Transcriptional Regulation in Normal and Malignant Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-28-SCI-28
Author(s):  
Koichi Akashi
Keyword(s):  
Transcription Factors ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Histone Variant ◽  
Lineage Commitment ◽  
Regulation Of Transcription ◽  
Hematopoietic Stem ◽  
Genome Wide ◽  
Regulated Expression ◽  
Transcription Factor Expression

Abstract SCI-28 Lineage commitment should involve selective and temporally-regulated expression of essential genes. In multi- or oligo-potent progenitors, the expression of oligo-lineage-affiliated genes is primed: oligo-lineage genes are co-expressed prior to the commitment at the single cell level, and once lineage fate is decided, genes of irrelevant lineages are immediately downregulated. For example, single common myeloid progenitors (CMP) co-express both granulocyte/monocyte-affiliated and megakaryocyte/erythroid-affiliated genes. The priming of these lineage-restricted genes could be dependent also upon the priming of lineage-specific transcription factors. Consistent with this hypothesis, a population with potent CMP activity that co-express both PU.1 (myeloid) and GATA-1 (erythroid) transcription factors was newly identified by using mice having PU.1 and GATA-1 reporters. In downstream of such “priming” stage, the order of expression as well as the level of expression of multiple transcription factors plays a critical role in reading-out specific lineages. The precise regulation of transcription factors should be critical to maintain hematopoietic homeostasis, and the deregulation of transcription factor expression could induce leukemic transformation. To understand the regulation machinery upstream of transcription factors, we are currently attempting to model an epigenetic landscape in hematopoietic development. Genome-wide analysis of histone positioning revealed that a histone variant marks hematopoietic transcription factors and other lineage-related genes prior to commitment, and therefore the variant can predict the actively-transcribed region in a later stage of hematopoiesis. For example, in hematopoietic stem cells, the histone marking was observed broadly in genes-related to myelo-erythroid and lymphoid genes, while in committed progenitors, it was restricted to specific lineages such as myeloid, erythroid and/or lymphoid genes. These results suggest that the genome marking by a histone variant should be a primary event for initiating lineage commitment. Disclosures: No relevant conflicts of interest to declare.

New Approaches for Mapping Epigenome Dynamics

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-21-SCI-21
Author(s):  
Steven Henikoff
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Conflicts Of Interest ◽  
Regulation Of Transcription ◽  
Base Pairs ◽  
Simple Method ◽  
Genome Wide ◽  
Resolution Mapping

Abstract The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely mapping RNAPII genome-wide. We have used this method to show exactly where RNAPII stalls as it unwraps a nucleosome in vivo, surprisingly in a different place in vivo from where it stalls in vitro. We also have discovered that a variant histone, H2A.Z, which is found in essentially all eukaryotes, helps to reduce the nucleosome barrier to transcription, and in this way may modulate transcription. Other protein components of the epigenome involved in dynamic processes are nucleosome remodelers, which use the energy of ATP to slide or even evict nucleosomes from DNA. Some remodelers help RNAPII get started and others help it overcome the nucleosome barrier to transcription, and by mapping them at base-pair resolution, we can gain insight into how they act. We have also applied our high-resolution mapping tools to transcription factors, which bind DNA at specific sites to regulate transcription and other processes. Our ability to achieve high spatial and temporal resolution mapping of the binding and action of nucleosomes, transcription factors, remodelers and RNAPII provides us with a detailed picture of epigenome dynamics. By using these tools we are beginning to understand how DNA sequence and conformation are recognized for regulation of transcription and other epigenomic processes. Disclosures No relevant conflicts of interest to declare.

CHD7 Deficiency Inhibits CBFB-MYH11 -Induced Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3662-3662
Author(s):  
Tao Zhen ◽  
Ling Zhao ◽  
R. Katherine Hyde ◽  
Lemlem Alemu ◽  
Pu Paul Liu
Keyword(s):  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Poly I:C ◽  
Luciferase Assay ◽  
Regulation Of Transcription ◽  
Mechanistic Studies ◽  
Spleen Cells ◽  
Hematopoietic Stem ◽  
Consistent Finding

Abstract Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. Mechanistic studies showed that the interaction between CBFbeta-SMMHC (encoded by CBFB-MYH11) and RUNX1 plays a critical role in the leukemogenesis of this kind of leukemia. Recently, it was shown that chromodomain-helicase-DNA binding protein 7 (CHD7) interacts with RUNX1 and suppresses RUNX1-induced expansion of hematopoietic stem and progenitor cells. These results suggest that CHD7 also plays a role in CBFB-MYH11 induced leukemogenesis. To test this hypothesis, we crossed Cre-based conditional Chd7 knockout mice (Chd7f/f) with Cre-based conditional Cbfb-MYH11 knockin mice (Cbfb+/56M) to generate Chd7f/f, Mx1-Cre, Cbfb+/56M mice, which express Cbfbeta-SMMHC but not CHD7 after poly I:C treatment to induce Cre expression. Three weeks after poly I:C treatment, the Lin- and the Lin-/Sca1-/c-Kit+ (LK) progenitor cell populations were significantly lower in Chd7f/f, Mx1-Cre, Cbfb+/56M mice than that in Mx1-Cre, Cbfb+/56M mice. In addition, there were fewer BrdU+ cells in the Lin- and LK populations in the Chd7f/f, Mx1-Cre, Cbfb+/56M mice. Importantly, Chd7f/f, Mx1-Cre, Cbfb+/56M mice developed leukemia with a much longer latency than the Mx1-Cre, Cbfb+/56M mice. Secondary transplantation assay demostrated that spleen cells isolated from Chd7f/f, Mx1-Cre, Cbfb+/56M and Mx1-Cre, Cbfb+/56M leukemic mice were transplantable, but recipients that received spleen cells from Chd7f/f, Mx1-Cre, Cbfb+/56M leukemic mice had a longer latency than those who received cells from Mx1-Cre, Cbfb+/56M mice. The above results suggest that CHD7 deficiency slows leukemia initiation and development by inhibiting the proliferation ability of the Lin- and LK populations. Further mechanistic studies showed that CHD7 interacted with RUNX1 but not CBFbeta-SMMHC by co-Immunoprecipitation and immunofluorescence staining, suggesting that CHD7 is a partner of the RUNX1-CBFbeta-SMMHC transcription complex. In vitro luciferase assay showed that CHD7 enhanced RUNX1 and CBFbeta-SMMHC's transcriptional activity on M-CSFR, a RUNX1 target gene. These data indicate that CHD7 is important for Cbfb-MYH11 induced leukemogenesis by facilitating RUNX1 regulation of transcription and cellular proliferation. Disclosures No relevant conflicts of interest to declare.

Genome-wide identification of AP2/ERF transcription factors in mungbean (Vigna radiata) and expression profiling of the VrDREB subfamily under drought stress

2018 ◽  
Vol 69 (10) ◽  
pp. 1009 ◽  
Author(s):  
Abdullahi Muhammad Labbo ◽  
Maryam Mehmood ◽  
Malik Nadeem Akhtar ◽  
Muhammad Jawad Khan ◽  
Aamira Tariq ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Drought Stress ◽  
Stress Tolerance ◽  
Vigna Radiata ◽  
Critical Role ◽  
Drought Stress Tolerance ◽  
Legume Crop ◽  
Genome Wide ◽  
Responsive Element ◽  
Major Factors

Mungbean (Vigna radiata L.) is a valuable legume crop grown in tropical and subtropical areas of Asia. Drought is one of the major factors hindering its growth globally. APETALA2/ethylene-responsive element factor binding proteins (AP2/ERF) are an important family of plant-specific transcription factors (TFs) involved in drought-stress tolerance. We identified 71 AP2/ERF TFs in the mungbean genome by using bioinformatics tools and classified them into subfamilies: AP2 (16 members), ERF (22), RAV (2), DREB (30) and soloist (other proteins with no domain, 1). Members of DREB play a critical role in drought-stress tolerance. Ten-day-old mungbean plants cv. AZRI-06 were exposed to drought stress by complete withholding of water for 7 days. Root samples were collected from control and drought-stressed plants, and the expression pattern of 30 identified VrDREB genes was determined by qPCR. Most VrDREB genes exhibited differential expression in response to drought. Five genes (VrDREB5, VrDREB12, VrDREB13, VrDREB22, VrDREB30) were highly expressed under drought stress and might be considered excellent candidates for further functional analysis and for improvement of mungbean drought tolerance.

Geminin Regulates Hematopoietic Stem Cell Proliferation and Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1478-1478
Author(s):  
Kathryn M. Shinnick ◽  
Kelly A. Barry ◽  
Elizabeth A. Eklund ◽  
Thomas J. McGarry
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Transcription Factors ◽  
Cell Division ◽  
Dna Replication ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Regulatory Protein ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation

Abstract Abstract 1478 Poster Board I-501 Hematopoietic stem cells supply the circulation with mature blood cells throughout life. Progenitor cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The mechanisms that control the choice between continued cell division and terminal differentiation are incompletely understood. The unstable regulatory protein Geminin is thought to maintain cells in an undifferentiated state while they proliferate. Geminin is a bi-functional protein. It limits the extent of DNA replication to one round per cell cycle by binding and inhibiting the essential replication factor Cdt1. Loss of Geminin leads to replication abnormalities that activate the DNA replication checkpoint and the Fanconi Anemia (FA) pathway. Geminin also influences patterns of cell differentiation by interacting with Homeobox (Hox) transcription factors and chromatin remodeling proteins. To examine how Geminin affects the proliferation and differentiation of hematopoietic stem cells, we created a mouse strain in which Geminin is deleted from the proliferating cells of the bone marrow. Geminin deletion has profound effects on all three hematopoietic lineages. The production of mature erythrocytes and leukocytes is drastically reduced and the animals become anemic and neutropenic. In contrast, the population of megakaryocytes is dramatically expanded and the animals develop thrombocytosis. Interestingly, the number of c-Kit+ Sca1+ Lin- (KSL) stem cells is maintained, at least in the short term. Myeloid colony forming cells are also preserved, but the colonies that grow are smaller. We conclude that Geminin deletion causes a maturation arrest in some lineages and directs cells down some differentiation pathways at the expense of others. We are now testing how Geminin loss affects cell cycle checkpoint pathways, whether Geminin regulates hematopoietic transcription factors, and whether Geminin deficient cells give rise to leukemias or lymphomas. Disclosures: No relevant conflicts of interest to declare.

The bHLH Transcription Factors SCL and LYL1 Awaken Hematopoietic Stem Cells by Repression of p21

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 214-214
Author(s):  
David J. Curtis ◽  
Nhu-Y Nguyen ◽  
Jessica Salmon
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Bhlh Factors ◽  
Mild Defect ◽  
Bhlh Transcription Factors

Abstract Abstract 214 The basic helix-loop-helix (bHLH) transcription factors SCL (TAL1) and LYL1 are regulators of adult hematopoietic stem cell (HSC) activity with significant functional redundancy: HSCs lacking SCL (SCLδ/δ) have a mild defect in short-term repopulating activity whilst HSCs lacking LYL1 (LYL1−/−) have normal repopulating activity. In contrast, we have shown previously that HSCs lacking both SCL and LYL1 (DKO) are unable to grow in vitro and have no in vivo repopulating activity. Phenotypic and expression analyses of SCLδ/δ, LYL1−/− and DKO mice were performed to determine how bHLH factors regulate HSC activity. Consistent with the short-term repopulating defects of SCLδ/δ HSC, Lineage negative Sca-1+ c-Kit+ (LSK) bone marrow cells from SCLδ/δ mice had reduced in vitro replating activity associated with increased quiescence – 90% in G0 compared with 70% in normal LSK. Increased quiescence was associated with delayed hematopoietic recovery following treatment of mice with 5-Fluorouracil. Consistent with the increased quiescence, expression of the cell cycle inhibitor, Cdkn1a (p21) was increased three-fold in SCLδ/δ and LYL1−/− LSK. Moreover, p21 levels in LSK isolated from DKO mice were increased 50-fold. To determine the functional relevance of the elevated levels of p21 in DKO HSCs, we generated DKO mice on a p21-deficient (p21−/−) background. Remarkably, loss of p21 rescued in vitro cell growth of DKO progenitors. More importantly, primary and secondary competitive repopulation assays demonstrated multi-lineage repopulating activity of p21−/− DKO HSCs. These results suggest the bHLH factors SCL and LYL1 function as repressors of p21, allowing HSCs to enter cell cycle during stress hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Innate Lymphoid Cell-Derived IL-22 Mediates Endogenous Thymic Repair Under the Control of IL-23

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 143-143
Author(s):  
Jarrod A Dudakov ◽  
Alan M Hanash ◽  
Lauren F. Young ◽  
Natalie V Singer ◽  
Mallory L West ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Thymic Epithelial Cells ◽  
Immune Competence ◽  
Strong Negative Correlation ◽  
Hematopoietic Stem ◽  
Stimulation Of

Abstract Abstract 143 Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. Endogenous regeneration of the thymus is a crucial function that allows for renewal of immune competence following infection or immunodepletion caused by cytoreductive chemotherapy or radiation. However, the mechanisms governing this regeneration remain poorly understood. Thymopoiesis is a highly complex process involving cross-talk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) that are crucial for T cell development. IL-22 is a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces. Here we demonstrate for the first time a critical role for IL-22 in endogenous thymic repair. Comparing IL-22 KO and WT mice we observed that while IL-22 deficiency was redundant for steady-state thymopoiesis, it led to a pronounced and prolonged loss of thymus cellularity following sublethal total body irradiation (SL-TBI), which included depletion of both thymocytes (p=0.0001) and TECs (p=0.003). Strikingly, absolute levels of IL-22 were markedly increased following thymic insult (p<0.0001) despite the significant depletion of thymus cellularity. This resulted in a profound increase in the production of IL-22 on a per cell basis (p<0.0001). These enhanced levels of IL-22 peaked at days 5 to 7 after SL-TBI, immediately following the nadir of thymic cellularity. This was demonstrated by a strong negative correlation between thymic cellularity and absolute levels of IL-22 (Fig 1a). In mucosal tissues the regulation of IL-22 production has been closely associated with IL-23 produced by dendritic cells (DCs) and ex vivo incubation of cells with IL-23 stimulates the production of IL-22. Following thymic insult there was a significant increase in the amount of IL-23 produced by DCs (Fig 1b) resulting in similar kinetics of intrathymic levels of IL-22 and IL-23. We identified a population of radio-resistant CD3−CD4+IL7Ra+RORg(t)+ thymic innate lymphoid cells (tILCs) that upregulate both their production of IL-22 (Fig 1c) and expression of the IL-23R (p=0.0006) upon exposure to TBI. This suggests that they are responsive to IL-23 produced by DCs in vivo following TBI and, in fact, in vitro stimulation of tILCs by IL-23 led to upregulation of Il-22 production by these cells (Fig 1d). We found expression of the IL-22Ra on cortical and medullary TECs (cTECs and mTECs, respectively), and uniform expression across both mature MHCIIhi mTEC (mTEChi) and immature MHCIIlo mTECs (mTEClo). However, in vitro stimulation of TECs with recombinant IL-22 led to enhanced TEC proliferation primarily in cTEC and mTEClo subsets (p=0.002 and 0.004 respectively). It is currently unclear if IL-22 acts as a maturation signal for mTECs, however, the uniform expression of IL-22Ra between immature mTEClo and mature Aire-expressing mTEChi, together with the preferential promotion of proliferation amongst mTEClo and cTEC seem to argue against IL-22 as a maturational signal but rather as promoter of proliferation, which ultimately leads to terminal differentiation of TECs. Of major clinical importance, administration of exogenous IL-22 led to enhanced thymic recovery (Fig. 1e) following TBI, primarily by promoting the proliferation of TECs. Consistent with this, the administration of IL-22 also led to significantly enhanced thymopoiesis following syngeneic BMT. Taken together these findings suggest that following thymic insult, and specifically the depletion of developing thymocytes, upregulation of IL-23 by DCs induces the production of IL-22 by tILCs and regeneration of the supporting microenvironment. This cascade of events ultimately leads to rejuvenation of the thymocyte pool (Fig. 1f). These studies not only reveal a novel pathway underlying endogenous thymic regeneration, but also identify a novel regenerative strategy for improving immune competence in patients whose thymus has been damaged from infection, age or cytoreductive conditioning required for successful hematopoietic stem cell transplantation. Finally, these findings may also provide an avenue of study to further understand the repair and regeneration of other epithelial tissues such as skin, lung and breast. Disclosures: No relevant conflicts of interest to declare.

Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3584-3584
Author(s):  
Takahiro Shima ◽  
Yoshikane Kikushige ◽  
Toshihiro Miyamoto ◽  
Koichi Akashi
Keyword(s):  
Stem Cells ◽  
Acute Myelogenous Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Myelogenous Leukemia ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Kit Mutation ◽  
Acute Myelogenous

Abstract Abstract 3584 The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo. Disclosures: No relevant conflicts of interest to declare.

Coexistance of Multiple Myeloma and Myelodysplastic Syndrome or Lymphoid Diseases At Diagnosis: Evidence for Two Clonal and Independent Chromose Abnormalities

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5063-5063
Author(s):  
Hossein Mossafa ◽  
Sabine Defasque ◽  
Christine Fourcade ◽  
JeanPierre Hurst ◽  
Bertrand Joly
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cells ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Chromosome 8 ◽  
Trisomy 8 ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Trisomy 12

Abstract Abstract 5063 Introduction, We describe the simultaneous presentation of multiple myeloma (MM) and yeloproliferative disorders (MPD) or lymphoid diseases (LD) at diagnosis. Therapy-related myelodysplasia (tMDS) occurring during the course of MM is generally believed as a result from hematopoietic stem cell-toxic therapies, such as ionizing radiation and alkylating agent-based chemotherapies (melphalan, nitrosoureas).Patients and methods, We study a total of 342 patients (151 F, 191 M; median age 68.1 years; range 42 to 93 Years), diagnosed with MM based on the International Staging System. The basis for inclusion of patients in this study was with previous untreated MM ones. The study was performed in accordance with the declaration of Helsinki. To determine whether chemotherapies for MM factors play the critical role in the development of secondary disease, simultaneously two different cultures were processed, an unstimulated 96 hours culture (U96HC) on whole BM(WBM), a short-time 24 hours culture (ST24HC) after CD138+ plasma cells (PCs) depleted on negative fraction (CD138- cells) of BM and the FISH was investigated on purified CD138+.All samples were enriched in PCs by the Automated Magnetic Cell Sorter (Miltenyi technology)proceeded with anti-CD138 specific antibodies applied. The CD138+ PCs and the CD138- cells were collected in different tubes. The CD138− cells were used for a ST24HC. FISH was performed on the purified CD138+, PCs with a recommended FISH panel (MM International Working Group). Screening was performed systematically for the following unbalanced alterations and reciprocal rearrangements: del(13)(q14)(D13S25), del(17)(p13)(TP53),+3(D3Z), +9(D9Z1), +15(D15Z14), t(4;14)(p16;q32)/IGH-FGFR3, t(11;14)(q13;q32)/IGH-CCND1 (Abbott).After observing the results of U96HC on whole BM (CD138+ and CD138− cells), ST24HC (CD138− cells) and FISH for each patient, two clone cytogenetically were distinct and unrelated chromosomal abnormalities were found in 40 (11.7%) of the 342 MM patients (6 F, 34 M; median age 74 years; range 42 to 87 Years) 34 had a MPD and 6 had a LD. A second immunophenotyping analysis confirmed the presence of those LD/MM simultaneous haematological malignancy. In the cases of the patients with MM/ MPD, the frequency of cytogenetic abnormality unrelated to the myeloma clone was respectively; the 20q deletion, detected for 13 the 34 patients, the 20q- is a sole abnormality for 12 cases and associated with a complex caryotype in 1 case. The trisomy of chromosome +8 was observed in 7 cases, the del(7q) or monosomy 7 in 5 cases, loss of gonosome Y in 4 cases, del(11) for 2 cases, translocation t(9;22) in one case, 5q abnormality in one case and trisomy 9 with JAK2 V617F mutation in one case. For the patients with MM/LD, 5 patients had a trisomy +12 and or trisomy +18 like sole abnormality or associated with others cytogenetics abnormalities and one patient had 6q deletion. Discussion, Whereas in the literature the most common cytogenetic abnormalities typifying MPD after alkylator-based therapy include partial or complete deletions of chromosomes 5, 7, and 20 as well as trisomy 8. In our study we observed those abnormalities with the same frequency for the patients had simultaneous MPD associated in untreated MM at diagnosis. Six patients had simultaneous LD and MM. The marginal zone lymphoma was confirmed for 3 patients. The CC observed a trisomy +12 for those three patients associated with +18 and +19 for 2 cases and del(13) and trisomy 3 for one among them. We demonstrated in untreated MM patients the coexistence of MM and MPD or LD at diagnosis with MPD-type or LD-type chromosome abnormalities within MM signature karyotype. We hence recommend that CC studies, 96 hours WBM, 24 hours on negative fraction CD138− cells and FISH on purified CD138+ PCs, the three should be an integral part of the evaluation of patients with MM at diagnosis into clinical trials using HDT is warranted to determine whether patients who are predisposed to developing tMDS/sAML, they can be identified prospectively. Disclosures: No relevant conflicts of interest to declare.

Failure of Erythropoiesis and Megakaryocytopoiesis in RASA3 Mutant Scat Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 680-680
Author(s):  
Lionel Blanc ◽  
Babette Gwynn ◽  
Steven L. Ciciotte ◽  
Luanne L. Peters
Keyword(s):  
Bone Marrow ◽  
Positional Cloning ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Inbred Mouse Strain ◽  
Recessive Mutation ◽  
Initial Increase ◽  
Severe Anemia ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 680 Scat (severe combined anemia and thrombocytopenia) is a spontaneous, autosomal recessive mutation coisogenic with the BALB/cBy inbred mouse strain. Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. As its name implies, crisis episodes are characterized by severe anemia and thrombocytopenia, but significant lymphocyte depletion occurs as well. The first crisis episode begins in utero, lasts until postnatal day (P) 9 on average, and is associated with 10–15% mortality. Remarkably, in homozygotes that survive the first crisis, a remission phase occurs wherein the disease phenotype reverts to normal. This remission is transient, however, and is followed by a second crisis episode during which 94% of scat/scat mice die by P30. Previously we showed that the scat phenotype is transferrable via the hematopoietic stem cells and is also recapitulated in scat/scat, Hox11−/− double homozygotes in which a spleen does not develop, indicating that the splenic micro-environment plays little or no role in disease appearance or progression. Positional cloning of scat revealed a missense mutation in Rasa3 encoding a GTPase activating protein (GAP) that negatively regulates Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. We further showed that Rasa3 is a conserved gene critical to vertebrate erythropoiesis via morpholino knockdowns in zebrafish which resulted in profound anemia. Here we report data that shed further light on RASA3 function during hematopoiesis. Overall, the data indicate that defects in RASA3 profoundly and negatively impact erythropoiesis and megakaryocytopoieis through, at least in part, a Ras-mediated mechanism. FACS analyses of scat spleen and bone marrow erythroid populations reveal a severe block in erythropoiesis during crisis periods. In the spleen, despite an initial increase in size due to expansion of Ter-119+ cells, there is ultimately a loss of compensatory erythropoiesis resulting in a return to normal cellularity and a striking loss of hemoglobinized cells as the crisis phenotype deepens. In addition, the bone marrow shows loss of Ter-119+ cells and overall cell depletion during crisis. Megakaryocyte numbers are increased in scat crisis BM and spleen. By transmission electron microscopy, scat crisis megakaryocytes display features characteristic of a significant developmental delay: a disorganized demarcation membrane system with no platelet forming areas and few granules with hypersegmented nuclei and excess rough endoplasmic reticulum. In addition to the severe anemia and thrombocytopenia, a significant lymphopenia occurs in scat crisis mice. However, the scat phenotype is not lymphocyte mediated, as the scat phenotype is completely recapitulated in mice doubly homozygous for scat and the immunodeficient mutations, scid and Rag1tm1Mom, in which B- and T-lymphocytes are completely depleted. Together these results suggest that lymphopenia is a secondary phenomenon in scat, and the severe anemia and thrombocytopenia aspect of the phenotype neither follows from nor is dependent upon loss of lymphocytes. Despite the delay observed in erythroid differentiation, some mature red cells are produced although ∼50% of these are reticulocytes. By confocal microscopy, we show that RASA3 protein localizes to the plasma membrane as well as internal membrane compartments in wild type reticulocytes, where it partially colocalizes with CD71. Western blot analyses of reticulocytes after Percoll gradient purification show that RASA3 is lost during the maturation step, both in vivo and in vitro. Interestingly, in scat, RASA3 is present in reticulocytes, but appears to be mislocalized, the protein being found in the cytosol. Preparation of ghosts from wild type and scat reticulocytes confirms that RASA3 is not attached to the membrane in scat animals during crisis. In pull-down assays active GTP-bound Ras is increased in scat crisis reticulocytes when compared to wild type, suggesting that scat is a RASA3 loss of function mutation due to its mislocalization and demonstrating a critical role for the RASA3-Ras axis during mammalian erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2332-2332
Author(s):  
Vitalyi Senyuk ◽  
Yunyuan Zhang ◽  
Yang Liu ◽  
Ming Ming ◽  
Jianjun Chen ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Myeloid Differentiation ◽  
Dna Hypermethylation ◽  
Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

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