Thrombopoietin is required for full phenotype expression in a JAK2V617F transgenic mouse model of polycythemia vera

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.

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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 ◽

10.1182/blood.v128.22.1958.1958 ◽

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.

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