Loss of pleckstrin-2 reverts lethality and vascular occlusions in JAK2V617F-positive myeloproliferative neoplasms
Baobing Zhao, … , Charles S. Abrams, Peng Ji
Baobing Zhao, … , Charles S. Abrams, Peng Ji
Published November 20, 2017
Citation Information: J Clin Invest. 2018;128(1):125-140. https://doi.org/10.1172/JCI94518.
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Loss of pleckstrin-2 reverts lethality and vascular occlusions in JAK2V617F-positive myeloproliferative neoplasms

Abstract

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.

Authors

Baobing Zhao, Yang Mei, Lan Cao, Jingxin Zhang, Ronen Sumagin, Jing Yang, Juehua Gao, Matthew J. Schipma, Yanfeng Wang, Chelsea Thorsheim, Liang Zhao, Timothy Stalker, Brady Stein, Qiang Jeremy Wen, John D. Crispino, Charles S. Abrams, Peng Ji

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Figure 2

Plek2 is a downstream target of the JAK2/STAT5 pathway.

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Plek2 is a downstream target of the JAK2/STAT5 pathway. (A and B) Wester...
(A and B) Western blot (A) and quantitative PCR (B) analyses of Plek2 expression in the cultured erythroblasts treated with indicated JAK2 inhibitors after 20 hours. Different concentrations of JAK2 inhibitor AZD1480 or ruxolitinib were added to the cultured erythroblasts in the presence of Epo (2 U/ml). Hsc70 was used as a loading control. (C and D) Western blot (C) and quantitative PCR (D) analyses of Plek2 expression in the cultured erythroblasts transduced with JAK2 wild-type (WT) and JAK2V617F mutant. (E and F) Western blot (E) and quantitative PCR (F) analyses of Plek2 expression in the cultured erythroblasts transduced with STAT5 wild-type (WT), dominant-negative (DN), and constitutively active (CA) mutants. (G and H) Western blot (G) and quantitative PCR (H) analyses of Plek2 expression in the cultured lineage-negative bone marrow progenitor cells exposed to thrombopoietin. D1 to D3 indicate different days of in vitro culture. (I and J) Same as G and H except the cells were exposed to GM-CSF. (K) ChIP–quantitative PCR assay showing STAT5 binding at the Plek2 promoter in freshly purified Ter119-negative E13.5 mouse fetal liver erythroblasts (D0) and cultured mouse fetal liver erythroblasts on day 1 (D1). P1 to P7 indicate fragments in the Plek2 promoter region amplified in ChIP–qPCR assays. (L) Luciferase reporter assay of STAT5 binding on the Plek2 promoter. HET293T cells were transfected with indicated constructs, together with a Plek2 promoter construct. Luciferase activity was measured at 48 hours after transfection. (M and N) Normalized ATAC-sequencing peaks in the Plek2 locus (M) and relative expression of Plek2 (N) in the indicated cell type. Ery, erythroid cells; Mono, monocyte. The y axis represents normalized arbitrary units. Boxed regions show cell type–specific peaks around the Plek2 gene. TSS, transcription start site. Data were obtained from Corces et al. (22).