miR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2
Enrique Fuentes-Mattei, Recep Bayraktar, Taghi Manshouri, Andreia M. Silva, Cristina Ivan, Diana Gulei, Linda Fabris, Nayra Soares do Amaral, Pilar Mur, Cristina Perez, Elizabeth Torres-Claudio, Mihnea P. Dragomir, Adriana Badillo-Perez, Erik Knutsen, Pranav Narayanan, Leonard Golfman, Masayoshi Shimizu, Xinna Zhang, Wanke Zhao, Wanting Tina Ho, Marcos Roberto Estecio, Geoffrey Bartholomeusz, Ciprian Tomuleasa, Ioana Berindan-Neagoe, Patrick A. Zweidler-McKay, Zeev Estrov, Zhizhuang J. Zhao, Srdan Verstovsek, George A. Calin, Roxana S. Redis
Enrique Fuentes-Mattei, Recep Bayraktar, Taghi Manshouri, Andreia M. Silva, Cristina Ivan, Diana Gulei, Linda Fabris, Nayra Soares do Amaral, Pilar Mur, Cristina Perez, Elizabeth Torres-Claudio, Mihnea P. Dragomir, Adriana Badillo-Perez, Erik Knutsen, Pranav Narayanan, Leonard Golfman, Masayoshi Shimizu, Xinna Zhang, Wanke Zhao, Wanting Tina Ho, Marcos Roberto Estecio, Geoffrey Bartholomeusz, Ciprian Tomuleasa, Ioana Berindan-Neagoe, Patrick A. Zweidler-McKay, Zeev Estrov, Zhizhuang J. Zhao, Srdan Verstovsek, George A. Calin, Roxana S. Redis
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Research Article Hematology Oncology

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

Abstract

Myelofibrosis (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 JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F 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-543 was significantly upregulated in nonresponders. 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 JAK2V617F 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.

Authors

Enrique Fuentes-Mattei, Recep Bayraktar, Taghi Manshouri, Andreia M. Silva, Cristina Ivan, Diana Gulei, Linda Fabris, Nayra Soares do Amaral, Pilar Mur, Cristina Perez, Elizabeth Torres-Claudio, Mihnea P. Dragomir, Adriana Badillo-Perez, Erik Knutsen, Pranav Narayanan, Leonard Golfman, Masayoshi Shimizu, Xinna Zhang, Wanke Zhao, Wanting Tina Ho, Marcos Roberto Estecio, Geoffrey Bartholomeusz, Ciprian Tomuleasa, Ioana Berindan-Neagoe, Patrick A. Zweidler-McKay, Zeev Estrov, Zhizhuang J. Zhao, Srdan Verstovsek, George A. Calin, Roxana S. Redis

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Figure 4

JAK2V617F mutation promotes increase in miR-543 levels.

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JAK2V617F mutation promotes increase in miR-543 levels. (A) Representat...
(A) Representative diagram of the JAK2V617F-transgenic mouse model of MF and the representative histology slides of the BM tissue. Scale bars: 100 μm. (B) Differential expression of miR-543 measured by RT-qPCR in wild-type control mice and in JAK2V617F-transgenic mice. (C) Differential expression of miR-543 measured by RT-qPCR in the HCD57 parental control cells and HCD57 erythroleukemia cells containing the JAK2V617F mutation (HCD-VF1). U6 levels were used as normalizer. (D) Relative luciferase activity in HEK293 cells transiently cotransfected with the control vector and miR-543 promoter sequence with putative STAT3 binding site. (E) Expression levels of STAT3 and p-STAT3 Tyr705 were determined by Western blot analysis in HCD57 and HCD-VF1 cells. β-Actin was used as a loading control. (F) Expression levels of STAT3 and p-STAT3 Tyr705 were determined by Western blot analysis in HCD-VF1 cells in which STAT3 was silenced by siRNA. β-Actin was used as a loading control. (G) Silencing of STAT3 in HCD-VF1 erythroleukemia cells led to decreased miR-543 expression levels as assessed by RT-qPCR. Two-tailed t test was used to determine statistical significance for B and D. Mann-Whitney-Wilcoxon nonparametric test was used for C and E. Each dot represents 1 animal. All experiments were performed independently 3 times, and representative blots are shown.