Thrombopoietin Receptor (MPL) Genotype Modifies the Myeloproliferative Phenotype in a JAK2 V617F Transgenic Mouse Model of Polycythemia Vera.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 964-964
Author(s):  
Jerry L. Spivak ◽  
Donna M Williams ◽  
Brady L. Stein ◽  
Ophelia Rogers ◽  
Tsivia Hochman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Polycythemia Vera ◽  
Transgenic Mouse ◽  
Gene Dosage ◽  
Jak2 V617f ◽  
Transgenic Mouse Model ◽  
Single Mutation ◽  
Hematopoietic Stem

Abstract Abstract 964 The JAK2 V617F mutation is primarily associated with three chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) but how a single mutation could be responsible for three different disorders is still unresolved. A gene dosage effect was proposed based on the MPD phenotypes in mice with differential expression of a JAK2 V617F transgene, where low expression correlated with an ET phenotype and high expression with a PV phenotype. However, quantitative studies of JAK2 V617F expression in humans revealed significant overlap between PV and ET. Since JAK2 is the cognate tyrosine kinase for the erythropoietin (EPO) and thrombopoietin (TPO) receptors, and JAK2 V617F is expressed in pluripotent hematopoietic stem cells, PV is the ultimate clinical phenotype of the mutation. Furthermore, TPO but not EPO promotes the survival and proliferation of pluripotent hematopoietic stem cells, suggesting that the TPO receptor (Mpl) is essential not only for generating thrombocytosis, but also the stem cell expansion that is characteristic of PV. To examine the role of Mpl in the genesis of the JAK2 V617F MPD phenotype, we manipulated the MPL genotype in a transgenic mouse expressing 13 copies of JAK2 V617F (V617Ftg) (Blood 111:5109, 2009) by breeding these mice with MPL knockout mice (Science265:1445, 1994), which are hematologically normal except for profound thrombocytopenia, to create three genotypes: V617Ftg/MPL wild type (wt); V617Ftg/MPL heterozygote (het), and V617Ftg/MPL knockout (ko). We compared the blood counts, spleen weights, plasma TPO levels, and bone marrow and spleen histology of these three genotypes with each other and with MPL wt, MPL het and MPL ko mice over a 33 week period. Crossbreeding gave the expected genotypes, JAK2 V617F transgene expression was stable in all groups, platelet Mpl expression by immunoblotting correlated with MPL genotype, there was no unexpected mortality, and body weights were not different for any of the genotypes during the observation period. As expected, in V617Ftg/MPL wt mice there was a robust and persistent thrombocytosis (2087 +/− 641 × 106/μL vs 1005 +/− 176 × 106/μL, p<0.001), an erythrocytosis (hemoglobin, 18.3 +/− 1.1 gm % vs 14.9 +/− 0.72 gm %, p <0.001) that peaked at 14-16 weeks but then diminished, and a leukocytosis (16.3 +/− 5.1 × 106/μL vs 12.9 +/−3.4 ×106/μL, p = 0.043) as compared to MPL wt mice. By contrast, in V617Ftg/MPL ko mice, the PV phenotype was virtually abrogated in all cell types as compared to V617Ftg/MPL wt (hemoglobin, 16.1 +/− 0.87 vs 18.3 +/− 1.1, p< 0.001; leukocyte count, 11.3 +/− 2.8 vs 16. 3 +/− 5.1 , p= 0.003, and platelet count, 293 +/− 102 vs 2087 +/− 641, p< 0.001), and not different than their MPL ko counterparts except for a mild erythrocytosis (16.1 +/− 0.9 vs 14.9 +/−, p < 0.001), while in V617Ftg/MPL het mice, erythrocytosis was comparable to the V617Ftg/MPL wt mice and higher than in MPL het controls (17.9 +/− 1.4 gm% vs 14.9 +/− 0.9 gm % p <0.001), but there was only minimal thrombocytosis (1310 +/− 274 × 106/μL vs 1021+/− 241 × 106/μL, p< 0.001), and no leukocytosis (14.8 +/− 4.0 106/μL vs 14.1 +/− 3.7 × 106/μL, p=0.4 ) as compared to the MPL het mice. Marrow and spleen histology reflected the genotype and blood counts and spleen weight was increased equally in all three V617Ftg/MPL genotypes as compared to controls. Plasma TPO was elevated in MPL ko (5530 +/− 1334 pg/mL, p =0.006) and V617Ftg/MPL ko (4201 +/− 736 pg/mL, p = 0.001 ), but not in MPL het mice (723 +/− 720 pg/mL), compared to MPL wt mice (323 +/− 62 pg/mL), while in V617Ftg/MPL wt (163 +/− 52 pg/mL, p < 0.001) and V617Ftg/MPL het mice (176 +/− 56 pg/mL, p < 0.001) plasma TPO was lower than in MPL wt mice. Based on these data, we conclude that MPL genotype is an important modifier of the MPD phenotype in a JAK2 V617F transgenic mouse model of PV, not only for thrombopoiesis but, importantly, also for erythropoiesis and myelopoiesis. We also infer from these data that the impaired Mpl expression observed in human PV may also be a significant modifier of the JAK2 V617F phenotype, either by acting as a dominant-negative with respect to JAK2 V617F activity, or possibly through impaired plasma TPO regulation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Description of a knock-in mouse model of JAK2V617F MPN emerging from a minority of mutated hematopoietic stem cells

Blood ◽  
2019 ◽  
Vol 134 (26) ◽  
pp. 2383-2387 ◽  
Author(s):  
Olivier Mansier ◽  
Badr Kilani ◽  
Amélie V. Guitart ◽  
Alexandre Guy ◽  
Virginie Gourdou-Latyszenok ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Polycythemia Vera ◽  
Jak2 V617f ◽  
Jak2v617f Mutation ◽  
Small Subset ◽  
Hematopoietic Stem ◽  
Key Points

Key Points PF4iCre;JAK2 V617F/WT mice develop a full MPN that mimics polycythemia vera. The PF4iCre system induces JAK2V617F mutation in a small subset of HSC.

High Mobility Group A1/2 Chromatin Remodeling Proteins Associate with Polycythemia Vera Transformation to Acute Leukemia in Humans and a JAK2 V617F Transgenic Mouse Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1958-1958
Author(s):  
Linda Resar ◽  
Donna Marie Williams ◽  
Zhizhuang Joe Zhao ◽  
Ophelia Rogers ◽  
Lingling Xian ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mouse Model ◽  
Acute Leukemia ◽  
Polycythemia Vera ◽  
Transgenic Mouse ◽  
Transgenic Mouse Model ◽  
Murine Models ◽  
Hematopoietic Stem ◽  
Epigenetic Regulators ◽  
Transgenic Murine Models

Abstract Introduction: The MPN are clonal hematopoietic stem cell (HSC) disorders characterized by an overproduction of blood cells and an increased risk of transformation to an aggressive phase with myelofibrosis (MF) and/or acute myeloid leukemia (AML). Polycythemia vera (PV) is the most common clinical subtype, and while PV starts as an indolent process, nearly 25% of patients will progress to MF and/or AML. PV is caused by acquired mutations of JAK2, yet JAK2 mutations alone do not account for MF or AML transformation. Mutations in genes encoding epigenetic regulators are associated with MPN transformation, but the mechanism of action is not understood. HMGA1/2 chromatin binding proteins are potent oncogenes that drive tumor progression by activating oncogenic and stem cell transcriptional networks. Both HMGA1/2 are overexpressed in acute leukemia and have been shown to be drivers of clonal expansion in myeloid disease in humans and in murine myeloproliferative disease models. We hypothesized that HMGA proteins could be critical drivers of transformation in PV and therefore tested the association of HMGA1/2 expression to transformation in human and murine PV. Methods: We examined the HSC genomic context and clonal evolution in 49 JAK2V617F-positive PV patients using standard and SNP-array karyotyping and a targeted resequencing panel of 163 genes associated with myeloid cancers. We examined HSC clonal burden by examining JAK2V617F HSC genotypes on a single cell basis. We measured HMGA1 and HMGA2 expression in a JAK2V617F positive human cell line, in isolated CD34+ HSCs from PV patients during chronic and transformation phases, in JAK2V617F transgenic murine models of PV (tgJAK2V617F) and PV-AML (tgJAK2V617F/MPLSV; Blood 2015;126:484) using a real-time quantitative RT-PCR (qRT-PCR) assay. Results: Both HMGA1 and HMGA2 mRNA were up-regulated in all JAK2V617F-positive contexts. In primary human PV CD34+ HSCs, HMGA1 and HMGA2 were found to be increased by 7 and 100 fold, respectively, compared to controls. Moreover, there was a dramatic up-regulation in both HMGA1/2 in patients who transformed from PV to MF or AML compared to chronic phase PV, whether analyzed cross-sectionally (Figure) or prospectively in selected patients. In addition to disease phase, over-expression of HMGA1/2 correlated with clonal dominance of JAK2V617F-homozygous stem cells, and additional mutations of epigenetic regulators including EZH2 and SETBP1. Similarly, when assessed in unfractionated bone marrow or in tumor samples in the two transgenic mouse models for PV and PV-AML, Hmga1/2 were overexpressed compared to wild-type littermates, with highest levels in the PV-AML transgenic mouse model. Conclusion: HMGA1 and HMGA2 are overexpressed in PV, and higher levels associate with disease progression to MF and AML, both in human PV and in transgenic murine models of PV. These data suggest HMGA proteins are critical drivers of PV transformation and that the mechanism of HMGA1/2 overexpression is a consequence of aberrant JAK/STAT signaling and epigenetic dysregulation. Our findings indicate that HMGA1/2 overexpression may function as a necessary molecular switch for PV leukemic transformation. Therefore, HMGA proteins and their transcriptional pathways offer novel therapeutic targets aimed at the prevention of PV progression to MF and AML. Disclosures No relevant conflicts of interest to declare.

Ifnα Attenuates the Disease Phenotype and Extends Survival in Mouse Models of MPN

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 53-53
Author(s):  
Harini Nivarthi ◽  
Andrea Majoros ◽  
Eva Hug ◽  
Ruochen Jia ◽  
Sarada Achyutuni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mouse Models ◽  
Jak2 V617f ◽  
Type I ◽  
Type I Ifn ◽  
Hematopoietic Stem ◽  
Exon 9 ◽  
Ifn Treatment

The curative potential of Type I interferons for patients suffering from Myeloproliferative Neoplasms (MPNs) has been reported and these are the only class of drugs that can lead to reduction of the mutant allelic burden in patients. However, modelling IFN treatment in mice has been challenging. Here, we report the use of murine pegylated IFNα (murine ropeginterferon-a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) to model IFN treatment in transgenic MPN mouse models. We started treating JAK2V617Ff/+;vavCre and control vavCre mice (n=6-8) with PBS or mRopeg (600 ng/mouse/week), by subcutaneous injections from the time they were 4 weeks old. The mice were bled every 2 weeks from the facial vein and the blood parameters were monitored. We observed significant normalization of platelet and WBC counts in Jak2-V617F fl/+ vavCre mice to wild type levels. No effect on hematocrit and hemoglobin level was observed in the Jak2-V617F fl/+ vavCre mice. VavCre control animals showed no sign of negative effect such as cytopenia during the entire treatment course. We observed a highly significant prolongation of the survival of mRopeg treated JAK2V617Ff/+;vavCre mice over a duration of 80 days of treatment. While all the PBS treated JAK2V617Ff/+;vavCre mice died within 60 days, all the mRopeg treated mice were still alive till the end of the treatment duration. We also generated a novel transgenic mouse model that conditionally expresses hybrid mutant CALR protein (murine exons 1-8 and human CALR del52 exon9) from the endogenous murine Calr locus. We bred them into vavCre background (in both heterozyhous and homozygous states) to induce expression of CALR-del52 in hematopoietic cells. Upon Cre recombinase expression, the endogenous murine exon 9 is replaced by the human del52 exon 9 and the expression of the humanized Calr-del52 oncoprotein is detectable by Western blot analysis using mutant CALR specific antibodies. Calr-del52 animals develop an essential thrombocythemia (ET) like phenotype when expressed in a heterozygous state with elevated number of hematopoietic stem cells and megakaryocytes in the bone marrow. In the homozygous state, the thrombocythemia is more severe with splenomegaly and older animals show anemia with increased WBC. Bone marrow histology shows megakaryocytic hyperplasia with no sign of fibrosis up to age of one year. We treated a cohort of animals with 600 ng mRopeg/PBS once a week for 4 weeks. Peripheral blood counts were determined at baseline and at regular intervals during treatment. At the end of treatment, mice were sacrificed, and splenic and bone marrow cells were immunophenotyped and quantified by FACS. We observed correction of thrombocythemia in the homozygous Calr-del52 mice but no unspecific decrease of platelet count in the vavCre mRopeg treated animals. We observed significant specific reduction of the long-term hematopoietic stem cells (LT-HSCs/fraction A) in homozygous CALR-del52 mice. In conclusion, Type I IFN treatment significantly reduces platelet counts to normal levels in both JAK2 and CALR mutant driven MPN mouse models. The prolongation of survival of JAK2V617F transgenic mice upon Type I IFN treatment is particularly remarkable; as no survival data is reported until now in any clinical trials or other animal models. Further experiments are required to understand the mechanism of action of this phenomenon. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

2021 ◽  
Vol 9 ◽  
Author(s):  
Wanbo Tang ◽  
Jian He ◽  
Tao Huang ◽  
Zhijie Bai ◽  
Chaojie Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Hematopoietic Stem ◽  
Genetic Lineage Tracing

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

Abstract 12653: The Therapy-related Clonal Hematopoiesis Driver Gene Ppm1d Promotes Inflammation and Non-ischemic Heart Failure in a Murine Model

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Yoshimitsu Yura ◽  
Emiri Miura-Yura ◽  
Kenneth Walsh
Keyword(s):  
Stem Cells ◽  
Angiotensin Ii ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Cancer Patients ◽  
Nlrp3 Inflammasome ◽  
Cardiac Dysfunction ◽  
Hematopoietic Stem ◽  
Cardiac Stress ◽  
Clonal Hematopoiesis

Background: Therapy-related clonal hematopoiesis in cancer patients is typically associated with somatic mutations in hematopoietic cell genes that encode regulators of the DNA-damage response (DDR) pathway. The Protein Phosphatase Mg2+/Mn2+ Dependent 1D ( PPM1D ) gene is the most frequently mutated DDR gene associated with therapy-related clonal hematopoiesis. While epidemiological evidence suggests an association between therapy-related clonal hematopoiesis and cardiovascular disease in cancer patients, causal and mechanistic relationships have never been evaluated in an experimental system. Methods: To test whether hematopoietic cell mutations in PPM1D can increase the susceptibility to cardiac stress, we evaluated cardiac dysfunction in response to angiotensin II infusion in a mouse model where clonal-hematopoiesis-associated mutations in Ppm1d were produced by CRISPR-Cas9 technology. Results: Mice transplanted with hematopoietic stem cells containing clinically relevant mutations in exon 6 of Ppm1d exhibited augmented cardiac remodeling following the continuous infusion of angiotensin II. Ppm1d -mutated macrophages showed impairments in the DDR pathway and had an augmented proinflammatory profile. Mice transplanted with Ppm1d mutated cells exhibited elevated IL-1β in the stressed myocardium, and bone marrow derived macrophages produced more IL-1β in response to LPS stimulation. The administration of an NLRP3 inflammasome inhibitor to mice reversed the cardiac phenotype induced by the Ppm1d -mutated hematopoietic stem cells under conditions of Angiotensin II-induced stress. Conclusions: A mouse model of Ppm1d -mediated clonal hematopoiesis was more susceptible to cardiac stress following of angiotensin II infusion. Mechanistically, disruption of the DDR pathway led to elevations in inflammatory cytokine production, and the NLRP3 inflammasome was shown to be essential for this augmented cardiac stress response. These data indicate that therapy-related clonal hematopoiesis involving mutations in PPM1D could contribute to the cardiac dysfunction observed in cancer survivors.

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