Ezh2 Loss Accelerates JAK2V617F-Driven Primary Myelofibrosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 110-110
Author(s):  
Takahisa Tomioka ◽  
Goro Sashida ◽  
Kotaro Shide ◽  
Kazuya Shimoda ◽  
Naoto Yamaguchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Primary Myelofibrosis ◽  
Polycomb Group ◽  
Conditional Knockout ◽  
Post Transplantation ◽  
Myeloid Malignancies

Abstract Polycomb group proteins are transcriptional repressors that epigenetically regulate transcription via histone modifications. There are two major polycomb-complexes, the Polycomb Repressive Complexes (PRC1 and PRC2). PRC2 contains SUZ12, EED, and EZH2 that catalyze the trimethylation of histone H3 at lysine 27 (H3K27me3) and silence target-genes expression. EZH2 is generally thought to act as an oncogene in lymphoma by silencing tumor suppressor genes through H3K27me3 modifications. However, loss-of-function mutations of EZH2 have been found in myeloid malignancies such as MDS and MPN including primary myelofibrosis (PMF). In a recent study, EZH2 mutations were independently associated with shorter survival in PMF patients, suggesting that EZH2 functions as a tumor suppressor in PMF. Although JAK2V617F mutant is found in approximately 50% of PMF patients, it remains obscure whether the presence of JAK2V617F mutant predicts survival of PMF patients, and the functional contribution of JAK2V617F to the development of PMF has not been fully delineated. JAK2 has been shown to directly phosphorylate H3Y41 (H3Y41p) and reduce HP1a binding, thereby activating expression of target genes. However, it is unknown how JAK2V617F epigenetically alter expression of target genes in the development of PMF. Given that JAK2V617F mutation is significantly associated with EZH2 mutations in PMF patients, in order to understand how EZH2 mutations contribute to the pathogenesis of JAK2V617F-positive PMF, we generated a novel mouse model of PMF utilizing H2K-JAK2V617F transgenic mice and Ezh2 conditional knockout mice. We first harvested 5x106 bone marrow cells from tamoxifen-inducible Cre-ERT;Ezh2wild/wild (WT), Cre-ERT;Ezh2flox/flox (Ezh2 cKO), JAK2V617F TG/Cre-ERT;Ezh2wild/wild (JAK2 TG) and JAK2V617F TG/Cre-ERT;Ezh2flox/flox (JAK2 TG/Ezh2 cKO) mice, and then transplanted into lethally irradiated recipient mice. At 4 weeks post transplantation, we deleted Ezh2 via administration of tamoxifen, and observed disease progression until 9 months post transplantation. WT and Ezh2 cKO mice did not develop myeloid malignancies. While all 11 JAK2 TG mice died due to PMF-like disease after a long latency as previously reported, 10 out of 10 JAK2 TG/Ezh2 cKO mice immediately developed PMF and died by approximately 50 days post-deletion of Ezh2. JAK2 TG/Ezh2 cKO mice showed a significantly shorter median survival than did JAK2 TG mice (36.5 days versus 245 days, p<0.01). In the peripheral blood, moribund JAK2 TG/Ezh2 cKO mice showed increased mature neutrophils, severe anemia, and thrombocytopenia, compared to WT or JAK2 TG mice at 2 months post transplantation. At the time of sacrifice, JAK2 TG/Ezh2 cKO mice showed a significant hypoplastic bone marrow without an increased myeloblast cells, but also had a marked splenomegaly due to infiltration of myeloid cells compared to JAK2 TG mice. In addition, JAK2 TG/Ezh2 cKO mice showed a severe myelofibrosis in both bone marrow and spleen, indicating that Ezh2 loss obviously promotes JAK2 V617F-driven PMF in vivo. To understand a molecular mechanism how Ezh2 functions as a tumor suppressor for PMF, we performed gene expression analysis in Lin-Sca1+c-Kit+ (LSK) cells. While Ezh2 cKO LSKs and JAK2 TG LSKs showed up-regulation (>2-fold) of 1044 and 861 genes, respectively, JAK2 TG/Ezh2 cKO LSKs showed up-regulation (>2-fold) of more genes (1306), compared to WT LSKs. As expected, H3Y41p and H3K27me3 target genes were significantly upregulated in JAK2 TG/Ezh2 cKO LSKs, whereas H3K27me3 targets were significantly repressed in JAK2 TG LSKs, consistent with the tumor suppressor role of Ezh2 in PMF. We are now working to understand how dysregulated genes are involved in the progression of JAK2V617F-induced PMF after deletion of Ezh2. In conclusion, we have successfully established the progressive PMF in mice reconstituted with Ezh2 null cells expressing JAK2V617F mutant, and demonstrated that Ezh2 functions as a tumor suppressor in this context. This model can be utilized for innovating new therapies for PMF. Disclosures: No relevant conflicts of interest to declare.

Loss of Ezh2 Promotes the Development of Mutant-RUNX1 Induced MDS

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 402-402
Author(s):  
Goro Sashida ◽  
Satomi Tanaka ◽  
Makiko Mochizuki-Kashio ◽  
Atsunori Saraya ◽  
Tomoya Muto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
The Self ◽  
Post Transplantation ◽  
Self Renewal ◽  
Myeloid Malignancies ◽  
Empty Vector ◽  
Marrow Cells

Abstract Abstract 402 Polycomb group proteins are transcriptional repressors that epigenetically regulate transcription via histone modifications. There are two major polycomb-complexes, the Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2). PRC2 contains SUZ12, EED, and EZH1/EZH2, and catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3), silencing target-genes. We have shown that the self-renewal of Ezh2-deficient HSCs is not compromised and H3K27me3 marks are not completely depleted in the absence of Ezh2, possibly as a result of Ezh1 complementation. EZH2 is generally thought to act as an oncogene in lymphoma and solid tumors by silencing tumor suppressor genes. Recently however, loss-of-function mutations of EZH2 have been found in myeloid malignancies such as AML, MDS and MPN, suggesting that EZH2 also functions as a tumor suppressor, although it remains unclear how EZH2 prevents the transformation of myeloid malignancies. RUNX1 is a critical transcription factor in the regulation of the self-renewal and differentiation of HSCs. RUNX1 mutations are frequently found in MDS, AML following MDS (MDS/AML) and de novo AML patients. One of the most frequent mutations, RUNX1S291fs, lacks the transactivation domain in C-terminus, but retains the RUNT DNA biding domain, resulting in a dominant negative phenotype. RUNX1S291fs-transduced bone marrow cells have been shown to generate MDS/AML in vivo. Given that RUNX1 and EZH2 mutations coexist in MDS and AML patients as reported recently, we generated a novel mouse model of MDS utilizing RUNX1S291fs retrovirus and Ezh2 conditional knockout mice in order to understand how EZH2 loss contributes to the pathogenesis of MDS upon genetic mutation of RUNX1. We first harvested CD34-Lin-Sca1+c-Kit+(LSK) HSCs from tamoxifen-inducible Cre-ERT;Ezh2wild/wild (EW) and Cre-ERT;Ezh2flox/flox (EF) mice (CD45.2) and transduced these cells with RUNX1S291fs retrovirus or an empty vector, which contains IRES-GFP. Then, we transplanted RUNX1S291fs-transduced Cre-ERT;Ezh2wild/wild (S291EW) or Cre-ERT;Ezh2flox/flox (S291EF) HSCs into lethally irradiated recipient mice (CD45.1) together with life saving dose 1×105 CD45.1 bone marrow cells. At 6 weeks post transplantation, we deleted Ezh2 via administration of tamoxifen, and observed disease progression until 12 months post transplantation. The empty vector transduced control mice with or without Ezh2 (EW and EF) did not develop myeloid malignancies. Two out of 16 S291EW mice died due to MDS progression, while 12 out of 16 and 1 out of 17 S291EF mice developed MDS and MDS/AML, respectively. S291EF mice showed significantly shorter median survival than S291EW mice (314 days versus undefined, p=0.037). In the peripheral blood, we observed significantly lower CD45.2+GFP+ chimerism in S291EF mice; however S291EF mice eventually showed macrocytic anemia and variable white blood cell counts accompanied with dysplastic features of MDS. Despite low CD45.2+GFP+ chimerism in peripheral blood, S291EF mice showed a higher chimerism of CD45.2+GFP+ cells in the bone marrow and had a significantly increased number of LSK and CD34-LSK cells compared to EW, EF, and S291EW mice, indicating that Ezh2 loss promoted HSCs/progenitors expansion, but impaired myeloid differentiation in the presence of RUNX1S291fs. We also saw enhanced apoptosis of CD71+Ter119+ erythroblasts in S291EF MDS mice, which may account for the anemia we observed. Since S291EF MDS bone marrow cells were transplantable in secondary experiments, we performed limiting-dilution assays to evaluate the frequency of MDS initiating cells and found that the frequency of MDS initiating cells was much higher in S291EF pre-MDS Lin-Mac1-Kit+ cells compared to S291EW pre-MDS Lin-Mac1-Kit+ cells. To understand this molecular mechanism, we performed gene expression analysis during MDS progression. S291EF MDS LSKs showed 1979 and 1875 dysregulated (>5-fold) genes, compared to EW LSK and S291EF pre-MDS LSK, respectively. We are now working to understand how these dysregulated genes are involved in the development of RUNX1S291fs-induced MDS after deletion of Ezh2. In summary, we have successfully recapitulated the clinical feature of MDS in mice reconstituted with Ezh2 null HSCs expressing a RUNX1 mutant, and demonstrated that Ezh2 functions as a tumor suppressor in this context. Disclosures: No relevant conflicts of interest to declare.

RXRα Null Haematopoietic Cells Fail To Reconstitute Haematopoiesis in Lethally Irradiated Recipient Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2669-2669
Author(s):  
Richard A. Wells ◽  
Kenneth R. Chien ◽  
Pei-Hua Yen
Keyword(s):  
Bone Marrow ◽  
Hormone Receptors ◽  
Bone Marrow Cells ◽  
Conditional Knockout ◽  
In Vitro Assays ◽  
Peroxisome Proliferator ◽  
Post Transplantation ◽  
Haematopoietic Cells

Abstract The retinoid X receptor (RXR) acts as an obligate heterodimeric partner for multiple nuclear hormone receptors (NRs), including the retinoic acid receptor (RAR), thyroid receptor (TR), vitamin D receptor (VDR), and peroxisome proliferator-activated receptor (PPAR). Targeted disruption of RXRα in the mouse yields an embryonic lethal phenotype due to impairment of cardiac development. We have utilized a conditional knockout approach to investigate the roles of NR signaling in haematopoiesis. Bone marrow cells were isolated from a mouse homozygous for a targeted mutation in which exon IV of RXRα is flanked by loxP sites (RXRαfl/fl). This mutation permits normal expression of RXRα, but expression of cre recombinase results in excision of exon IV, abrogating expression of functional RXRα (RXRαko/ko). We employed a retrovirus to deliver cre to conditionally targeted haematopoietic cells. Lineage-depleted RXRαfl/fl bone marrow (BM) cells were transduced with a retrovirus that expresses a GFP-cre fusion, or with a control retrovirus expressing only EGFP. Transduced cells were isolated to >97% purity by FACS. The effect of RXRα disruption on haematopoiesis was assessed by in vitro assays and by transplantation into strain-matched lethally irradiated recipient mice. Progenitor assays performed in methylcellulose medium supplemented with haematopoietic growth factors revealed that GFP-cre - transduced (RXRαko/ko) grafts contain slightly fewer BFU-E and CFU-GM per 10,000 cells (60% and 80% of EGFP - transduced RXRαfl/fl cells, respectively). Long-term culture initiating cells (LTCIC) were enumerated for RXRαko/ko and RXRαfl/fl grafts. RXRα excision resulted in a moderate (25%) reduction in LTCIC. RXRαko/ko HSCs grown in suspension culture (IMDM supplemented with 10% foetal bovine serum, IL3, IL6, and kit ligand) for two weeks show reduced proportions of Mac1 positive (5% vs 27%) and Gr-1 positive (5% vs 12%) cells and strikingly increased CD117 positive cells (84% vs 49%). In vivo function of RXRαko/ko HSCs was evaluated by transplantation into lethally irradiated mice. Recipients were analyzed at 2, 4, and 6 weeks post-transplantation. Two weeks after transplantation, RXRαko/ko and RXRαfl/fl HSCs showed similar patterns of engraftment, with GFP-positive erythroid and myeloid cells found mainly in the spleen. At 4 weeks, recipients of RXRαfl/fl grafts showed significant BM engraftment of myeloid and erythroid lineages, while RXRαko/ko recipients exhibited minimal BM engraftment. At 6 weeks post-transplant, engraftment of RXRαfl/fl cells was well-established in both BM and spleen. RXRαko/ko HSCs showed minimal myeloid engraftment, but both spleen and BM were populated predominantly by Ter119 positive erythroid cells, which exhibit markedly dyserythropoietic morphology. This difference was reflected in peripheral blood counts; recipients of RXRαko/ko grafts were profoundly anaemic, thrombocytopenic, and neutropenic, and pronounced RBC polychromasia and poikilocytosis. These data indicate that disruption of RXRα in adult HSCs results in a modest reduction in early and committed progenitors in vitro, but profoundly disrupts ability to reconstitute haematopoiesis in a lethally irradiated recipient. Myeloid and megakaryocyte lineages do not engraft in recipients of RXRαko/ko HSCs. RXRαko/ko erythropoiesis is dysplastic and yields markedly abnormal erythrocytes. Further investigation is required to elucidate the multiple roles of RXRα in haematopoiesis.

Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Primary Myelofibrosis (PMF) Bone Marrow Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1742-1742
Author(s):  
Srdana Grgurevic ◽  
Srdan Verstovsek ◽  
Zhiming Liu ◽  
Taghi Manshouri ◽  
David Harris ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Alternative Pathway ◽  
Primary Myelofibrosis ◽  
Canonical Pathway ◽  
Lymphocytic Leukemia ◽  
Janus Kinase ◽  
Low Density ◽  
Iκb Kinase

Abstract Abstract 1742 Primary myelofibrosis (PMF) is a stem cell–derived hematologic malignancy, characterized by an expansion of one or more myeloid lineage resulting in bone marrow (BM) hypercellularity, magakaryocyte proliferation with atypia, granulocytic proliferation, and reticulin and/or collagen fibrosis. An acquired activating mutation in Janus kinase 2 at codon V617F (JAK2V617F) is detected in BM cells of the majority of patients with PMF. Constitutively activated JAK2 induces phosphorylation and activation of STAT3. Phosphorylated STAT3 forms heterodimers, translocates to the nucleus, binds to DNA, activates STAT3-target genes, and induces production of cytokines that interact with the BM microenvironment. Hematopoietic stroma derived soluble factors provide PMF cells with survival advantage (Manshouri et al. Cancer Res 71: 3831, 2011) and, as reported previously, most of these factors activate NF-κB in a variety of cell types. NF-κB plays an important role in the survival and proliferation of normal and neoplastic cells. In several hematologic malignancies, the NF-κB p65/p50 dimers were found to be activated to variable degrees. The activation of NF-κB is mediated by either the canonical pathway or the alternative pathway. The canonical pathway is typically activated by extracellular signals that activate the β subunit of the IκB kinase (IKK) complex (IKKβ) that induces the phosphorylation and degradation of the NF-κB inhibitor IκBα. Following IκBα degradation, NF-κB heterodimers translocate to the nucleus and bind to DNA. We have recently found that in chronic lymphocytic leukemia (CLL) constitutively activated STAT3 induces the production of unphsophorylated (U) STAT3. U-STAT3 binds to the NF-κB dimers p65/p50 in competition with IκB and the U-STAT3/NF-κB complex shuttles to the nucleus where NF-κB binds to DNA and activates NF-κB-regulated genes (Liu et al. Mol Cancer Res 9: 507, 2011). Because in PMF constitutively activated JAK2 induces phosphorylation of STAT3 and this activated form of STAT3 induces the production of U-STAT3, we wondered whether, like in CLL, U-STAT3 activates NF-κB in PMF. To determine whether NF-κB is constitutively activated in PMF we obtained BM low density cells from untreated patients with PMF. First we studied low-density BM cells of 11 patients with PMF using the electrophoretic mobility shift assay (EMSA). Cells of all samples bound to a p65/NF-κB DNA-labeled probe and the addition of an unlabelled (cold) p65/NF-κB probe attenuated or completely eliminated the binding. Typically, NF-κB-DNA binding appears and disappears due to repeated degradation and re-synthesis of IκB and the consequent activation and inactivation of NF-κB, respectively. Because we found that NF-κB is constitutively activated in all PMF BM samples we hypothesized that, like in CLL cells, activation of NF-κB in PMF cells is induced by an IκB-unrelated mechanism as reported by Yang J et al. (Cancer Res 65:939, 2005). By using immunoprecipitation of two different PMF BM samples we determined that STAT3 binds to the RelA/p65 NF-κB protein, and by using EMSA we found that anti-STAT3, similar to anti- NF-κB p65 antibodies, attenuated the binding of PMF BM cell extract to the NF-κB DNA probe. Taken together, our data suggest that U-STAT3 binds the NF-κB dimers p65/p50 and constitutively activates NF-κB in PMF. Disclosures: No relevant conflicts of interest to declare.

Conditional TRF1 Knockout in Haematopoietic Progenitor Cells Causes Telomere Shortening and Bone Marrow Failure by Induction of Cellular Senescence

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 49-49
Author(s):  
Fabian Beier ◽  
Miguel Foronda ◽  
Jose A Palacios ◽  
Paula Martinez ◽  
Maria A Blasco
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Conditional Knockout ◽  
Telomere Maintenance ◽  
Loss Of Function ◽  
Telomere Shortening ◽  
Exon 1

Abstract Abstract 49 Introduction: Mutations in the telomerase complex may cause bone marrow failure syndromes due to loss-of-function and consecutive telomere shortening. In addition to the telomerase complex, the six “shelterin” proteins (TRF1, TRF2, TIN2, RAP1, POT1 and TPP1) are required for telomere maintenance. TRF1 has a prominent role in chromosome capping function and prevents the recognition of telomeres by DNA repair mechanisms. At the moment, only TIN2 mutations have been linked to bone marrow failure. Here we aimed to identify other shelterin proteins might cause bone marrow failures. A previous study reported an clinical association between TRF1 mutations and acquired aplastic anemia, however the proof-of-principle that TRF1 can cause bone marrow failure is still missing (Savage SA Exp Hematol 2006). Material and Methods: To address this issue, we used the Mx1-Cre system in combination with the recently generated TRF1 allele in which the exon 1 of TRF1 is flanked by floxP (Martinez P Gen Dev 2009). The bone marrow of the bitransgenic mice was transplanted into B6 wildtype mice and poly (P:I) injections allowed the conditional knockout of TRF1. Results: Initiation of poly (P:I) injections 4 weeks after transplantation resulted in a failure of all three haematopoietic lineages after 17 days and histopathology revealed massive hypocellular bone marrow consistent with a bone marrow failure. Transplanted control animals showed normal histopathology and even increased neutrophil and thrombocyte counts. Further detailed FACS analysis 7 days after initiation of poly (P:I) injections showed a significant depletion of common myeloid, megakaryocte-erythocyte and common lymphoid progenitor cells, but only a slight decrease of lin-, c-kit+,Sca-1+ haematopoietic stem cells. Interesting, we found no increased rate of apoptosis for the decrease of the progenitor cells, but ß-galactosidase staining showed significant higher amounts of senescent cells in the bone marrow. Further detailed analysis of FACS sorted bone marrow cells showed that especially the c-kit positive progenitor fraction underwent senescence and cell cycle analysis showed an increased G2-M phase indicating a G2-M arrest. In line with these findings RT-PCR of FACS sorted BM revealed increased levels of p21 in the c-kit positive fraction. In addition BrdU injections into the mice on day 7 after poly (P:I) initiation showed increased incorporation and telomere length analysis of transplanted animals with and without poly (P:I) injections revealed massive telomere shortening on day 17. Conclusions: Our data indicates that TRF1 knockout especially affects haematopoietic progenitor cells by inducing G2-M arrest, induction of p21, and subsequent senescence. Further, compensation of the progenitor cell depletion leads to higher cell turnover and consecutively massive telomere shortening. Taken together this is the first report proving that TRF1 can cause a bone marrow failure and is accompanied with significant telomere shortening. Disclosures: No relevant conflicts of interest to declare.

SDF-1 Signaling in Myeloid Malignancies,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3824-3824
Author(s):  
Rita de Cassia Carvalho Melo ◽  
Carolina Bigarella ◽  
Matheus Manolo Arouca ◽  
Mariana Ozello Baratti ◽  
Fabiola Traina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Leukemic Cells ◽  
Myeloid Malignancies ◽  
Cxcr4 Signaling ◽  
Pkc Ζ ◽  
Worse Prognosis

Abstract Abstract 3824 The factor SDF-1 (stromal derived factor-1) was identified as an important chemoattractant factor produced by bone marrow cells. SDF-1 acts on its receptor CXCR4 and plays primordial function in migration, retention and development of hematopoietic progenitors in bone marrow. CXCR4 is expressed in leukemic cells and enables them to access marrow niches that normally are restricted to quiescent stem cells, thereby ensuring its protection from cell death resulting in a worse prognosis. In addition, it may induce activity of metalloproteinases (MMPs), which are enzymes that digest the extracellular matrix, making tumor cells more infiltrating. Moreover, higher levels of TIMP-2, an inhibitor of metalloproteinase 2 (MMP2), correlates with better prognosis in solid tumors. Recently, CXCR7 was identified as another SDF-1-binding receptor and the involvement of CXCR7 with tumor progression is well established in non-hematopoietic malignancies. Since it is well established that CD34 + progenitor cells from patients with myelodysplastic syndromes (MDS) are not attracted by gradient of SDF-1 despite of having CXCR4 normal expression, we addressed if MDS cells have an abnormal localization of CXCR4 or association with CXCR7. P39 and U937 cell line were used as a model of MDS and AML, respectively. Western blot analysis showed similar expression levels of CXCR4 and CXCR7 in both cell lines however we found, by confocal microscopy and flow cytometry, that CXCR4 was localized in the cytoplasm) of P39 cells while it was was in the membrane of U937 cells. Since the protein quinase C (PKC z) is related to the SDF-1/CXCR4 signaling by increasing CXCR4 expression and its membrane availability, we overexpressed HA-tagged PKCz in P39 cells.This procedure resulted in translocation of CXCR4 to the membrane of P39 cells but did not change the CXCR7 subcellular localization. Transwell chemotaxis assay showed that P39 cells overexpressing PKCz displayed higher chemotactic ability upon SDF-1 treatment compared with control P39 (35 fold increase pcDNA3-PKCz-HA vs pcDNA3-HA transfected P39 cells, p=0.0032; x2 test), suggesting that PKCz restored the chemotactic capacity of P39 cells. RNA expression of CXCR7 and TIMP-2 was analyzed by Real-time PCR (normalized by GAPDH and HPRT) in bone marrow samples from 50 MDS (FAB= 22 RA, 8 RARS, 20 RAEB), 29 acute myeloid leukemia (AML) patients and 11 healthy donors. CXCR7 mRNA expression did not differ significantly comparing all groups: controls, MDS (low or high risk) and AML whereas the expression of TIMP- 2 mRNA was significantly decreased in high risk MDS (p=0.0033) and AML (p=0.0003), (Mann-Whitney test) compared with normal controls. Taken together, our results suggest that a defect in the PKC ζ/CXCR4 pathway is involved with the unresponsiveness of MDS cells to SDF-1 and TIMP-2 downregulation could play a role in worse prognosis of myeloid malignancies. The role of CXCR7 is still undefined in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

Benzene-Induced Leukemogenesis: Irreversible Inhibition of PTPN2 and Subsequent STAT1 Signaling Alteration By the Hematotoxic Metabolite Benzoquinone

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3671-3671
Author(s):  
Romain Duval ◽  
Linh-Chi Bui ◽  
Cécile Mathieu ◽  
Emile Petit ◽  
Jean-Marie Dupret ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Tyrosine Phosphatase ◽  
Hematopoietic Cells ◽  
Loss Of Function ◽  
Irreversible Inhibitor ◽  
Irreversible Inhibition ◽  
Stat Signaling

Abstract Benzene (BZ) is a chemical compound of industrial and toxicological interest classified as a class I human carcinogen. Environmental and occupational exposure to BZ lead to bone marrow malignancies such as leukemia. The leukemogenic effects of BZ relies on its metabolization in bone marrow cells into reactive metabolites, in particular benzoquinone (BQ) that can react with macromolecules (arylation) and/or induce oxidative stress. Although BZ is well recognized as a leukemogenic chemical, most of the key molecular and cellular mechanisms underlying its hematotoxicity are not fully understood. PTPN2 is a protein tyrosine phosphatase (PTP) mainly expressed in hematopoietic cells and playing a key role in the homeostasis of the hematopoietic system. In particular, this PTP is an important modulator of growth factors and JAK/STAT signaling pathways. Loss of function analyses in patients with mutation/deletion of the PTPN2 gene and knock-out mouse models indicate that PTPN2 acts as a tumor suppressor in haematologic disorders such as leukemia. We found that BQ, the prime hematotoxic metabolite of BZ, is an irreversible inhibitor of human PTPN2. Kinetic and biochemical analyses using purified PTPN2 indicated that the irreversible inhibition of the enzyme by BQ is mainly due to arylation of its active site cysteine. Exposure of immortalized human hematopoietic cells (Jurkat T and THP-1 lines) to BQ leads to the irreversible inhibition of endogenous PTPN2 activity with a concomitant over activation of JAK/STAT signaling pathway. Irreversible BQ-dependent inhibition of PTPN2 in cells was found to be mainly due to overoxydation of its catalytic cysteine into sulfinic and/or sulfonic forms. In Vivo experiments conducted in mice confirmed that exposure to BZ leads to irreversible inhibition of PTPN2 in bone marrow and spleen cells. Our data provide the first mecanistic evidence that irreversible inhibition of PTPN2, a tumor suppressor tyrosine phosphatase, may contribute to benzene-dependent leukemogenesis. Disclosures No relevant conflicts of interest to declare.

A Patient-Derived EZH2 Mutant Induces MDS-like Diseases with Derepressed ABCG2 Expression in Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4116-4116
Author(s):  
Kimihito Cojin Kawabata ◽  
Daichi Inoue ◽  
Jiro Kitaura ◽  
Yuka Harada ◽  
Susumu Goyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Target Genes ◽  
De Novo ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Short Form ◽  
Physiological Role ◽  
Aberrant Expression ◽  
H3k27 Trimethylation ◽  
Marrow Cells

Abstract A histone H3 Lysine 27 (H3K27)-methyltransferase, enhancer of zeste homolog 2(EZH2) is known as a tumor-associated gene. Physiological role of EZH2 is an enzymatic component of polycomb repressive complex 2 (PRC2) to inhibit expression of target genes. While EZH2 plays oncogenic roles by repressing the expression of tumor suppressors in solid tumors and some lymphomas, it plays rather tumor-suppressive roles in myeloid malignancies. We have generated a short-form EZH2 that lacks the catalytic SET domain (EZH2-dSET). Using this EZH2 mutant we could produce serially transplantable MDS-like diseases. Microarray analysis using the MDS-like bone marrow cells enabled us to identify novel targets of EZH2 in MDS tumorigenesis, including ATP-binding cassette (ABC) transporters. Derepression of Abcg2 via decreased H3K27-trimethylation was confirmed. Retroviral transduction of EZH2-dSET to MDS-like cell lines increased surface ABCG2-high populations and those cells functioned to exclude anticancer drugs as expected. Intriguingly, with Abcg2 expression alone, primary bone marrow cells could produce an MDS-like cytopenic disease in our BMT model. In our clinical specimens, ABCG2 high expressions were observed in MDS samples but not in de novo AML and CML samples. In two MDS patients, ABCG2 expression decreased along with leukemic transformation. Interestingly, two out of 33 MDS patients with extremely high expression of ABCG2 harbored the same U2AF1 mutation (Q157P). In addition, somatic mutations of EZH2 and those of either U2AF1 or SRSF2 were mutually exclusive in all investigated cases. Interestingly, U2AF1 mutants (S34F and Q157P) reduced EZH2 expression, leading the derepression of ABCG2 via decreased H3K27-trimethylation. These results indicate a link between U2AF1 mutations and ABCG2 expression via disrupted EZH2. In conclusion, different mechanisms are supposed to converge at dysregulated EZH2 in MDS. And a short form of EZH2 upregulates ABCG2 expression resulting in MDS advancing to secondary leukemia. Thus, either mutations affecting the EZH2 function or mutations of EZH2 itself could play an important role in MDS, and one of the downstream targets of EZH2 suppression in MDS pathogenesis is aberrant expression of ABCG2. Disclosures No relevant conflicts of interest to declare.

Unraveling STAT Signaling in NPM/ALK-Induced Lymphoma: STAT3 but Not STAT5 Is Required for Lymphomagenesis in a Bone Marrow Transplantation Model Using Conditional Knockout Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1969-1969
Author(s):  
Tobias Dechow ◽  
Elisa Amon ◽  
Cornelius Miething ◽  
Rebekka Grundler ◽  
Nicole Schatz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Conditional Knockout ◽  
Prolonged Survival ◽  
Control Group ◽  
Wild Type ◽  
Post Transplantation ◽  
Knock Out ◽  
Alk Positive ◽  
Transplantation Model

Abstract Abstract 1969 Poster Board I-992 Introduction: NPM/ALK is a constitutively activated, oncogenic kinase leading to the development of anaplastic large cell lymphoma in humans. Multiple pathways have been implicated in NPM/ALK-dependent signaling including STAT3 and STAT5. In a transgenic mouse model it could be demonstrated that STAT3 is critical for NPM/ALK-driven lymphomagenesis. However, deletion of STAT3 did not lead to prolonged survival of the mice. In addition, the role of STAT5 in NPM/ALK-positive lymphoma in vivo is largely unclear. Methods: We used the established murine bone marrow (BM) transplantation model using BM derived from STAT3 and STAT5 conditional knock-out (fl/fl) mice. Precisely, we harvested BM from STAT3fl/fl and STAT5fl/fl mice and expressed NPM/ALK-Cre/EGFP in the BM cells by using retroviral gene transfer. BM cells were transplanted in lethally irradiated wild-type mice. Expression of NPM/ALK and deletion of STAT3 and STAT5 was determined in BM cells. Results: BM from STAT3fl/fl and STAT5fl/fl mice was successfully harvested, retrovirally infected, and transplanted in recipient mice. Analysis of BM cells revealed expression of NPM/ALK and deletion of STAT3 or STAT5 in NPM/ALK-Cre/EGFP-infected STAT3fl/fl and STAT5fl/fl BM respectively. Within the STAT3 group, control mice transplanted with NPM/ALK-EGFP-infected STAT3fl/fl BM died after 2-3 weeks from a NPM/ALK-positive lymphoma. An additional control group transplanted with NPM/ALK-Cre/EGFP-transduced wild-type BM also died from a NPM/ALK lymphoma but with an increased latency. Notably, mice transplanted with NPM/ALK-Cre/EGFP-infected STAT3fl/fl (STAT3-deleted) BM showed a significantly prolonged survival compared to either control groups including 5 mice that survived at least 300 days after transplantation. Interestingly, only about 60% of these mice died from an NPM/ALK-positive high-grade lymphoma or myeloma-like disease, whereas the remaining animals died from other (irradiation-induced) diseases. In contrast, 150 days post transplantation STAT5 deletion in NPM/ALK expressing BM cells did not increase survival of the transplanted mice compared to the control group with wild-type BM using NPM/ALK-Cre/EGFP retrovirus. Conclusion: Specific deletion of STAT3 but not STAT5 in NPM/ALK-expressing cells markedly attenuates disease progression. The described transplantation model using BM from conditional knock-out mice and an Cre-containing expression vector is extremely helpful to specifically study signaling in oncogene-driven hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

The Role of JARID2 in Normal and Malignant Hematopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 793-793
Author(s):  
Hamza Celik ◽  
Kramer C Ashley ◽  
Martens Andy ◽  
Elizabeth Eultgen ◽  
Cates Mallaney ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Median Survival ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Kaplan Meier ◽  
Marrow Cells

Abstract Despite the increasing availability of targeted therapies for myeloproliferative neoplasms (MPNs), there remains a subset of these patients that transform to secondary acute myeloid leukemia (sAML). MPN patients who develop sAML have a dismal outcome, with a median survival of six months. The mechanisms and pathways that contribute to transformation from MPN to sAML have not been well delineated. The most commonly mutated genes found in the MPN initiating clones include JAK2, MPL and CALR. Transformation to sAML however requires acquisition of additional co-operating mutations such as TET2, IDH1/2, and NRAS. Recent genome sequencing studies identified deletions of JARID2, a gene associated with the Polycomb Repressive Complex 2 (PRC2) involved in implementing global H3K27me3 in post-MPN sAML. Mutations in JARID2 are found only in the blast phase of MPNs, but not in chronic phase of the disease. This data suggests that a JARID2 deletion could be a sAML-specific transforming event by acting as a tumor suppressor in HSCs. To investigate the role of Jarid2 as a tumor suppressor, we utilized an inducible mouse model of the prototypical MPN driver mutation Jak2V617F. We have established our model system by crossing Mx1-CRE:Jarid2fl/fl (Jarid2KO) or Mx1-CRE:Jarid2fl/+ (Jarid2HET) with JAK2V617F mice to generate a Mx1-CRE:Jarid2fl/fl Jak2V617F/+ or Mx1-CRE: Jarid2fl/+Jak2V617F/+ strain. Mx1-CRE mediates both activation of Jak2V617Fand deletion of Jarid2 simultaneously in adult hematopoietic compartment upon injection of the double-stranded RNA analog polyinosinic:polycytidylic acid (pIpC). In all cases, the absence of Jarid2 in Jak2V617F/+ background accelerated MPN progression, characterized by elevated hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, and splenomegaly compared to the control groups. Median survival of Jarid2KO-Jak2V617F/+ and Jarid2HET-Jak2V617F/+ strains also revealed a dose-dependence of Jarid2 on life expectancy with a median of 27 and 56 days post pIpC treatment, respectively (Figure 1). Together, these data suggest that loss of Jarid2 in Jak2V617F/+ background accelerates the progression of MPN. To more completely understand the tumor suppressor function of Jarid2 in MPN, we aimed to define its role in normal hematopoiesis. Jarid2 is highly expressed in myeloid-biased compared to lymphoid-biased HSCs, suggestive of a role in HSC subtype determination. Moreover, conditional knock-out studies show that each core component of PRC2 (EED, SUZ12 and EZH2) has distinct as well as overlapping functional properties in hematopoiesis. To study the function of Jarid2 in normal hematopoiesis, we crossed Jarid2fl/fl mice to the Vav-CRE strain to facilitate conditional inactivation of Jarid2 in hematopoietic cells. Vav1-CRE is expressed throughout life in definitive HSCs and all hematopoietic lineages starting at E10.5. Analysis of eight-week old Vav1-CRE:Jarid2fl/fl mice showed that complete loss of Jarid2 induced a significant compromise in hematopoiesis with a robust reduction in phenotypically-defined bone marrow HSCs, a defective B-cell generation in the bone marrow (BM), a differentiation block in T-cell development in thymus, and a significant reduction in peripheral blood counts. A competitive transplantation strategy was also employed to assess the stem cell potential of Jarid2-KO HSCs. 2.5 x 105 whole bone marrow cells isolated from adult mice were transplanted into lethally irradiated recipient mice along with 2.5x105 whole bone marrow cells from congenic wild-type mice. Peripheral blood analysis of these mice over the period of 16-weeks post-transplant has shown that the loss of Jarid2 disrupts HSC function, leading to enhanced myeloid and reduced lymphoid output. Collectively, these data illustrate that Jarid2 is required for hematopoietic hemostasis including appropriate lineage fate determination of HSCs. The loss of Jarid2 in a Jak2V617F background promotes acceleration of MPN and implicates Jarid2 as a hematopoietic tumor suppressor. Figure 1. Kaplan-Meier analysis of a cohort of Jarid2KO-Jak2V617F (n = 5) and Jarid2HET -Jak2V617F (n = 6) and littermate controls (n = 4-8 each). Figure 1. Kaplan-Meier analysis of a cohort of Jarid2KO-Jak2V617F (n = 5) and Jarid2HET -Jak2V617F (n = 6) and littermate controls (n = 4-8 each). Disclosures No relevant conflicts of interest to declare.

Regulation of Myelopoiesis by Differentiation Stage-Specific Activities of HOXA9 and HOXA10

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-29-SCI-29
Author(s):  
Elizabeth A. Eklund
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Gene Networks ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Murine Bone Marrow

Abstract Abstract SCI-29 HOXA9 and HOXA10 are homeodomain (HD) transcription factors that are implicated in control of myelopoiesis and contribute to myeloid leukemogenesis. These proteins are expressed coordinately during hematopoiesis, with maximal expression in granulocyte/monocyte progenitor (GMP) cells. Engineered overexpression of Hoxa9 or Hoxa10 in primary bone marrow cells expands the GMP population in vitro, and results in myeloproliferation in murine bone marrow transplant experiments. Mice transplanted with Hoxa9- or Hoxa10-overexpressing bone marrow develop acute myeloid leukemia (AML) over time. Consistent with this, increased and sustained expression of a set of HD proteins, including HOXA9 and HOXA10, is found in a subset of human AML, including AML with MLL gene translocations (11q23-AML). Since the DNA-binding HDs of HOXA9 and HOXA10 are highly conserved, we hypothesize that they recognize a common set of target genes. However, since HOXA9 and HOXA10 diverge outside the HD, we considered the unexplored possibility that they perform different functions in regulating such genes. To identify molecular mechanisms for HOX-induced GMP expansion and leukemogenesis, we performed a chromatin immunoprecipitation-based screen for HOXA10 target genes. Gene ontology studies determined that the identified set is enriched for genes encoding growth factors and receptors, including fibroblast growth factor 2 (FGF2). We found that production of FGF2 by Hoxa10-overexpressing GMP stabilizes β-catenin and induces proliferation in an autocrine manner. We also found that HOXA9 and HOXA10 activate common FGF2 cis elements. The Hoxa10-target-gene set is also enriched for HD-transcription factors, including CDX4. We determined that Cdx4 transcription is activated by HOXA10 in GMP, but repressed by HOXA9 in differentiating myeloid cells. CDX4 activates transcription of both Hoxa9 and Hoxa10, identifying a HOX-CDX cross-regulatory mechanism. This mechanism may be influenced by Fgf2, since Hoxa10 and Cdx4 are β-catenin target genes, but β-catenin activity decreases Hoxa9 expression. Gene expression profiling studies indicate that HOXA9, HOXA10, CDX4, and FGF2 are increased in 11q23-AML, suggesting clinical relevance. Arih2 (encoding the E3 ligase Triad1) is another common HOXA9 and HOXA10 target gene that may influence Fgf2 activity. We found that Arih2 transcription is repressed by HOXA9 in myeloid progenitors, but activated by HOXA10 in differentiating phagocytes. FGF receptors are destabilized by ubiquitination, and we found increased FGF-R ubiquitination in Hoxa10-overexpressing cells. Therefore, Triad1-dependent regulation of FGF-R stability is another mechanism for control of FGF2 activity and myeloproliferation by HOXA9 and HOXA10. Therefore, HOXA9 and HOXA10 regulate a common set of target genes that control GMP expansion in a manner that is antagonistic for some genes and cooperative for others. Clinical correlative studies suggest that coordinate control of these genes by HOXA9 and HOXA10 is dysregulated in HOX-overexpressing leukemia. Understanding HOX-regulated gene networks may identify therapeutic targets for HOX-overexpressing leukemias. For example, blocking FGF-related signaling pathways may ameliorate cytokine hypersensitivity in such leukemias, and would be a topic of interest for additional studies. Disclosures: No relevant conflicts of interest to declare.

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