In this report we describe a somatic mutation in the transmembrane region of
MPLW515L) in a subset of
JAK2V617F-negative MF. Expression of MPLW515L transforms hematopoietic cells to cytokine-independent proliferation, and results in constitutive activation of JAK-STAT signaling. The relevance of this mutation is underscored by its effect in the murine bone marrow transplant assay, as expression of MPLW515L induces myeloproliferation characterized by splenomegaly, leukocytosis, marked thrombocytosis, extramedullary hematopoeisis, and myelofibrosis. In addition, a small-molecule JAK kinase inhibitor inhibits proliferation of MPLW515L-transformed cells, suggesting that targeted inhibition of JAK-STAT signaling may be an effective therapy for both
Although the constitutively active JAK2V617F mutant kinase is present in many patients with MPD, there are a significant proportion of MF and ET patients who are
JAK2V617F-negative. We hypothesized that activation of JAK–STAT signaling in these patients might result from activating mutations in cytokine receptors, which led to the discovery of a novel mutation in
MPLW515L) in patients with
JAK2V617F-negative MF. The mutation is present in granulocytes but not in non-hematopoietic germline DNA, demonstrating that
MPLW515L is a somatic mutation present in clonally derived hematopoietic cells. Moreover, the
MPLW515L allele was not observed in a panel of normal individuals, demonstrating that it is not a common single nucleotide polymorphism. These data provide genetic evidence that
MPLW515L is a pathogenetic mutation in this subset of
In contrast to previous reports [
], we have identified a mutant MPL receptor that results in constitutive activation of downstream signal transduction pathways. MPL signals via its association with JAK2 and TYK2, resulting in phosphorylation of both MPL and JAK2/TYK2 and consequent activation of the STAT3, STAT5, ERK/MAPK, and PI3K/AKT signaling pathways. MPLW515L activates JAK-STAT signal transduction independently of ligand binding, and, in contrast with MPLWT, confers cytokine-independent growth to 32D, Ba/F3, and UT7 cells. Moreover, Western blot analysis shows ligand-independent phosphorylation of downstream signaling proteins, which may effect both proliferation and differentiation. Finally, hematopoietic cells that express MPLW515L are hypersensitive to stimulation with TPO as assessed by cell proliferation and Western blot analysis, suggesting MPLW515L-positive hematopoietic progenitors may have a selective proliferative advantage as compared with wild-type progenitors in MF patients.
The discovery of this mutation provides a novel mechanism for activation of signal transduction in hematopoietic malignancies. Somatic mutations in MPL have not previously been described, though previous genetic and biochemical data suggest that the cytoplasmic-transmembrane junction (which includes W515) is important in its activation. These data include the germline S505N allele in
associated with familial ET [
], the W515S mutation that has been reported as a spontaneous mutation in Ba/F3 cells stably transduced with MPL[
], and the observation that in vitro deletion of the RWQFP domain that includes W515 results in MPL activation [
]. The mechanism of activation of MPL by these alleles is not fully understood, and it will be of value to obtain structural insight into the role of the cytoplasmic transmembrane–junction region in the regulation of MPL signal transduction.
Expression of MPLW515L in murine bone marrow recapitulated many of the phenotypic characteristics of MF in humans, including atypical megakaryocytic hyperplasia, splenomegaly due to extramedullary hematopoiesis, and thrombocytosis that may be attenuated in humans by splenic sequestration [
]. This is in contrast to murine bone marrow transplant experiments with constitutively activated tyrosine kinase alleles evaluated in our laboratory and by others, including TEL-JAK2, FLT3-ITD, and FIP1L1-PDGFRA [
], that each result in a neutrophilic MPD without involvement of the megakaryocyte lineage. It is of interest that JAK2V617F, which is associated with ET and MF in humans, results in polycythemia and leukocytosis, but not thrombocytosis, in the same murine BMT model [
]. Of note, absolute leukocytosis in both the JAK2V617F and the MPLW515L-mediated BMT models is higher than is typically observed in human PV and MF, respectively. Also, MPLW515L mice develop rapidly fatal disease. These differences between the murine model and the human phenotype are likely accounted for by the retroviral transduction model of disease, and is similar to what has been observed for the rapidly fatal BCR-ABL-mediated disease in the murine BMT model as compared with human CML [
MPLW515L-expressing bone marrow and spleen cells exhibit cytokine-independent colony formation, another feature of clinical ET and MF. In addition, the MPLW515L murine bone marrow transplant model shares some phenotypic characteristics with other murine models of MF, including that induced by overexpression of thrombopoietin, either in transgenic [
] or in bone marrow transplant models [
], and in the transgenic
], though the severity and chronicity of the MF phenotype varies in these different models, which, unlike the current model, are not based on somatic disease alleles observed in human MF.
MPLW515L is an attractive target for therapeutic intervention with small molecule inhibitors. Current treatments for MF include empiric therapies such as hydroxyurea or interferon, and splenectomy for symptomatic splenomegaly [
]. Allogeneic stem cell transplantation is a viable option for some patients with MF, but most patients are diagnosed at an advanced age and are not candidates for stem cell transplantation. Recent clinical trials with thalidomide demonstrated modest efficacy in MF, largely by slowing the rate of fibrosis in these patients rather than by reversal of fibrosis [
]. Allogeneic BMT data does indicate that eradication of the malignant hematopoietic clone in MF does result in complete reversal of fibrosis [
], and suggests that drugs that target mutant alleles such as MPLW515L may be effective disease-remitting agents. JAK2 inhibitor development is well under way as a potential therapeutic strategy for treating MPD patients with the
allele. Data presented here indicate that the subset of
negative MF patients with the
mutation will also be sensitive to inhibition with small molecule JAK2 inhibitors. These findings also suggest that a more expansive genomic screen for involvement of other components of the JAK-STAT signal transduction pathway is warranted in MPD patients.