JAK2V617F and Loss of Ezh2 in Hematopoietic Cells Contribute Synergistically to Myeloproliferative Neoplasm Initiation Potential, and Accelerate Progression of Disease
Authors: Shimizu Takafumi , Hao-Shen Hui , Kubovcakova Lucia , Lundberg Pontus , Dirnhofer Stephan , Orkin Stuart H. , Grisouard Jean , Shimizu Yukiko , Pianta Annalisa , Szczerba Barbara , Nienhold Ronny , Skoda Radek C. 
Published: October 2019

Background ;The gain-of-function JAK2 mutation, JAK2V617F, is the most common molecular abnormality in myeloproliferative neoplasms (MPNs) and appears in patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Ezh2 is a component of PRC2, catalyzing methylation of H3K27, and frequently mutated in MPN patients. Loss-of-function mutation of Ezh2 was reported as a poor prognostic marker in myelofibrosis patients. Many JAK2V617F-positive MPN patients harbor other mutations, but combination effects of JAK2V617F and Ezh2 mutation have not been analyzed.

Methods & Results ; To investigate the interaction between the two kinds of mutation in hematopoiesis, we studied transgenic mice with conditional expression of JAK2V617F, and inducible loss-of-function of Ezh2, both singly and in combination. Conditional expression of Cre was achieved using the SclCreER system with four weeks of tamoxifen injection, leading to excision of loxP-flanked alleles of Ezh2, and simultaneously induced expression of JAK2V617F, via the Flip-Flop recombination system (FF1). Mice with heterozygous deletion of Ezh2 showed no changes in peripheral blood, but homozygous deleted mice displayed slightly increased platelet counts compared to control mice. JAK2V617F-expressing (FF1) mice showed a typical PV phenotype, including erythrocytosis, thrombocytosis and neutrophilia. Mice expressing JAK2V617F with heterozygous deletion of Ezh2 (FF1;Ezh2+/-) also showed typical PV, but with more profound thrombocytosis and neutrophilia, and faster progression to fibrosis, than FF1 mice. Mice expressing JAK2V617F with homozygous deletion of Ezh2 (FF1;Ezh2-/-) had even shorter survival and showed more profound thrombocytosis and myelofibrosis than FF1;Ezh2+/- mice, but without erythrocytosis. These mice had dacryocytes and mobilized c-kit positive progenitor cells in peripheral blood. Bone marrow histology revealed mild fibrosis (grade: 1) in FF1 mice; more excessive fibrosis (grade: 1-2) in FF1;Ezh2+/- mice; and obvious fibrosis associated with collagen fiber formation and osteosclerosis (grade: 2) in FF1;Ezh2-/- mice. Cell compartment analysis in FF1 mice revealed expansion of hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MegP) in bone marrow and spleen, while common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP) and megakaryocyte erythrocyte progenitor (MEP) were expanded principally in spleen. The expansion of HSCs and all progenitors were accelerated in FF1;Ezh2+/- mice and FF1;Ezh2-/- mice. Especially MegP and mature megakaryocytes were further expanded in FF1;Ezh2-/- mice compared to FF1;Ezh2+/-. To clearly estimate disease progression, transgenic bone marrow cells were transplanted to lethally irradiated recipient mice with WT competitor bone marrow cells and tamoxifen was administrated after transplantation. FF1 bone marrow cells clearly showed outcompeting potential to WT bone marrow cells. FF1;Ezh2+/- and FF1;Ezh2-/- bone marrow cells showed more stronger outcompeting potential than FF1 bone marrow cells. Finally, disease initiative potential was evaluated by limiting dilution transplantation. Total bone marrow cells, with heterozygous deletion of Ezh2 (Ezh2+/-), or expressing JAK2V617F (FF1), or the combination of the two mutations were transplanted (following Cre induction and recombination) to lethally irradiated recipient mice, mixed with competitor cells (control or Ezh2+/- bone marrow cells were transplanted by 1:100 dilution and FF1 or FF1;Ezh2+/- bone marrow cells were transplanted by 1:250 dilution against WT competitor cells). Bone marrow cells with Ezh2+/- showed higher reconstitution capacity than control (35% vs 10%; Ezh2+/- vs control) without MPN phenotype. Bone marrow cells with FF1;Ezh2+/- showed stronger reconstitution capacity (39% vs 15%; FF1;Ezh2+/- vs FF1), and greater disease initiative potential (50% vs 33%; FF1;Ezh2+/-vs FF1) than FF1.

Summary; We clarified that loss-of-function of Ezh2 accelerated JAK2V617F-induced MPNs. Especially JAK2V617F with Ezh2 homozygous loss-of-function induced excessive megakaryopoiesis and resulted in PMF. JAK2V617F with heterozygous deletion of Ezh2 synergistically enhanced bone marrow reconstitution and disease initiative potential.

Disclosures

Skoda: Novartis: Consultancy; Sanofi: Consultancy.