MiR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2

Myelofibros is (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAK(V617F) mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK(V617F) inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-S43 was significantly upregulated in non responders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2(V617F) mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options

Generation and characterization of a JAK2V617F-containing erythroleukemia cell line.

The JAK2V617F mutation is found in the majority of patients with myeloproliferative neoplasms (MPNs). Transgenic expression of the mutant gene causes MPN-like phenotypes in mice. We have produced JAK2V617F mice with p53 null background. Some of these mice developed acute erythroleukemia. From one of these mice, we derived a cell line designated J53Z1. J53Z1 cells were stained positive for surface markers CD71 and CD117 but negative for Sca-1, TER-119, CD11b, Gr-1, F4/80, CD11c, CD317, CD4, CD8a, CD3e, B220, CD19, CD41, CD42d, NK-1.1, and FceR1. Real time PCR analyses demonstrated expressions of erythropoietin receptor EpoR, GATA1, and GATA2 in these cells. J53Z1 cells grew rapidly in suspension culture containing fetal bovine serum with a doubling time of ∼18 hours. When transplanted into C57Bl/6 mice, J53Z1 cells induced acute erythroleukemia with massive infiltration of tumor cells in the spleen and liver. J53Z1 cells were responsive to stimulation with erythropoietin and stem cell factor and were selectively inhibited by JAK2 inhibitors which induced apoptosis of the cells. Together, J53Z1 cells belong to the erythroid lineage, and they may be useful for studying the role of JAK2V617F in proliferation and differentiation of erythroid cells and for identifying potential therapeutic drugs targeting JAK2

Moving on

This report details our journey in producing the short documentary Moving on; 回娘家. From the initial stages of idea formation to post production, this report reflects our experience, the challenges we met with and the rationale behind decisions made.Bachelor of Communication Studie

Inhibition of J53Z1 cells by selective protein kinase inhibitors.

<p>J53Z1 cells were cultured in the presence of various concentrations of indicated protein kinase inhibitors. <b>Top and middle panels.</b> Cell viability was assessed by XTT assays after 72 hr of incubation. Control experiments were performed in the presence of 0.1% DMSO. Error bars denote standard deviation (n = 3). *P<0.001 in reference to control. Note that MV-4-11 cells were analyzed for comparison (middle panel). <b>Bottom panel.</b> Apoptosis assays were performed with J53Z1 cells after 24 hr of incubation with 0.2 µM of ruxolitinib or AZD1480. Cells were stained with FITC-annexin V and propidium iodide. Percentages of annexin V-positive cells are indicated.</p

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

Surface Markers on J53Z1 Cells.

<p>Surface Markers on J53Z1 Cells.</p

Development of erythroleukemia phenotypes in mice receiving implantation of J53Z1 cells.

<p>Cultured J53Z1 cells (1×10⁶) were implanted into 12-week-old wild type C57Bl/6 mice through retro-orbital injections. <b>Upper panel.</b> Red blood cells, spleen, and liver were analyzed 2 to 4 weeks after implantation. Error bars denote standard deviation (n≥4), *P<0.001. <b>Lower panel.</b> Paraffin sections of spleen and liver from representative control and J53Z1-implanted mice were subjected to H&E staining. Note the loss of normal tissue architecture and infiltration of densely stained erythroleukemia cells in tissues from J53Z1-transplanted mice. Photos were taken with a 40x objective lens.</p

Morphology of J53Z1 cells.

<p>Normal growing J53Z1 cells were attached to glass slides by cytospin and then subjected to Wright-Giemsa staining. HCD-57 erythroid leukemia cells were stained for comparison. Photos were taken with a 100x objective lens.</p

Flow cytometric analysis of CD71 and TER-119 expression on J53Z1 cells.

<p>J53Z1, HCD-57, and normal mouse bone marrow cells were labeled with anti-CD71 and TER-119 monoclonal antibodies or with a nonspecific isotype control mouse IgG before flow cytometric analysis.</p