Takefumi Ishii

Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth.

JAK2V617F, a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva™) is a potent inhibitor of JAK2V617F activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2V617F-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2V617F-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2V617F-positive PV and other myeloproliferative disorders.

Recurrence of clonal hematopoiesis after discontinuing pegylated recombinant interferon-α 2a in a patient with polycythemia vera [11]

Recurrence of clonal hematopoiesis after discontinuing pegylated recombinant interferon- α 2a in a patient with polycythemia vera

Haematopoietic cell lineage distribution of MPLW515L/K mutations in patients with idiopathic myelofibrosis

Idiopathic myelofibrosis (IM) is a clonal haematological malignancy that is thought to originate at the level of the pluripotent haematopoietic stem cell (Baxter et al, 2005). The JAK2V617F mutation has been documented in 50% of patients with IM (Baxter et al, 2005). Pikman et al (2006) and Pardanani et al (2006) have recently defined two gain of function mutations, MPLW515L and MPLW515K (MPLW515L/K), in patients with IM and shown that these mutations play a role in the development of IM. Like JAK2V617F, the MPLW515L/K mutations also lead to activation of JAK-STAT signalling in these IM patients (Pikman et al, 2006). MPLW515L/K mutations are much less common than JAK2V617F but are disease-specific since they solely occur in 5 c. 10% of patients with IM (Lasho et al, 2006; Moliterno et al, 2006; Pardanani et al, 2006; Pikman et al, 2006). MPLW515L/K mutations have been previously detected in DNA from granulocytes of patients with IM (Lasho et al, 2006; Moliterno et al, 2006; Pardanani et al, 2006; Pikman et al, 2006). It is, however, unclear what other haematopoietic cell lineages are involved by these mutations. In the current study, we screened the granulocytes of 38 patients with IM for the MPLW515L/K mutations by DNA sequencing and allele-specific polymerase chain reaction (PCR). All patients met the World Health Organization diagnostic criteria for IM and all human tissue samples were obtained after informed consent was provided. Of the 38 patients, three patients had MPL515 mutations, two

Involvement of mast cells by the malignant process in patients with Philadelphia chromosome negative myeloproliferative neoplasms.

The Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematologic malignancies frequently characterized by a mutation in JAK2 (JAK2V617F). Peripheral blood (PB) CD34+ cells from patients with polycythemia vera (PV) and primary myelofibrosis (PMF) generated in vitro significantly fewer mast cells (MCs) than normal PB CD34+ cells. The numbers of MC progenitors assayed from MPN CD34+ cells were, however, similar to that assayed from normal CD34+ cells. A higher percentage of the cultured MPN MCs expressed FcɛRIα, CD63 and CD69 than normal MCs, suggesting that cultured MPN MCs are associated with an increased state of MC activation. Further analysis showed that a higher proportion of cultured PV and PMF MCs underwent apoptosis in vitro. By using JAK2V617F, MplW515L and chromosomal abnormalities as clonality markers, we showed that the malignant process involved MPN MCs. JAK2V617F-positive MC colonies were assayable from the PB CD34+ cells of each of the 17 JAK2V617F positive MPN patients studied. Furthermore, erlotinib, a JAK2 inhibitor, was able to inhibit JAK2V617F-positive PV MC progenitor cells, indicating that malignant MC progenitor cells are a potential cellular target for such JAK2 inhibitor-directed therapy.

Circulating angiogenic monocyte progenitor cells are reduced in JAK2V617F high allele burden myeloproliferative disorders

The clinical course of patients with Philadelphia chromosome negative myeloproliferative disorder is frequently complicated by thrombotic events. Post-natal vasculogenesis has been proposed to play a critical role in angiogenesis by acting through a hierarchy of endothelial progenitor cells. Some endothelial progenitor cells have been shown to share a number of features associated with monocytes while other more primitive progenitor cells produce endothelial cells in vitro exclusively. The cells which share features of monocytes and endothelial cells have been termed angiogenic monocytes. Reduced levels of angiogenic monocyte progenitor cells have been reported to be predictive of atherosclerotic disease progression. Angiogenic monocyte progenitor cells were assayed in vitro from the peripheral blood mononuclear cells of myeloproliferative disorder patients. Angiogenic monocyte colonies were plucked and analyzed for endothelial cells and hematopoietic cell markers, JAK2V617F and their ability to incorporate into vascular endothelium following their transplantation into non-obese diabetic, severe combine immunodeficient mice. Myeloproliferative disorder angiogenic monocyte colonies that were detected were uniformly JAK2V617F positive and produced cells that expressed phenotypic markers characteristic of both monocytes and endothelial cells. Reduced numbers of angiogenic monocyte colonies were present in the blood of myeloproliferative disorder patients with a high JAK2V617F burden (>50%), (p<0.01). Transplanted angiogenic monocytes were able to contribute to the vascular endothelium of non-obese diabetic, severe combine immunodeficient mice. These studies suggest that reduced numbers of circulating angiogenic monocyte progenitors contribute to the propensity to develop thrombotic complications in myeloproliferative disorder patients.

T cells from patients with polycythemia vera elaborate growth factors which contribute to endogenous erythroid and megakaryocyte colony formation

In the present study, we report that media conditioned by polycythemia vera (PV) CD3+ cells promote BFU-E and CFU-Mk colony formation by both cord blood and PV peripheral blood CD34+ cells in the absence of exogenous cytokines and promoting megakaryocyte proplatelet formation. CD3+ cells constitutively produce elevated levels of IL-11, while stimulation with the addition of phytohemagglutinin (PHA) increased GM-CSF levels in most of the patients with PV. Anti-IL-11-neutralizing antibody partially inhibited the formation of BFU-E and CFU-Mk colonies promoted by PV CD3+ cell-conditioned media. Although IL-11 is not produced by normal T cells, real-time PCR and flow cytometric analysis showed that IL-11 was upregulated in the CD3+ cells of most PV patients as compared to normal CD3+ cells. In addition, a greater percentage of BFU-E colonies formed by PV CD34+ cells in the presence of PV CD3+ cell-conditioned media alone were JAK2V617F-positive as compared with that induced by EPO. We conclude that dysregulated production of soluble growth factor(s), including IL-11 and GM-CSF by PV T cells, contributes to the in vitro formation of erythroid colonies in the absence of exogenous cytokines by PV CD34+ cells and likely plays a role in sustaining hematopoiesis in PV.