Expression of HMGA2 Collaborates with JAK2V617F to Progress Myeloproliferative Neoplasms

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 JAK2V617F (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 that represses expression of HMGA2 (Ikeda et al, Blood, 2011). ΔHmga2 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 JAK2V617F⁺ hematopoiesis, we crossed ΔHmga2^+/- mice with JAK2V617F^+/- Tg mice (Shide et al, Leukemia, 2008). ΔHmga2^-/-JAK2V617F^-/- wild type (WT), ΔHmga2^+/-JAK2V617F^-/- (ΔHmga2-Tg), ΔHmga2^-/-JAK2V617F^+/- (JAK2V617F-Tg) and ΔHmga2^+/-JAK2V617F^+/- (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 JAK2V617F-Tg or ΔHmga2-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 JAK2V617F-Tg mice, respectively. Mean spleen weights were 0.067, 0.10, 0.83 and 2.8 g in WT, ΔHmga2-Tg, JAK2V617F-Tg and double-Tg mice, while BM cell counts were 2.4, 2.8, 0.4 and 1.2 x 10⁷ /femur, respectively. However, JAK2V617F-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 JAK2V617F, HMGA2 augments lineage^-Sca1⁺Kit⁺ cells (WT: ΔHmga2-Tg: JAK2V617F-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 JAK2V617F⁺ hematopoiesis. Most ΔHmga2-Tg and JAK2V617F-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 JAK2V617F⁺ HSPC activity, we performed BMT with 0.25 x 10⁶ Ly5.2⁺ ΔHmga2-Tg, JAK2V617F-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 ΔHmga2-Tg than double-Tg cells, while JAK2V617F-Tg cells were almost rejected at 8 weeks after BMT. To confirm role of HMGA2 without let-7 repression in JAK2V617F⁺ hematopoiesis, we performed another BMT with 1 x 10⁴ KIT⁺ cells of JAK2V617F-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 JAK2V617F-Tg cells with Hmga2-m7 developed MPN-like disease, whereas donor cells were rejected in recipients of JAK2V617F cells with empty vector. In conclusion, HMGA2 may play a crucial role in hematopoiesis harboring JAK2V617F by expanding HSPC, leading to disease progression.