The High Mobility Group A1 Chromatin Regulator Is Required for Pathologic Megakaryocyte Development and Progression to Myelofibrosis in JAK2V617F Murine Models

Introduction:JAK2^V617F-positive myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by unregulated JAK2/STAT signaling and increased risk of transformation to myelofibrosis (MF). Pathologic JAK2/STAT signaling of the thrombopoietin/thrombopoietin receptor pathway in HSC, progenitors, and megakaryocytes drives megakaryocytic proliferation, megakaryocytic hypertrophy, thrombocytosis, HSC niche damage, osteosclerosis, myelofibrosis, and extramedullary hematopoiesis (EMH). The High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins that foster aberrant STAT3 signaling in diverse contexts. In murine models, Hmga1/2 overexpression drives clonal expansion and deregulated proliferation. In patients with MPN, HMGA1/2 genes are overexpressed with disease progression to MF. We therefore sought to: 1) elucidate mechanisms mediated by HMGA1/2 in JAK2^V617F-associated MF transformation and 2) test the hypothesis that HMGA proteins are rational therapeutic targets for MF progression.

Methods: We used a JAK2^V617F transgenic murine model (VF) in which 13 copies of the human JAK2^V617F cDNA are driven by the Vav promoter (Blood 2008; 111:5109-5117). To elucidate the function of Hmga1 or Hmga2 in the JAK2^V617F context, we crossed VF mice onto a background deficient for Hmga1 or Hmga2 to generate VF/Hmga1^+/- and VF/Hmga2^+/- progeny. We also generated a tissue specific model whereby Hmga1 was deleted in HSC via Vav-cre (Vav-cre Hmga1^+/-) and crossed these mice onto the VF model (VF/Vav-cre Hmga1^+/-). Serial blood counts, histology of marrow and spleen, and immunohistochemistry with CD61 antibodies were compared in each model at 8, 16, and/or 40 weeks of age. We also performed flow cytometric analysis of HSC and progenitor populations in mice with these genotypes.

Results: The VF mice develop an MPN phenotype by 8 weeks with marked thrombocytosis and erythrocytosis. By 33 weeks, VF mice progress to MF characterized by anemia, splenomegaly due to extramedullary hematopoiesis (EMH), marked megakaryocytic hyperplasia, megakaryocytic hypertrophy, increased platelet size, and osteosclerosis with reticulin fibrosis (Figure A and B). ). The VF mouse also develops an expansion of megakaryocyte-erythroid progenitors (MEP) compared to wildtype mice (P<0.01). Neither mice with Hmga1deficiency (Hmga1^+/- or Hmga1^-/-) nor Hmga2^+/- mice develop thrombocytopenia or other blood count abnormalities at one year of age, and preliminary data suggests that the MEP compartment is normal in Hmga1^+/- and Hmga1^-/- mice. However, VF/Hmga1^+/- mice had reduction of both megakaryocyte hypertrophy and hyperplasia, bone marrow fibrosis, and osteosclerosis compared to VF mice at corresponding age ranges (Figure A). Further, there was mitigation of thrombocytosis, reduction in platelet size, and decreased spleen weight in VF/Hmga1^+/- mice compared to VF mice at corresponding age ranges (Figure B). Importantly, VF/Hmga1^+/- mice fail to develop anemia which occurs after 33 weeks in the VF model (VF hemoglobin 12.9 g/dl, VF/Hmga1^+/- hemoglobin 15.1 g/dl, P<.05). In VF/Hmga1^-/- mice, there was no expansion in MEP. The MPN progression to MF was prevented by both global or HSC-specific heterozygous Hmga1 deficiency, demonstrating that the effects of Hmga1 are specific to the hematopoietic cell context rather than the bone marrow microenvironment. In addition, similar degrees of mitigation were observed in VF mice with heterozygous or homozygous Hmga1 deficiency. In contrast, Hmga2 deficiency failed to prevent progression in the VF model. Unexpectedly, Hmga2 deficiency exacerbated megakaryocyte hypertrophy and reticulin fibrosis in VF mice.

Conclusions: In a JAK2^V617F murine model, heterozygous deficiency of Hmga1 prevents MPN progression to MF, interrupting both the development of megakaryocytic hyperplasia and fibrosis, which are hallmarks of murine and human JAK2^V617F disease. While Hmga1 is not required for megakaryocyte or platelet development under steady state conditions, it is critical to the amplification of aberrant signaling in MF associated VF. Further, our findings underscore a key role for HMGA1 as a mediator of aberrant JAK2/STAT3 signaling and a therapeutic target to quell myeloproliferation and prevent MF progression.

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