in the SRI domain, can express SETD2 mutant protein but completely lose the interaction with RNA pol II. Setd2Exon6-Δ/WT allele results in a frame shift and nonsense mediated decay of Setd2 mRNA and protein. After induction of excision with pIpC injection, Setd2-exon6flox/flox/Mx1-Cre+ (Setd2Exon6-Δ/Δ) mice showed severe anemia, increased platelet count, and a reduction in bone marrow (BM) cellularity compared to wild-type (WT) mice, while Setd2F2478/WT and Setd2Exon6-Δ/WT mice did not show any obvious hematological changes. The Lin- Sca-1+ c-Kit+ (LSK) population in Setd2Exon6-Δ/Δ mice was 2.5-fold decreased compared to those in WT, while the LSK populations in Setd2F2478/WT and Setd2Exon6-Δ/WT mice were comparable with those in WT. Interestingly, all three of these Setd2 mutant alleles showed a higher frequency of Lin- Sca-1- c-Kit+ (LK) cells in the BM. In the LK populations, we found an increased CMP population in Setd2F2478/WT and Setd2Exon6-Δ/WT mice; of note, the CMP population in the Setd2Exon6-Δ/Δ mice had disappeared while the MEP population expanded with higher expression of CD16/32. Next, to assess the function of the HSPCs, we performed CFU assays and competitive bone marrow transplantations (CBMT). Consistent with our phenotypic findings, the number of colonies derived from Setd2F2478/WT and Setd2Exon6-Δ/WT BM cells was increased in the first two passages, while the number of colonies derived from Setd2Exon6-Δ/Δ mice was significantly decreased. In CBMT, we found that mice transplanted with Setd2Exon6-Δ/Δ BM cells showed anemia and an impaired BM reconstitution, compared to the control (p = 0.0002). On the other hand, the Setd2F2478/WT and Setd2Exon6-Δ/WT models showed comparable capabilities of BM reconstitution. Taken together, these results suggest that Setd2 has an essential role in the maintenance of adult hematopoiesis. SETD2 mutations (mainly one allele mutation) have been frequently identified in acute leukemia, especially in about 22% of MLL leukemia. To understand the role of SETD2 in leukemic stem cells, Setd2 mutant mice were bred with the Mll-AF9 knock-in mouse. The Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice showed higher frequencies of LK and LSK populations compared to Mll-AF9 mice, indicating that Setd2 mutations may increase the stemness of leukemia stem cells (LSCs). The cells derived from Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice resulted in a significantly higher yield of colonies and growth advantage in serial replating CFU assay compared to the cells derived from Mll-AF9 mice. After BMT of equal numbers of cells from Mll-Af9 or Mll-AF9/ Setd2F2478/WT mice into recipient mice, the Mll-AF9/ Setd2F2478/WTBMT mice developed leukemia with significantly shortened latencies compared with MLL-Af9 BMT mice. In conclusion, our data suggests that Setd2 plays an important role in maintaining normal HSPCs. Half the doses of Setd2 can still maintain the normal hematopoiesis while a total loss of Setd2 leads to a failure of hematopoiesis. In leukemia, heterozygous mutants of Setd2 can accelerate leukemogenesis by expanding LSCs. Whether the remaining WT allele is required for leukemia maintenance is unclear. Further reduction of Setd2 levels, or complete deletion of the other WT allele, may diminish SETD2-mutated leukemia. Such tumor vulnerability can be explored as a therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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S1P1 Agonists for Use as Adjunct Mobilizing Agents

Blood ◽

10.1182/blood.v116.21.826.826 ◽

2010 ◽

Vol 116 (21) ◽

pp. 826-826 ◽

Cited By ~ 3

Author(s):

Nadia Harun ◽

Marilyn Thien ◽

Julius G Juarez ◽

Kenneth Francis Bradstock ◽

Linda J. Bendall

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Receptor Expression ◽

Hematopoietic Stem ◽

Knock Out ◽

S1p Receptor ◽

Knock Out Mouse

Abstract Abstract 826 Harvesting hematopoietic stem cells (HSC) mobilised into peripheral blood (PB) for transplantation is mediated through bone marrow (BM) retentive and egress factors. Factors that retain hematopoietic stem cells (HSC) in the BM are well defined, with CXCL12 and VCAM1 playing major roles. However, the factors involved in the egress of HSC from the BM into the peripheral blood (PB) are currently uncharacterised. Sphingosine-1-Phosphate (S1P) is a lymphoid organ egress factor for lymphocytes, mediated through the S1P1 receptor, which is also expressed on HSC. We hypothesised that S1P mediates the egress of HSC out of the BM and into the PB. Our laboratory used a number of different mouse models with various S1P levels or S1P receptor expression to elucidate the role of the S1P gradient between the BM and PB. Sphingosine kinase-1 knock-out (SK1KO) mice were utilized for their reduced PB S1P levels. A sphingosine lyase inhibitor 4′deoxypyridoxine (DOP) was used to increase BM S1P levels. Mice treated with FTY720 for 14h had suppressed S1P1 expression and an S1P1 conditional knock-out mouse was also generated by our group. Animals were also treated with S1P receptor agonists such as SEW2871. Mobilisation experiments, competitive repopulation assays and chemotaxis assays were performed utilizing the various models. Plasma from SK1KO mice had a reduced capacity to induce migration in haematopoietic progenitor cells (HPC), confirming the chemokine activity of S1P. Consistent with this, AMD3100 induced mobilization was inhibited in SK1KO mice and DOP treated mice, demonstrating the role of an S1P gradient in HPC mobilization. Mice treated with FTY720 significantly inhibited AMD3100, although not G-CSF, mediated mobilisation of HPC in mice. No HPC accumulation was detected in secondary lymphoid organs such as lymph nodes or spleen. Most importantly, FTY720 treatment reduced the number of transplantable HSC in the blood following AMD3100-mediated mobilisation using a competitive repopulation assay. Our laboratory also generated an S1P1 conditional knock-out mouse. When mobilised with AMD3100, these S1P1 knock-out animals displayed a marked reduction in HPC mobilisation compared to wild-type animals. Finally, the S1P1 agonist SEW2871 increased HPC mobilisation synergistically, by approximately 2 fold when combined with AMD3100, but not G-CSF. S1P supports the egress of HSC from the BM into the PB following inhibition of the CXCL12/CXCR4 axis. S1P1 conditional knock-out mice display a significantly reduced mobilising capacity. S1P receptor agonist, SEW2871, acts synergistically with AMD3100 to increase HPC mobilisation in vivo, raising the possibility that such a combination may increase the efficiency of HSC collection for transplantation purposes. Disclosures: No relevant conflicts of interest to declare.

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Dnmt3a Deletion Predisposes Hematopoietic Stem Cells To Malignant Transformation

Blood ◽

10.1182/blood.v122.21.4198.4198 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4198-4198

Author(s):

Allison Mayle ◽

Liubin Yang ◽

Grant A Challen ◽

Ting Zhou ◽

Vivienne I. Rebel ◽

...

Keyword(s):

Malignant Transformation ◽

Dna Methyltransferase ◽

De Novo ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Hematologic Malignancies ◽

Myeloproliferative Disease ◽

Loss Of Function ◽

Hematopoietic Stem ◽

Knock Out

Abstract DNA methyltransferase 3A (DNMT3A), a de novo DNA methyltransferase, is mutated in various hematological malignancies affecting both myeloid (20%), mixed (50%), and lymphoid (18%) malignancies and is associated with poor prognosis. The most frequently reported DNMT3A mutation is R882 in acute myeloid leukemia (AML), which results in altered enzyme activity, but various missense and nonsense mutations have also been found throughout the gene, suggesting that loss-of-function mutations in DNMT3A may also contribute to leukemogenesis. Our group recently showed that transplantation of HSCs from Dnmt3a knock-out (KO) mice led to increased hematopoietic stem cell (HSC) self-renewal and inhibition of differentiation, but was insufficient to cause transformation. However, in these experiments, Dnmt3a-KO HSCs were transplanted alongside wild-type whole bone marrow to quantitate HSC function, potentially protecting against malignant transformation. We hypothesized that if Dnmt3a-KO HSCs were transplanted alone, a predisposition to transformation would be uncovered. We established a large non-competitive transplantation cohort receiving 500 control or Dnmt3a-KO HSCs and monitored the mice closely for disease. Strikingly, mice with Dnmt3a-KO HSCs had significantly shorter survival (246d vs 467d, p<0.0001, Figure 1). As mice succumbed to disease, we analyzed histological changes in hematopoietic organs and performed CBCs and immunophenotyping to diagnose the diseases. We identified multiple disease classes within the Dnmt3a-KO recipients, including T-cell acute lymphoblastic leukemia, myeloproliferative disease (MPD), myelofibrosis (MF), and myelodysplastic syndromes (MDS). The relatively long disease latency suggests that acquisition of secondary hits promotes disease; identification of these secondary mutations is ongoing.Figure 1Mice transplanted with Dnmt3a-KO HSCs succumb to hematologic malignanciesFigure 1. Mice transplanted with Dnmt3a-KO HSCs succumb to hematologic malignancies Here, we show that Dnmt3a deletion in noncompetitive transplanted HSCs leads to an array of hematologic disorders that models the spectrum of disorders seen in human malignancies. Since DNMT3A mutations are known early genetic lesions in leukemia development, mutations that cooperate with DNMT3A might influence the type of disease developed. This mouse model serves to validate an important role for Dnmt3a in the development of hematologic malignancies, and is also valuable for the study of future targeted therapies. Mice transplanted with Dnmt3a-KO HSCs died significantly earlier than mice transplanted with control HSCs (median survival 246 days and 467 days, p<0.0001). Fifty and 20 female mice were transplanted with 500 Dnmt3a-KO or control HSCs respectively, all at 6-8 weeks of age. Censored points indicate mice that were euthanized for unrelated reasons. Disclosures: No relevant conflicts of interest to declare.

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Role of the Ether-a-gò-gò-Related Gene 1B Isoform in Hematopoiesis

Blood ◽

10.1182/blood.v120.21.1222.1222 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1222-1222

Author(s):

Serena Pillozzi ◽

Marika Masselli ◽

Marinella Veltroni ◽

Emanuele De Lorenzo ◽

Antonella Fiore ◽

...

Keyword(s):

Ion Channels ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Membrane Receptors ◽

Stress Tests ◽

Related Gene ◽

Hematopoietic Stem ◽

Knock Out ◽

Functional Defect

Abstract Abstract 1222 Normal hematopoiesis is characterized by the tuned regulation of self renewal, proliferation, differentiation and migration of hematopoietic stem cells and HSC-derived multipotent and lineage-committed hemopoietic progenitor cells. This regulation is a complex process, which requires several levels of control provided by the activity of numerous membrane receptors and soluble proteins, which mediate the communication among hematopoietic cells, and between the cells and the microenvironment. In this contest, ion channels must be mentioned. Indeed, besides their canonical role in cell excitability, they can also modulate different cellular functions, such as proliferation, apoptosis and differentiation, in both excitable and non excitable cells. This role is also relevant in hematopoietic cells, where ion channels have a clear role in different functions of fully differentiated cells (Int Rev Cell Mol Biol. 2010;279:135–190). On these bases, we analyzed the role of ether a gò-gò-related gene 1 (ERG1) channels in normal hematopoiesis. In particular, we performed lack of function studies using a murine ERG1 knock out (KO) model (in SV129 strain). Since mice with a general and complete KO of the whole ERG1 gene die during early development, we analyzed mice with a selective deletion of the ERG1B isoform (ERG1B−/− mice, Mol Cell Biol. 1003;23(6):1856–1862), which is the ERG1 isoform mostly expressed in leukemic blasts (Blood. 2007;110(4):1238–1250). Such mice are viable and do not show any life threatening physical or behavioral abnormalities. First, we verified ERG1 transcripts expression in wild type SV129 mice (WT): both transcripts were expressed in spleen and thymus with higher values for the ERG1A isoform; ERG1B isoform presented a good expression level in bone marrow (BM) especially in the Sca-1+ population. Consequently we performed experiments to evaluate the role of ERG1B in normal hematopoiesis. Young (0–3 months old) KO mice presented a reduced number of CFUs (colony forming units) in the BM. CFUs levels were restored in adult mice. BM of KO mice showed hypocellularity and an increased number of megakaryocytes intriguingly associated with a reduction of erythrocytes (Ter119+). As evidenced by the histological analysis, splenomegaly of KO mice could be traced back to a great amount of mature red blood cells, filling the interfollicular space of the red pulp and subcapsulary space. Such splenic congestion in ERG1B−/− mice is accompanied by a relative decrease in the number of megakaryocytes, as well as by a reduced capacity to develop CFUs. On the whole, these data are suggestive of a failure of spleen hemopoiesis, with a concomitant red cell engulfment that lead to a putative erythropoiesis that occurs locally in the spleen maybe due to reprogrammation of hematopoietic cells of different lineage. To better characterize hematopoiesis in ERG1B−/− mouse model we performed two different stress tests: myelotoxicity and acute hemolytic anemia induction. Myelotoxicity was induced by single dose injection of cyclophosphamide (450 mg/Kg) in both WT and KO mice. From this induction we expected a decreased myelopoiesis, mostly affecting granulocytes and monocytes, followed by a rebound due to the capacity of the mice to undertake myelopoiesis. In KO model this compensation was absent suggesting a functional defect into the myeloid lineage correlated with ERG1B deletion. Finally, we induced acute anemia in mice testing their response to phenylhydrazine (PHZ, 60 mg/Kg). As expected, in WT mice, RBCs value rapidly declined followed by a compensatory erythropoiesis. In ERG1B−/− mice, we observed a reduced capacity to recover physiological RBCs values. Such results suggest that a functional defect occurred also into the erythroid lineage. On the whole, the present study provides evidence that the ERG1B isoform exerts a relevant role in hematopoiesis, driving the commitment and maturation of different hematopoietic cell populations. Disclosures: No relevant conflicts of interest to declare.

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Displaced Niche Location and Decreased Quiescence Maintenance of Hematopoietic Stem Cells Due to Dysregulation of Notch Adhesive Interaction with Stromal Environment in Mice with Notch O-Fucosylation Deficiency

Blood ◽

10.1182/blood.v120.21.29.29 ◽

2012 ◽

Vol 120 (21) ◽

pp. 29-29

Author(s):

Weihuan Wang ◽

Xiaoran Huang ◽

Jay Myers ◽

Yiwei Wang ◽

Wei Xin ◽

...

Keyword(s):

Stem Cell ◽

Notch Signaling ◽

Conflicts Of Interest ◽

Loss Of Function ◽

Ligand Interaction ◽

Hematopoietic Stem ◽

Notch Ligand ◽

Endosteal Niche

Abstract Abstract 29 O-fucose modification and Fringe mediated O-fucose extension of Notch EGF-like repeats is essential for Notch binding with Notch ligand and Notch signaling transactivation. Previously we have shown that mice with conditional deficiency of Notch O-fucose modification develop a myeloproliferative disorder (MPD) with some mice displaying features of MPD-like leukemia. We disclosed that this MPD is mainly contributed by the cell-autonomous loss of response of myeloid progenitors bearing non-fucosylated Notch to Notch ligand induced suppression of granulo-monocytic differentiation. More recently, several Notch loss-of-function mutations have been identified in human chronic myelomonocytic leukemia (CMML). To gain a better insight of the role of Notch loss-of-function in stem cell dysfunction and MPD progression, here we studied the significance of O-fucose deficiency of Notch on progenitor proliferation and survival, and on HSC quiescence maintenance and niche location. We used a mouse model of pan-Notch signaling loss-of-function by Mx-Cre1 induced deficiency of Pofut1, an enzyme that modifies EGF O-fucosylation of all 4 Notch receptors. Pofut1-null hematopoietic stem and progenitor cells (HSPCs) had enhanced myeloid specification and proliferation in vitro, and displayed an increased activation of ERK and Stat5 in response to IL3 and GM-CSF when compared to the control HSPCs. The enhanced myeloid specification of Pofut1-null HSCs could be rescued by either activated Notch1 or Notch2. In addition, the HSPCs from Pofut1-null marrow and spleen displayed a 30% reduction of apoptosis. However, the increased proliferation and survival of Pofut1-null HSPCs were only partially reversed by the blocking of G-CSF, a cytokine that was up-regulated in the serum of Pofut1-null mice, supporting a role of cell-autonomous mechanism in its contribution to the increased proliferation and survival of Pofut1-null HSPCs. In line with this notion, we found that Pofut1-null mice had ∼ 50% increase in frequencies of the multi-potential progenitors (MPP) and the short-term self-renewable HSC (ST-HSC) but a 70% reduction of the more primitive long-term self-renewable HSC (LT-HSC). This change of HSC frequency was accompanied by an increased HSC cell cycling and a loss of adhesion to Notch ligand-expressing stromal cells despite that the Pofut1-null HSCs had a normal chemotactic response to SDF-1 and normal expression of CXCR4 as well as integrin adhesion molecules. Consistent with these findings, frequencies of circulating and splenic-residing HSCs were increased in Pofut1-null mice. To explore the mechanism by which loss of O-fucose of Notch regulates the stem cell activity in the bone marrow niches, we performed two-photon intravital microscopy to visualize the niche location of transplanted HSCs. We found that the Pofut1-null HSCs were positioned further from the endosteal niche and the niche supporting osteoblasts, when compared to control HSCs. In addition, Pofut1-null HSCs were not responsive to the inhibition of HSC expansion imparted by the osteoblasts in an in vitro co-culture assay. In summary, loss of O-fucosylation of Notch not only results in skewed myeloid specification and differentiation, but also promotes HSC proliferation and suppresses HSC quiescence. We conclude that the HSC phenotypes observed in mice with Pofut1 deficiency result as a consequence of the displacement of HSCs expressing non-fucosylated Notch from the suppressive endosteal niche that is otherwise enhanced by the adhesion between HSCs with the niche supporting cells through Notch and Notch ligand interaction. Disclosures: No relevant conflicts of interest to declare.

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Aberrant Bone Marrow Perivascular Niches are Involved in Multiple Myeloma Progression: Role of Notch–Delta Pathway.

Blood ◽

10.1182/blood.v114.22.2800.2800 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2800-2800

Author(s):

Sara Lamorte ◽

Marta Costa ◽

Giovanni Camussi ◽

Sergio Dias

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Annexin V ◽

Pcr Analysis ◽

Adherent Cells ◽

Feeder Layer ◽

Hematopoietic Stem ◽

Lower Chamber

Abstract Abstract 2800 Poster Board II-776 Bone marrow (BM) angiogenesis is implicated in Multiple Myeloma (MM) progression. In this study, we tested the hypothesis that MM progression occurs when aberrant BM perivascular niches are established. We isolated BM endothelial cells derived from MM patients (MM-BMECs) from BM aspirates using anti-CD31Ab coupled to magnetic beads. FACS analysis showed that of all the cell lines isolated were endothelial: more than 95% expressed Ulex Europaeus Agglutinin-1 and Factor VIII and were negative for monocyte-macrophage (CD14) and plasma cell markers (CD38). To test the hypothesis that in MM patients BM perivascular niches are aberrant we analyzed how MM-BMECs modulate hematopoietic stem cells (HSCs) properties using a BM microvascular endothelial cell line isolated from a healthy donor (BMECs) as control. We co-cultured cord blood cells CD34+ HSCs in the presence of MM-BMECs or BMECs feeder layer and we analyzed the ability of MM-BMECs compared with BMECs to modulate HSCs adhesion, chemotaxis and apoptosis. The results show that MM-BMECs promote CD34+ HSCs adhesion, recruitment and protect them from apoptosis. In detail, we showed that after 24h of co-culture there was a significant increase in the number of adherent HSCs on MM-BMECs than on BMECs: 43±9% versus 25±6%. Moreover, when HSCs were cultured for 48 hours in 1% of serum in the presence of MM-BMECs they were less sensitive to apoptosis (9±11% of Annexin V+ cells) than HSCs cultured in the presence of BMECs (14±1% of Annexin V+ cells) or without a feeder layer, as control (17±3% of Annexin V+ cells). For the migration assay a transwell chamber system, in which the upper and the lower chambers were separated by 5-μm pore-size filter, was used. BMECs, MM-BMECs or nothing was plated in the lower chamber, while HSCs were seeded into the upper chamber. Both chambers were loaded with unsupplemented EBM-2 plus 2% of serum. Cell migration was studied over a 6-8 hours period and evaluated as number of cells migrated into the lower chamber. The results showed a significantly greater migration of HSCs in the presence of MM-BMECs than BMECs: 12±2% versus 5±1% of migrated cells. Taken together, these data showed that MM-BMECs promoted HSCs migration, adhesion and survival. Next we evaluated how MM-BMECs modulate the hemopoiesis recovery after irradiation in a NOD-SCID mouse model. When injected into sub-lethally irradiated (3 Grey) NOD-SCID mice MM-BMECs were detected in the BM integrated within the murine BM vessels and promoted hematopoietic recovery. In detail, MM-BMECs provided signals favoring the commitment towards lymphoid lineage. In fact, 7 days after injection, the BM of mice injected with MM-BMECs showed an increase in the percentage of lymphoblast (2.7%), compared with mice injected with BMECs or PBS, as control (respectively, 1.5% and 1.4%); followed, 14 days after injection, by a significant increase in the percentage of peripheral blood lymphocytes in mice injected with MM-BMECs (75±6%) versus mice injected with BMECS and PBS (respectively 60±0.5% and 47±7%). Since MM is a plasma cells disorder and the Notch-Delta pathway has been shown to play a central role in regulating HSCs properties, including the decisions of HSCs to undergo T- or B-cell differentiation, we investigated the involvement of this pathway in MM-BMECs and HSCs interaction. As determined by FACS and RT-PCR analysis, MM-BMECs, compared to BMECs, over expressed Delta-like Notch ligand 4 (DII4). Thus, we investigated the role of DII4 in the MM-BMECs/BMECs-HSCs adhesion. The first results showed that the expression of DII4 by MM-BMECs is necessary to promote HSCs adhesion. In fact, using a blocking antibody against DII4 (AbαDII4) at 50ug/ml there was an impairment in HSCs adhesion to MM-BMECs (43±9% versus 24±2% of adherent cells without and with AbαDII4 treatment), but not on BMECs (25±6% versus 26±1.4% of adherent cells without and with AbαDII4 treatment). Ongoing experiments are focusing on the role of DII4 in the modulation of HSCs proliferation, protection against apoptosis and in vitro-in vivo B commitment by MM-BMECs. Taken together, all these data suggest that BMECs in MM may function as “aberrant perivascular niches”, modulating HSCs properties. This aberrant phenotype could be due to an alteration of the Notch-Delta pathway in BMECs that favors malignant clonal growth by protecting it from apoptosis, favoring migration, adhesion and providing self-renewing and/or proliferative cues. Disclosures: No relevant conflicts of interest to declare.

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Identification of Rab5 as a Gene Involved in Chronic Myeloid Leukemia (CML) Progression.

Blood ◽

10.1182/blood.v114.22.3470.3470 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3470-3470

Author(s):

Daniela Cilloni ◽

Monica Pradotto ◽

Francesca Messa ◽

Francesca Arruga ◽

Enrico Bracco ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Western Blot ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Small Gtpases ◽

Loss Of Function ◽

Imatinib Resistance ◽

Protein Levels

Abstract Abstract 3470 Poster Board III-358 The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood. By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. Methods Rab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. Results Rab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p<0.001 and p<0.0001) as compared to healthy subjects. Furthermore, in blast crisis samples we have found Rab5 transcripts levels to be further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Our preliminary analysis of samples from TOPS trial have shown a trend that Rab5 levels are lower among those patients achieving MMR by 12 months, when compared to the group of patients non achieving MMR on 400 mg, but that difference was not statistically significant (p=0.2). Among those randomized to receive imatinib 800 mg the difference was statistically significant with a median value among those achieving MMR of 1.27 vs 2.14 in the group without MMR (p=0.04). The protein levels have been analyzed by Western Blot and immunofluorescence and allow us to show detectable levels of Rab5 in samples collected at remission, but undetectable levels in course of active CML disease. Although preliminary, our results show a significant decrease of Rab5 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Rab5 in slowing down or suppressing a progression. Surprisingly, among CP CML patients the responders to TKI therapy have been detected to express a lower level of Rab5 than non responders. We are conducting further studies to better explain these data, which we find intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures No relevant conflicts of interest to declare.

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Mobilization Studies in Complement-Deficient Mice Reveal That AMD3100 Mobilization Depends On Complement Cascade Activation and Suggest Involvement of “Membrane Attack Complex - MAC” in Egress of Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.367.367 ◽

2009 ◽

Vol 114 (22) ◽

pp. 367-367

Author(s):

Marcin Wysoczynski ◽

HakMo Lee ◽

Rui Liu ◽

Wan Wu ◽

Janina Ratajczak, ◽

...

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Membrane Attack Complex ◽

Classical Pathway ◽

Compensatory Mechanism ◽

Complement Cascade ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Abstract 367 We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway as seen during G-CSF- or Zymosan mobilization, respectively. As a result, several potent bioactive CC anaphylatoxins (C3 and C5 cleavage fragments) are released that regulate egress of HSPCs (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40, Leukemia 2009; in press.). This explains why: i) NOD/SCID and RAG-/- animals that do not activate the Ig-dependent CC classical pathway; ii) C2fB-/- and C3-/- mice that do not activate the classical and alternative CC pathways; and iii) C5-/- mice that do not activate the distal pathway of CC are all poor G-CSF- and/or Zymosan mobilizers. In this study, we evaluated the role of CC in mobilization induced by CXCR4 antagonist AMD3100. We noticed that all CC activation-deficient mice mentioned above, except C5-/- mice, mobilize normally in response to AMD3100 administration. Accordingly, the number of mobilized CD34- SKL cells, leucocytes, and CFU-GM clonogeneic progenitors in mutant mice was similar to wt littermates. More important we observed that AMD3100 mobilization of HSPCs was preceded by a massive egress of leucocytes from BM and that AMD3100 was able to stimulate in these cells i) phosphorylation of MAPKp42/44 and ii) secretion of MMP-9. At the same time, ELISA data to detect CC activation revealed that serum levels of CC cleavage fragments, which were low in the initial phase of AMD3100 mobilization during granulocyte egress, become elevated later during HSPC egress. Thus, our data show that despite a fact that G-CSF and AMD3100 mobilize HSPCs by involving different mechanisms, activation of CC is a common phenomenon occurring during mobilization induced by both compounds. This further supports a pivotal role of CC activation in the egress of HSPCs from BM; however, both compounds activate CC differently. While G-CSF administration initiates CC activation at its proximal C1q-C3 level, AMD3100 induces CC activation at the distal C5 level, pointing to a crucial role of C5 cleavage in executing mobilization. To support this, all mice employed in our studies that display defects in activation of proximal stages of CC (NOD/SCID, RAG, C2fB-/-, and C3-/-) are normal AMD3100 mobilizers. However, C5 is cleavage required for mobilization occurs in the plasma of these animals latter on - directly by proteases released from AMD3100-stimulated granulocytes that egress from the BM as a first wave of mobilized cells. This compensatory mechanism cannot occur from obvious reasons in C5-/- mice. We conclude that AMD3100-directed mobilization similarly as G-CSF-induced one depends on activation of CC; however, AMD3100 in contrast to G-CSF activates CC at distal stages – directly by proteases released from mobilized/activated granulocytes. Cleavage of C5 and release of C5a and desArgC5a create a sinusoid-permissive environment in BM for HSPCs egress. This suggests involvement of both C5 cleavage fragments as well as a potential role of downstream elements of CC activation - membrane attack complex - MAC (C5b-C9) in stem cell mobilization. Therefore, some poor AMD3100 patient responders could possess a defect in activation of the distal steps of CC. Disclosures: No relevant conflicts of interest to declare.

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Flt3 Kinase Regulates Microvesicle Transfer of miRNA Between AML and Stromal Cells

Blood ◽

10.1182/blood.v118.21.1492.1492 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1492-1492

Author(s):

Noah Hornick ◽

Jianya Huan ◽

Jeffrey W Tyner ◽

Peter Kurre

Keyword(s):

Cell Lines ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Membrane Vesicles ◽

Mirna Expression Profile ◽

Cell Phenotype ◽

Cell Of Origin ◽

Hematopoietic Stem ◽

Global Comparison

Abstract Abstract 1492 The presence of an internal tandem duplication in the receptor tyrosine kinase Flt3 (Flt3-ITD) is found in 25–30% of cytogenetically normal AML and confers a worsened prognosis, including an increased likelihood for relapse after hematopoietic stem cell transplantation (HSCT). This tendency toward relapse, combined with the improved capacity of Flt3-ITD+ disease to resist chemotherapy, may imply mechanisms of resistance beyond those present in leukemias lacking this mutation. Microvesicles and exosomes, membrane-bound extracellular vesicles that capture cell-specific protein and RNA, have previously been isolated from the serum of CLL patients (Ghosh et al., Blood 2010 Mar 4;115(9): 1755–64). We recently observed that vesicles are constitutively produced by both AML cell lines and by AML blasts isolated from patients. Using light scatter analysis and transmission electron microscopy, we found that vesicles produced by the HL60 (Flt3-ITD-) and Molm14 (Flt3-ITD+) cell lines, as well as by AML patient cells, predominantly fall within the 30–100nm range, generally considered to specify exosomes. To demonstrate vesicle transfer to neighboring cells, we labeled vesicles produced by the HL-60 cell line with the fluorescent membrane dye PKH-26 and imaged their uptake by stromal cells. Internalization of labeled exosomes was detectable within 15 minutes following exposure, and occurred at approximately 27 particles per cell by 2 hours (n=20). This result indicates rapid uptake of exosomes by non-phagocytic bystander cells, and supports a potential role for the vesicle content in altering the cell phenotype. We next tested for the presence of certain candidate mRNAs in the AML cell lines HEL, HL-60, MOLM-14, and U937, and in vesicles produced by those cell lines. We detected several relevant mRNAs, including nucleophosmin-1 and Flt3-ITD, in the vesicle preparations. During a more global comparison of vesicle and cell-of-origin RNA spectra using bioanalyzer analysis, we found that the RNA in microvesicles from (Flt3-ITD+) MOLM14 cells contained no detectable ribosomal RNA, but a markedly increased proportion of small RNA transcripts, suggesting that miRNA content might be increased. An initial screen of cultured MOLM-14 cells in the presence and absence of a small-molecule Flt3 inhibitor (AC220) and microvesicles isolated from the culture supernatant revealed several candidate miRNAs, including let-7a, miR-99b, and miR-155, whose expression varied with the presence of inhibitor. Our results not only support a role for Flt3 kinase activation in determining the miRNA expression profile, but provide evidence for the kinase-regulated incorporation of miRNA into cell membrane vesicles. The recent description of several AML subtypes, including Flt3-ITD+ disease with unique miRNA profiles, and the role of miRNA as potent regulators of both microenvironmental function and immune responses provides strong motivation to evaluate the role of vesicles in AML therapy evasion. Disclosures: No relevant conflicts of interest to declare.

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