Comparative Analysis of the Constitutively Active MPLW515L and JAK2V617F Alleles in a Murine Bone Marrow Transplant Model of Myeloproliferative Disease.
Authors: Pikman Y. , Levine R.L. , Lee B.H. , Tothova Z. , Mercher T. , Wadleigh M. , Lee S.J. , Gilliland D.G. , Wernig G. 
Published: October 2019

Abstract
Although acquisition of JAK2V617F mutation is an important pathogenetic event in many patients with polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF), the genetic events that contribute to most cases of JAK2V617F-negative MPD are not known. We used high throughput DNA sequence analysis of cytokine receptors critical for erythroid, megakaryocytic, and granulocytic proliferation and identified a somatic mutation in the thrombopoietin receptor (MPLW515L) in patients with JAK2V617F-negative MF. Expression of MPLW515L transforms hematopoietic cells to cytokine-independent growth, and activated components of the JAK-STAT signal transduction pathway including JAK2, STAT5, STAT3, ERK and AKT. We compared the disease phenotype induced by MPLW515L with that we have previously reported for the JAK2V617F allele in a murine bone marrow transplant model of myeloproliferative disease. In Balb/C mice, expression of MPLW515L caused a rapidly fatal, fully penetrant MPD (median latency of ~18 days) that was characterized by marked leukocytosis and thrombocytosis, with average platelet count of 3.4 million/ul as well as bone marrow reticulin fibrosis and thrombotic complications. A similar phenotype was observed in MPLW515L C57/Bl6 recipients, but with a longer median latency of ~50 days. In a subset of C57/Bl6 mice, the acute MPD was followed by a progressive reduction in platelet count and an increase in bone marrow fibrosis that occurred over 3 months, and was reminiscent of human MF. These findings stand in contrast to MPD induced by the JAK2V617F allele in C57/Bl6 mice, in which there is marked erythrocytosis, but not thrombocytosis or thrombotic complications or leukocytosis. Analysis of myeloid progenitor populations using multiparameter flow cytometry demonstrated an expansion of the common myeloid progenitor (CMP), the granulocyte-monocyte progenitor (GMP) population, and the megakaryocytic-erythroid progenitor (MEP) population in MPLW515L-expressing bone marrow and spleen, with up to a 10-fold increase in CMPs and an 8-fold increase in MEPs, whereas there were no differences observed in the relative proportion of CMP, GMP or MEP populations in JAK2V617F mice. These data demonstrate important phenotypic differences between disease induced by the MPLW515L or JAK2V617F alleles in C57/Bl6 mice. These include (i) marked differences in the degree of leukocytosis, with MPLW515L induced marked leukocytosis compared with JAK2V617F and (ii) the effect on the megakaryocyte lineage in which JAK2V617F induces megakaryopoiesis, but with reduced megakaryocyte ploidy with no thrombocytosis or thrombotic complications, whereas MPLW515L enhances megakarypoiesis and induces thrombocytosis with thrombotic complications. These findings suggest that JAK2V617F and MPLW515L - each of which is characterized by activation of JAK-STAT signaling in hematopoietic progenitors - have qualitative and quantitative differences in their ability to impact proliferation and/or survival of hematopoietic progenitors that presumably are the consequence of differences in signal transduction, and result in disparate phenotypes.