Stat3 Negatively Regulates Myeloproliferative Neoplasm Induced By Jak2V617F

Abstract The JAK2V617F mutation has been found in most patients with Ph-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of several signaling molecules/pathways, such as Stat5, Stat3, Akt and Erk. Unraveling the contribution of these signaling pathways in MPNs will improve our understanding of the pathogenesis of MPNs and allow us to develop more effective targeted therapies. We have previously reported the generation of a conditional Jak2V617F knock-in mouse, which exhibits all the clinical features of human PV. Using this mouse model, we have demonstrated that Stat5 is absolutely required for the pathogenesis of PV induced by Jak2V617F. However, the contribution of other signaling molecules activated by Jak2V617F in the development and progression of MPNs still remains elusive. Stat3, a member of the family of signal transducer and activator of transcription (Stat), is often found activated in solid tumors and hematologic malignancies including MPNs. Although Stat3 is known to play a tumor-promoting function in various human malignancies, recent studies also have found a tumor suppressive function of Stat3 in certain malignancies. For instance, Stat3 negatively regulates BRAFV600E-induced thyroid tumorigenesis (Couto et al., Pro Natl Acad Sci USA 2012) or suppresses PTEN loss-induced malignant transformation of astrocytes (Iglesia et al., Genes Dev 2008). Thus, Stat3 can positively or negatively regulate cell growth and tumor progression. Here, we sought to determine the role of Stat3 in Jak2V617F-evoked MPN using conditional Stat3 knock-out (Stat3 floxed) and Jak2V617F knock-in mice. Whereas expression of Jak2V617F resulted an increase in red blood cells (RBC), hemoglobin, hematocrit, white blood cells (WBC), neutrophils and platelets in the peripheral blood of the Jak2V617F knock-in mice, deletion of Stat3 did not cause any significant change in RBC, hemoglobin, hematocrit and platelet numbers in Jak2V617F knock-in mice. Strikingly, Stat3 deficiency significantly increased nertrophil counts in mice expressing Jak2V617F. Flow cytometric analysis showed that deletion of Stat3 increased the hematopoietic stem cell (HSC) compartments (LSK, LT-HSC, ST-HSC) and GMP populations in the bone marrow (BM) and spleens of mice expressing Jak2V617F. However, MEP population was unaffected by Stat3 deletion. Cell cycle analysis using Hoechst/Pyronin Y staining revealed that Jak2V617F expression alone resulted in increased cycling of HSC-enriched LSK cells, and Stat3-deficiency further enhanced the cycling of Jak2V617F-expressing LSK cells. Stat3-deficiency also caused a marked expansion of Gr-1+/Mac-1+ population in the BM and spleens of mice expressing Jak2V617F. As a consequence, CD71+/Ter119+ population was proportionally reduced in Stat3-deficient Jak2V617F-expressing mice BM. Histopathologic analysis showed marked increase in granulocytes in the BM and spleens of Stat3-deficient Jak2V617F-expressing mice compared with mice expressing Jak2V617F. Stat3-deficient Jak2V617F-expressing mice also exhibited marked infiltration of neutrophils in their livers. Furthermore, deletion of Stat3 significantly reduced the survival of Jak2V617F knock-in mice. Together, these results suggest a negative role for Stat3 in Jak2V617F-induced MPN. Thus, Stat3 may not be a suitable therapeutic target for treatment of PV and other JAK2V617F-positive MPNs. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v124.21.159.159 ◽

2014 ◽

Vol 124 (21) ◽

pp. 159-159

Author(s):

Yue Yang ◽

Hajime Akada ◽

Dipmoy Nath ◽

Robert E Hutchison ◽

Golam Mohi

Keyword(s):

Peripheral Blood ◽

Blood Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Flow Cytometric Analysis ◽

Gene Set Enrichment Analysis ◽

Jak2v617f Mutation ◽

Interferon Response ◽

Hematopoietic Stem ◽

Erythroid Precursors

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

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Critical requirement for Stat5 in a mouse model of polycythemia vera

Blood ◽

10.1182/blood-2011-03-345215 ◽

2012 ◽

Vol 119 (15) ◽

pp. 3539-3549 ◽

Cited By ~ 77

Author(s):

Dongqing Yan ◽

Robert E. Hutchison ◽

Golam Mohi

Keyword(s):

Polycythemia Vera ◽

Blood Cells ◽

Myeloproliferative Neoplasms ◽

White Blood Cells ◽

Strong Support ◽

Primary Myelofibrosis ◽

Blood Parameters ◽

Critical Function ◽

Critical Requirement ◽

Multiple Signaling

The JAK2V617F mutation has been identified in most cases of Ph-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of multiple signaling molecules/pathways. However, the key signaling downstream of JAK2V617F required for transformation and induction of MPNs remains elusive. Using a mouse genetic strategy, we show here that Stat5 is absolutely required for the pathogenesis of PV induced by Jak2V617F. Whereas expression of Jak2V617F in mice resulted in all the features of human PV, including an increase in red blood cells, hemoglobin, hematocrit, white blood cells, platelets, and splenomegaly, deletion of Stat5 in the Jak2V617F knockin mice normalized all the blood parameters and the spleen size. Furthermore, deletion of Stat5 completely abrogated erythropoietin (Epo)–independent erythroid colony formation evoked by Jak2V617F, a hallmark feature of PV. Re-expression of Stat5 in Stat5-deficient Jak2V617F knockin mice completely rescued the defects in transformation of hematopoietic progenitors and the PV phenotype. Together, these results indicate a critical function for Stat5 in the pathogenesis of PV. These findings also provide strong support for the development of Stat5 inhibitors as targeted therapies for the treatment of PV and other JAK2V617F-positive MPNs.

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Combination approach to diagnosis and treatment of an elderly patient with chronic Ph-negative myeloproliferative neoplasm and concomitant surgical pathology. Clinical observation

MD-Onco ◽

10.17650/2782-3202-2021-1-1-61-65 ◽

2021 ◽

Vol 1 (1) ◽

pp. 61-65

Author(s):

Yu. E. Ryabukhina ◽

P. A. Zeynalova ◽

O. I. Timofeeva ◽

F. M. Abbasbeyli ◽

T. V. Ponomarev ◽

...

Keyword(s):

Elderly Patient ◽

Essential Thrombocythemia ◽

Early Stage ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Left Femur ◽

Hematopoietic Stem ◽

Chronic Myeloproliferative Neoplasms

Chronic myeloproliferative neoplasms (CMPN), Ph-negative, are of clonal nature, develop on the level of hematopoietic stem cell and are characterized by proliferation of one or more hematopoietic pathways. Currently, the group of Ph-negative CMPN includes essential thrombocythemia, primary myelofibrosis, polycythemia vera, myeloproliferative neoplasm unclassifiable.Identification of mutations in the Jak2 (V617F), CALR, and MPL genes extended understanding of biological features of Ph-negative CMPN and improved differential diagnosis of myeloid neoplasms. Nonetheless, clinical practice still encounters difficulties in clear separation between such disorders as primary myelofibrosis, early-stage and transformation of essential thrombocythemia into myelofibrosis with high thrombocytosis. Thrombocytosis is one of the main risk factors for thromboembolic complications, especially in elderly people.A clinical case of an elderly patient with fracture of the left femur developed in the context of Ph-negative CMPN (myelofibrosis) with high level of thrombocytosis is presented which in combination with enforced long-term immobilization and presence of additional risk created danger of thrombosis and hemorrhage during surgery and in the postoperative period.

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Incorporation of Clinical Information to Decipher Driver Mutations in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-118546 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5489-5489

Author(s):

Murtadha K. Al-Khabori ◽

Shoaib Al-Zadjali ◽

Mohamed Al-Rawahi ◽

Iman Al Noumani ◽

Khalil Al Farsi ◽

...

Keyword(s):

Platelet Count ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Clinical Information ◽

Primary Myelofibrosis ◽

Mutation Load ◽

Female Ratio ◽

Epigenetic Regulators ◽

The Impact

Abstract Introduction: Mutations in the epigenetic regulators are commonly found in myeloid disorders including Myeloproliferative Neoplasm (MPN). Primary myelofibrosis, dysplastic changes and severity of the disease were associated with the mutation load. Most of the studies had a limited number of targeted genes and included a mixture of JAK2 positive and negative disease. The objective of this study is to assess the impact of mutations in the epigenetic regulators on the presentation of patients with JAK2 V617F positive MPN. Methods: We retrieved the clinical and laboratory information on 61 consecutive eligible patients. Mutation analysis of the entire coding regions of ASXL1, ASXL2, CBL, CEBPA, CSF3R, DNMT3A, EZH2, IDH2, TET1 and TET2 genes was performed using next generation sequencing (NGS; Ion PGM Sequencer; Thermo Fisher ScientificÒ). The library was constructed and the templates were prepared using the PGM tool. The variants were annotated using the ClinVar database and the prediction from the Scale-Invariant Feature Transform (SIFT) and or Polymorphism Phenotyping (Polyphen) algorithms. Alignment, variant filtering and annotation were performed using Ion Torrent Suite. Standard descriptive and analytical statistics were used as appropriate to describe and compare different groups. The MPN subtype, bleeding, thrombosis, hemoglobin, platelet count, White Cell Count (WBC), Lactate Dehydrogenase (LDH) and erythropoietin level were compared for each candidate variant. An alpha threshold of 0.05 was used with no adjustment for multiple comparisons as the analyses were considered exploratory. All statistics were performed using R program. Variants were selected for further experimentation based on their frequency and association with the clinical information at diagnosis. Results: Sixty-one patients were included (Polycythemia Vera: 29, Essential Thrombocythemia: 21, Primary Myelofibrosis: 9, MPN unclassifiable: 2) with a median age of 62 years (Interquartile Range [IQR]: 44 - 70). Male to female ratio was 35:26. The median hemoglobin, WBC, platelet count, LDH and erythropoietin were 14.6 g/dL (IQR: 12.8 - 16.8), 11.5 *109/L (IQR: 11.5 - 14.4), 507 *109/L (IQR: 391 - 779), 265 mU/mL (IQR: 231 - 409) and 1.2 mU/mL (IQR: 1.0 - 4.8) respectively. At presentation, 54% had splenomegaly, 23% had an arterial or venous thrombosis, and 5% had bleeding. Sixty-three variants were found in the samples tested. The median mutation load was 13 variants (Range: 11-14). Patients with higher than the median mutation load had higher mean erythropoietin (7.8 vs. 0.9 g/dL; p = 0.02016). ASXL1 p.Leu815Pro variant was found in all patients. Only three variants were found in the ClinVar database. Seven variants were predicted to be pathogenic (ASXL1: 1, EZH2: 1, IHD1: 1, TET1: 1 and TET2: 3). Patients with TET2: p.Leu1721Trp variant had 6.4 higher odds of bleeding (p = 0.04345). Patients with TET2: p.His1778Arg variant had a lower WBC (9.1 vs. 13.9 *109/L; p = 0.01699) and LDH (213 vs. 348 mU/mL; p = 0.0006528) while those with TET1: p.Lle1123Met variant had a higher WBC (13.5 vs. 8.3 *109/L; p = 0.02756). None of the remaining comparisons were statistically significant. Conclusions: Incorporation of clinical information facilitates the prioritization of variants from DNA sequencing experiments. In MPN, we recommend ASXL1: p.Leu815Pro (expressed in all patients), TET2: p.Leu1721Trp (associated with bleeding), TET2: p.His1778Arg (correlated with the WBC and LDH) and TET1: p.Lle1123Met (correlated with LDH) for further functional experimentation. Disclosures No relevant conflicts of interest to declare.

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Plasma EDA Fibronectin in Primary Myelofibrosis Correlates with Bone Marrow Fibrosis and Predicts Splenomegaly

Blood ◽

10.1182/blood-2019-130735 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1688-1688

Author(s):

Alessandro Malara ◽

Cristian Gruppi ◽

Margherita Massa ◽

Vittorio Rosti ◽

Giovanni Barosi ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Myeloproliferative Neoplasm ◽

Primary Myelofibrosis ◽

Enzyme Linked Immunosorbent Assay ◽

Costal Margin ◽

Cell Transplant ◽

Healthy Controls ◽

Hematopoietic Stem

Introduction: Primary myelofibrosis (PMF) is a Philadelphia chromosome negative myeloproliferative neoplasm with adverse prognosis characterized by bone marrow (BM) fibrosis and extramedullary hematopoiesis. Fibronectin (FN) is an extracellular matrix glycoprotein that plays vital roles during tissue repair and regeneration. It exists in different forms. Plasma FN is synthesized by hepatocytes and secreted into the blood plasma, where circulates at a concentration of 300-600 μg/ml in a soluble, compact form. Differently, cellular FN is synthesized by several cell types, such as fibroblasts, endothelial cells, chondrocytes and myocytes. The alternative splicing of EDA and EDB and more complex splicing of the V domain, during transcription of FN1 gene, allows different isoforms of FN to be expressed in a tissue-dependent and temporally regulated manner. Very low levels (1.3-3 μg/ml) of FN containing EDA and/or EDB are present in plasma. Although its function is not well understood, EDA containing FN (EDA-FN) is known to agonize Toll like receptor 4 (TLR4), resulting in NF-κβ-dependent cytokine release; to induce myofibroblast differentiation during wound healing; and to increase agonist-induced platelet aggregation and thrombus formation in vivo. We previously showed that EDA-FN levels are increased in plasma and BM biopsies of PMF patients. Mechanistically, BM EDA-FN sustains megakaryocyte proliferation through TLR4 binding and confer a pro-inflammatory phenotype to cell niches promoting fibrosis progression in Romiplostim-treated mice. In this work we measured the plasma levels of EDA-FN in 104 well characterized patients with PMF to determine whether elevated levels of EDA-FN predict the occurrence of disease-related events. Methods: Plasma circulating EDA FN was measured with an enzyme linked immunosorbent assay developed at the University of Pavia, by our group. We obtained plasma EDA-FN concentration values and health care data of persons with PMF from the data-base of the Centre for the Study of Myelofibrosis at the IRCCS Policlinico S. Matteo Foundation in Pavia. We sequentially excluded persons treated with disease-modifying drugs at any time before or on the date of base-cohort entry, and those who had been splenectomized or had received a stem cell transplant. We also excluded persons with acute inflammatory diseases, autoimmune diseases, other neoplasms, and severe liver or renal dysfunction. For this study we selected everyone giving written informed consent and the study was approved by the local Ethic Committee. Immunofluorescence was performed on spleen sections from PMF patients who underwent splenectomy either because of anemia or symptomatic splenomegaly, or both; and healthy controls that were splenectomized following traumatic lesion of the spleen. Data were analyzed using STATISTICA software. Results: A homozygous JAK2V617F genotype was the major determinant of elevated plasma EDA-FN. Elevated EDA-FN levels were associated with anemia, increased levels of high-sensitivity C-reactive protein, BM fibrosis and splanchnic vein thrombosis at diagnosis. We interpreted these associations as reflecting the role EDA-FN plays in tissue remodeling, inflammation and vascular injury. Interestingly, EDA-FN levels resulted also associated with spleen size, and elevated levels of EDA-FN at diagnosis predicted large splenomegaly (more than 10 cm from the left costal margin) outcome. The evidence that plasma EDA-FN levels were not associated with the CD34+ hematopoietic stem cells mobilization, drove us to hypothesize that EDA-FN could reflect spleen endothelial cell activation and/or neoangiogenesis. Immunofluorescence analysis of spleen specimens from PMF patients and healthy controls revealed that high levels of EDA-FN were present in pathological spleens in strong association with endothelial neoangiogenesis. Conclusions: Quantification of EDA-FN level in PMF strongly correlates with BM fibrosis and may be the first marker of an altered spleen microvasculature that contributes to splenomegaly. Understanding the role of this FN isoform in PMF would be useful for testing new mechanisms of disease progression and new hypotheses about the treatment of splenomegaly in PMF. Disclosures No relevant conflicts of interest to declare.

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Expression of HMGA2 Collaborates with JAK2V617F to Progress Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v126.23.482.482 ◽

2015 ◽

Vol 126 (23) ◽

pp. 482-482

Author(s):

Koki Ueda ◽

Kazuhiko Ikeda ◽

Kazuei Ogawa ◽

Akiko Shichishima-Nakamura ◽

Kotaro Shide ◽

...

Keyword(s):

Disease Progression ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Extramedullary Hematopoiesis ◽

Hematopoietic Stem ◽

Cell Counts ◽

Epigenetic Modifiers ◽

One Year

Abstract Myeloproliferative neoplasms (MPN) are characterized by chronic proliferation of myeloid cells, extramedullary hematopoiesis and occasional leukemic transformation. Mutations in JAK2, CALR and MPL have been established as drivers of myeloproliferative phenotype, but their roles in disease progression with clonal expansion remain unclear. In addition, studies have shown mutations in epigenetic modifiers including TET2, DNMT3A, ASXL1 and EZH2, and aberrant expressions of microRNAs in MPN, but downstream of these changes is also largely unknown. Recently, we showed high expression of HMGA2 mRNA partly correlated with reduced microRNA let-7 in granulocytes of patients with MPN, including 100% patients with primary myelofibrosis (MF) and 20% polycythemia vera and essential thrombocythemia (Harada-Shirado et al, Brit J Haematol, 2015). In mice, loss of epigenetic modifiers such as BMI1 and EZH2, along with the Arf/Ink4a knockout (Oguro et al, J Exp Med, 2012) or the JAK2 V617F (Sashida et al, ASH, 2013), leads to overexpression of HMGA2 with accelerating MPN. We have generated transgenic (Tg) mice of Hmga2 cDNA with truncated 3'UTR (ΔHmga2) lacking binding sites of let-7 thatrepresses expression of HMGA2 (Ikeda et al, Blood, 2011). Δ Hmga2 mice overexpress HMGA2 and develop MPN-like disease, and represent a clonal advantage in competitive repopulations with serial bone marrow (BM) transplants (BMT). Here, to clarify if HMGA2 affect JAK2 V617F+ hematopoiesis, we crossed Δ Hmga2+/- mice with JAK2 V617F+/- Tg mice (Shide et al, Leukemia, 2008). Δ Hmga2-/-JAK2 V617F-/- wild type (WT), Δ Hmga2+/-JAK2 V617F-/- (Δ Hmga2 -Tg), Δ Hmga2-/-JAK2 V617F+/- (JAK2 V617F-Tg) and Δ Hmga2+/-JAK2 V617F+/- (double-Tg) mice were born at expected Mendelian ratios and we could analyze 5 - 6 of each. At 3 months old, leukocytosis, thrombocytosis, anemia and splenomegaly were most severe in double-Tg compared with JAK2 V617F-Tg or Δ Hmga2 -Tg mice. Relative to WT, peripheral leukocyte and platelet counts were nearly 16- and 4-fold higher in double-Tg, while 3- and 2-fold higher in JAK2 V617F-Tg mice, respectively. Mean spleen weights were 0.067, 0.10, 0.83 and 2.8 g in WT, Δ Hmga2 -Tg, JAK2 V617F-Tg and double-Tg mice, while BM cell counts were 2.4, 2.8, 0.4 and 1.2 x 107/femur, respectively. However, JAK2 V617F-Tg and double-Tg equally showed MF whereas no MF was detected in WT and DHmga2-Tg, suggesting that HMGA2 partly recovers cellularity in fibrotic BM. In the absence and presence of JAK2 V617F, HMGA2 augments lineage- Sca1+ Kit+ cells (WT: Δ Hmga2-Tg: JAK2 V617F-Tg: double-Tg= 0.17%: 0.19%: 0.17%: 0.27% in BM cells), endogenous erythroid colonies (1: 11: 13: 21 CFU-E/104 BM cells) and CD71+ Ter119+ erythroblasts (23%: 29%: 5.7%: 10% in BM and 2.0%: 4.4%: 7.9%: 16% in spleen cells), indicating HMGA2 contributes to expansion of hematopoietic stem/progenitor cells (HSPC) and erythroid commitment in JAK2 V617F+ hematopoiesis. Most Δ Hmga2-Tg and JAK2 V617F-Tg survived for over one year, but all double-Tg mice died within 4 months after birth due to severe splenomegaly and MF with no acute leukemia. To study the effect of HMGA2 on JAK2 V617F+ HSPC activity, we performed BMT with 0.25 x 106 Ly5.2+Δ Hmga2-Tg, JAK2 V617F-Tg or double-Tg cells with 0.75 x 106 Ly5.1+ competitor WT cells to lethally irradiated Ly5.1+ WT mice. Proportions of Ly5.2+ cells were higher in recipients of Δ Hmga2 -Tg than double-Tg cells, while JAK2 V617F-Tg cells were almost rejected at 8 weeks after BMT. To confirm role of HMGA2 without let-7 repression in JAK2 V617F+ hematopoiesis, we performed another BMT with 1 x 104 KIT+ cells of JAK2 V617F-Tg mice transduced with retroviral vector of Hmga2 with each let-7 -site-mutated full-length 3'UTR (Hmga2-m7) to sublethally irradiated WT mice. Recipients of JAK2 V617F-Tg cells with Hmga2-m7 developed MPN-like disease, whereas donor cells were rejected in recipients of JAK2 V617F cells with empty vector. In conclusion, HMGA2 may play a crucial role in hematopoiesis harboring JAK2 V617F by expanding HSPC, leading to disease progression. Disclosures No relevant conflicts of interest to declare.

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Critical Role for Stat5 in the Initiation and Maintenance of Polycythemia Vera in a Jak2V617F Knock-In Mouse Model

Blood ◽

10.1182/blood.v118.21.121.121 ◽

2011 ◽

Vol 118 (21) ◽

pp. 121-121 ◽

Cited By ~ 2

Author(s):

Dongqing Yan ◽

Golam Mohi

Keyword(s):

Polycythemia Vera ◽

Blood Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Critical Role ◽

Flow Cytometric Analysis ◽

Strong Support ◽

Blood Parameters ◽

Cyclin D2 ◽

P70s6 Kinase

Abstract Abstract 121 Version:1.0 StartHTML:0000000207 EndHTML:0000006199 StartFragment:0000002599 EndFragment:0000006163 SourceURL:file://localhost/Users/mohim/Desktop/ASH%202011/Dongqing%20Yan%202011%20ASH%20Abstract.doc The JAK2V617F mutation has been identified in most cases of Ph-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of multiple signaling molecules/pathways. However, the key signaling downstream of JAK2V617F required for transformation, induction and maintenance of MPNs remains elusive. Using a mouse genetic strategy, we found that Stat5 is absolutely required for the pathogenesis of PV induced by Jak2V617F. Whereas inducible expression of Jak2V617F in mice resulted in all the features of human PV, including increase in red blood cells, hemoglobin, hematocrit, white blood cells, platelets, and splenomegaly, deletion of Stat5 in the Jak2V617F knock-in mice normalized all the blood parameters and the spleen size. Histopathologic analyses revealed that Stat5 deficiency blocked the development of PV in mice expressing Jak2V617F. In addition, deletion of Stat5 completely abrogated erythropoietin (Epo)-independent erythroid colony formation evoked by Jak2V617F, a hallmark feature of PV. Flow cytometric analysis revealed that concomitant deletion of Stat5 reduced the Jak2V617F-induced expansion of LSK (lin−Sca-1+c-kit+) and MEP (megakaryocyte-erythroid progenitors) as well as CD71+Ter119+ and Gr-1+Mac-1+ populations to normal levels. Unlike Jak2V617F knock-in mice, which developed myelofibrosis at old age, Stat5-deficient Jak2V617F-expressing mice failed to develop myelofibrosis. Re-expression of Stat5 in Stat5-deficient Jak2V617F knock-in mice bone marrow by retroviral transduction completely rescued the defects in transformation of hematopoietic progenitors and the PV phenotype. Furthermore, deletion of Stat5 after establishment of PV disease in the transplanted animals expressing Jak2V617F by injection with polyinosine:polycytosine (pI:pC) normalized the blood parameters and inhibited the progression of the disease. Together, these results indicate a critical function for Stat5 in the induction and maintenance of PV. Biochemical analyses revealed that Stat5 deficiency significantly inhibited constitutive phosphorylation of p70S6 kinase and markedly reduced expression of Bcl-xL, Cyclin-D2 and Pim-1 mediated by Jak2V617F. These suggest that p70S6 kinase, Bcl-xL, Cyclin-D2 and Pim-1 are downstream targets of Jak2V617F-Stat5 signaling, and they may play a role in hematopoietic transformation induced by Jak2V617F. These findings provide strong support for the development of Stat5 inhibitors as targeted therapies for the treatment of PV and other JAK2V617F-positive MPNs. Disclosures: No relevant conflicts of interest to declare.

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46/1 Haplotype Permits to Follow JAK2 Homologous Recombination: Modeling JAK2V617F clonal Architecture in PV Patients

Blood ◽

10.1182/blood.v120.21.1757.1757 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1757-1757

Author(s):

Salma Hasan ◽

Jean Pierre Le Couedic ◽

Fabrizia Favale ◽

Barbara Monte-Mor ◽

Catherine Lacout ◽

...

Keyword(s):

Homologous Recombination ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Clonal Structure ◽

Hematopoietic Stem ◽

Clonal Architecture ◽

Specific Pcr ◽

Allele Specific ◽

Proliferative Advantage

Abstract Abstract 1757 Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders characterized by excess proliferation of one or several myeloid lineages. More than 95% polycythemia vera (PV) and 50–60% essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients harbor a somatic 1849 G>T mutation in JAK2 gene. Moreover about 30% of PV patients are homozygous for this mutation due to a loss of heterozygosity after a mitotic homologous recombination (HR). Among 92 haplotypes of chromosome 9p, 46/1 haplotype is strongly associated with the cis-aquisition of JAK2V617F mutation. The purpose of this study was to estimate the clonal frequency of WT, JAK2V617F/+ and JAK2V617F/V617F in progenitors compartments. Here, we have modeled the JAK2V617F clonal architecture in 9 PV patients heterozygous for the 46/1 haplotype by using the level of JAK2 and the 46/1 haplotype as a marker to follow HR. First we measured the global JAK2V617F and 46/1 allele burden in CD34+ cells either by allele-specific PCR or by Ion Torrent sequencing in order to calculate the expected WT, JAK2V617F/+ and JAK2V617F/V617F clones. Next, we compared the results with the experimental clonal frequency of WT, JAK2V617F/+ and JAK2V617F/V617F cells in individual colonies derived from the CD34+CD38+ compartment. In majority of patients, the observed values corresponded to the expected values suggesting that JAK2 46/1 haplotype can be used to estimate JAK2V617F clonal structure in PV patients. In three JAK2 46/1 heterozygous hemochromatosis patients used as controls, no JAK2 46/1 homozygous clone was observed showing that 46/1 haplotype itself was not responsible for HR. Furthermore, we have studied the proliferative advantage of the mutated clones in patients. No proliferative advantage of JAK2V617F clone has been observed in between CD34+CD38− and CD34+CD38+ progenitors stages whereas strong amplification of JAK2V617F clone was found in terminally differentiated polynuclear neutrophils (PNN). Moreover, during evolution of MPN in one patient, we observed an amplification of the JAK2V617F/V617F clone in both the CD34+CD38− and CD34+CD38+cell compartments suggesting acquisition of a proliferative advantage of the homozygous clone over time. This simple modeling could help to understand the effect of treatments on the JAK2V617F clonal structure without working at the unicellular level. Disclosures: No relevant conflicts of interest to declare.

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Ineffective Erythropoiesis With Increased Neutrophils In Old Mice Overexpressing Hmga2 cDNA

Blood ◽

10.1182/blood.v122.21.2469.2469 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2469-2469

Author(s):

Kazuhiko Ikeda ◽

Philip J. Mason ◽

Kayo Shirado Harada ◽

Kazuei Ogawa ◽

Monica Bessler ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Annexin V ◽

Primary Myelofibrosis ◽

Erythroid Cells ◽

Ineffective Erythropoiesis ◽

Hematopoietic Stem ◽

Cell Counts ◽

Young Mice

Abstract HMGA2 is frequently overexpressed in hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and primary myelofibrosis (PMF), and occasionally in myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). We recently reported that transgenic mice overexpressing truncated Hmga2 cDNA (ΔHmga2 mice) showed increased numbers in all lineages of peripheral blood (PB) cells, hypercellular bone marrow (BM), and splenomegaly in young adults of 3 months old (Ikeda et al, Blood, 2011). ΔHmga2 mice also showed growth advantage of hematopoietic stem cells (HSCs) in serial BM transplants and of committed progenitors in colony-replating assays. Thus, overexpression of HMGA2 may contribute to clonal expansion in PNH or myeloproliferation in PMF and MDS/MPN. Although these disorders show disease progression and development of leukemia in the long term, consequences of long-term overexpression of HMGA2 in hematopoiesis is unknown. Therefore, in this study, we investigated hematopoiesis in old ΔHmga2 mice at 15 months old. We found an increase in PB neutrophils in 16 old ΔHmga2 mice compared with 8 old wild-type (WT) mice (mean ± SD; 4.3 ± 1.7 vs. 2.4 ± 0.6 x109/L, p<0.01) as well as young mice. Strikingly, in contrast to young mice, we found a decrease in PB red blood cells of older ΔHmga2 mice compared with WT mice (8.5 ± 0.5 vs. 9.3 ± 0.4 x1012/L, p<0.01), which was associated with an increase in the mean corpuscular volume (MCV, 48.5 ± 3.7 vs. 42.1 ± 1.8 fL, p<0.01). PB total leukocyte and platelet counts were similar between old ΔHmga2 mice and old WT mice. Total BM cell counts were higher in 11 old ΔHmga2 mice compared with 12 WT mice (2.1 ± 0.4 vs. 1.4 ± 0.5 x107/femur, p<0.01). Spleens were 2-fold heavier in old ΔHmga2 mice (n=11) compared with old WT mice (n=12, p<0.01). Histology showed hypercellular BM with erythroid dysplasia, but neither fibrosis nor leukemia, in 5 old ΔHmga2 mice. Absolute numbers of BM lineage-Sca-1+c-kit+ HSCs were higher in 5 old ΔHmga2 mice compared with 3 old WT mice (73 ± 31 vs. 23 ± 10 x103/femur, p=0.036). In addition, proportions of CD71+Ter119+ erythroblasts were higher in 3 old ΔHmga2 mice compared with 3 WT mice (11.8 ± 1.2 vs. 5.1 ± 1.1%, p<0.01), although absolute number of megakaryocyte-erythrocyte progenitors did not show a statistical difference (137 ± 36 vs. 96 ± 18 x103/femur, p=0.1). Progenitor assay also showed increased numbers of BFU-E colonies in 3 old ΔHmga2 mice compared with 3 WT mice (25 ± 2 vs. 13 ± 2, p<0.01), but new BFU-E colonies did not grow from replating of these colonies of old ΔHmga2 mice, in contrast to young ΔHmga2 mice. Annexin V staining showed increased spontaneous apoptosis after 24-hour incubation in Ter119+CD71+ cells of 3 old ΔHmga2 mice compared with 3 old WT mice (26.6 ± 2.7 vs. 14.3 ± 7.0%, p=0.033). In MACS-sorted Ter119+ erythroid cells, expression of anti-apoptotic Bcl-xl mRNA in old ΔHmga2 mice was lower compared with old WT mice (3.3 ± 1.2 vs. 5.3 ± 0.3, p=0.016), while that in young ΔHmga2 mice were higher than young WT mice (6.9 ± 0.4 vs. 3.5 ± 0.3, p<0.01), suggesting the possibility that some changes, including expression of Bcl-xl affected sensitivity of erythroid cells to apoptosis during aging, resulted in ineffective erythropoiesis. In conclusion, long-term expression of HMGA2 may lead to neutrophilia with ineffective erythropoiesis, which may mimic MDS/MPN rather than myelofibrosis, possibly through impairment of Bcl-xl in erythroid cells during a long course. Ineffective erythropoiesis may also in part explain BM failure in PNH. Disclosures: No relevant conflicts of interest to declare.

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Calr Mutants Retroviral Mouse Models Lead to a Myeloproliferative Neoplasm Mimicking an Essential Thrombocythemia Progressing to a Myelofibrosis

Blood ◽

10.1182/blood.v124.21.157.157 ◽

2014 ◽

Vol 124 (21) ◽

pp. 157-157 ◽

Cited By ~ 4

Author(s):

Caroline Marty ◽

Nivarthi Harini ◽

Christian Pecquet ◽

Ilyas Chachoua ◽

Vitalina Gryshkova ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

In Vivo Model ◽

Common Mechanism ◽

Loss Of Function ◽

Hematopoietic Stem ◽

Platelet Counts

Abstract Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are malignant homeopathies resulting from the transformation of a multipotent hematopoietic stem cell (HSC). The common mechanism of transformation is the constitutive activation of the cytokine receptor/JAK2 pathway that leads to the myeloproliferation. The acquired point mutation JAK2V617F is the most prevalent (95% of PV and 60% of ET or PMF). In addition, other mutations affecting the same signaling pathway have been described such as JAK2 exon 12 mutations, mutations of MPL affecting W515, and loss-of-function mutations of LNK and also mutations of c-Cbl in 3% of PMF. Recently, whole exome sequencing allowed identifying a new recurrent genetic abnormalities in the exon 9 of the calreticulin gene (CALR) in about 30% of ET and PMF patients. All CALR mutants induce a frameshift of the same alternative reading frame and generate a novel C-terminus tail. To address the role of these new mutants in the pathophysiology of MPN, the goal of this study was to investigate the effect of the CALR mutant (del52 and ins5) expression by a retroviral mouse modeling. For that purpose, we transduced bone marrow cells with retrovirus expressing either CALRdel52, CALRins5, CALRWT or CALRDexon9 and performed a transplantation in lethally irradiated recipient mice (10 mice / group), which were then followed over one year. CALRdel52 expressing mice showed a rapid and strong increased in platelet counts (over 5 x106/mL) without any other changes in blood parameters during 6 months. In contrast, CALRins5 expressing mice presented platelet counts much lower than CALRdel52 but significantly higher than CALRWT or CALRDexon9 expressing mice. After 6 months, CALRdel52 expressing mice showed a decreased in platelets count associated with anemia and development of splenomegaly suggesting the progression to a myelofibrosis. Importantly, the disease was transplantable to secondary recipient for both CALRdel52 and CALRins5 mutants. The bone marrow and spleen were also analyzed over time. We observed a progressive increased in immature progenitors (SLAM cells) as well as a hypersensitivity of the megakaryocytic progenitors (CFU-MK) to thrombopoietin. Altogether, these results demonstrate that CALR mutants are able and sufficient to induce a thrombocytosis progressing to myelofibrosis in retroviral mouse model, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPN. Disclosures No relevant conflicts of interest to declare.

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