Targeting methyltransferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia
Yanli Jin, … , Ruibao Ren, Jingxuan Pan
Yanli Jin, … , Ruibao Ren, Jingxuan Pan
Published October 3, 2016; First published September 19, 2016
Citation Information: J Clin Invest. 2016;126(10):3961-3980. https://doi.org/10.1172/JCI85239.
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Research Article Hematology

Targeting methyltransferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia

Abstract

Imatinib-insensitive leukemia stem cells (LSCs) are believed to be responsible for resistance to BCR-ABL tyrosine kinase inhibitors and relapse of chronic myelogenous leukemia (CML). Identifying therapeutic targets to eradicate CML LSCs may be a strategy to cure CML. In the present study, we discovered a positive feedback loop between BCR-ABL and protein arginine methyltransferase 5 (PRMT5) in CML cells. Overexpression of PRMT5 was observed in human CML LSCs. Silencing PRMT5 with shRNA or blocking PRMT5 methyltransferase activity with the small-molecule inhibitor PJ-68 reduced survival, serial replating capacity, and long-term culture-initiating cells (LTC-ICs) in LSCs from CML patients. Further, PRMT5 knockdown or PJ-68 treatment dramatically prolonged survival in a murine model of retroviral BCR-ABL–driven CML and impaired the in vivo self-renewal capacity of transplanted CML LSCs. PJ-68 also inhibited long-term engraftment of human CML CD34+ cells in immunodeficient mice. Moreover, inhibition of PRMT5 abrogated the Wnt/β-catenin pathway in CML CD34+ cells by depleting dishevelled homolog 3 (DVL3). This study suggests that epigenetic methylation modification on histone protein arginine residues is a regulatory mechanism to control self-renewal of LSCs and indicates that PRMT5 may represent a potential therapeutic target against LSCs.

Authors

Yanli Jin, Jingfeng Zhou, Fang Xu, Bei Jin, Lijing Cui, Yun Wang, Xin Du, Juan Li, Peng Li, Ruibao Ren, Jingxuan Pan

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Figure 2

PRMT5 positively regulates BCR-ABL expression in CML cells.

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PRMT5 positively regulates BCR-ABL expression in CML cells. (A) Western ...
(A) Western blot analysis of PRMT5 and BCR-ABL protein levels in K562 cells stably expressing pCMV-2B-Flag (Vector) or pCMV-2B-Flag-PRMT5 (PRMT5) (#16 and #19). (B and C) Levels of BCR-ABL protein and mRNA were analyzed by Western blot (B) and qRT-PCR (C) in K562 cells electrotransfected with pCMV-2B-Flag-PRMT5 (PRMT5) or mutant pCMV-2B-Flag-PRMT5 (R368A) [PRMT5 (R368A)]. (D) Luciferase assay of 293T cells transiently cotransfected with the BCR-ABL promoter reporter and PRMT5 or mutant PRMT5 (R368A) constructs for 24 hours. (E) Western blot analysis of BCR-ABL and PRMT5 and its histone methylation marks in K562 with stable PRMT5 knockdown or Scramble control. (F) qRT-PCR analysis of BCR-ABL mRNA levels in K562 cells with stable PRMT5 knockdown. (G) Chemical structure of PJ-68. (H) Histone methyltransferase activity was measured in the presence of PJ-68 and different PRMT enzymes, including type I PRMT1, -3, -4, -6, and -8 and type II PRMT5. PJ-68 potently inhibited PRMT5 (IC50= 517 nM). (I) Western blot analysis of BCR-ABL and PRMT5 and its epigenetic marks in K562 cells treated with increasing concentrations of PJ-68. (J) PJ-68 inhibited BCR-ABL gene transcription as measured by qRT-PCR analysis. (K and L) PRMT5 inhibition reduced mRNA and protein levels of BCR-ABL in CML CD34+CD38– cells and CD34+CD38+ cells (n = 3). (M) qRT-PCR analysis of Mir203 level in K562 cells treated with PJ-68 for 24 hours. (N and O) PRMT5 inhibition by PJ-68 (N) or PRMT5 knockdown by shRNA (O) in K562 cells led to loss of recruitment of PRMT5 and its epigenetic marks to the promoter of Mir203 as determined by ChIP assays. Two-tailed Student’s t test was used for K, M, and N; 1-way ANOVA, post hoc intergroup comparisons, Tukey’s test were performed for C, D, F, and J. *P < 0.05, **P < 0.01, ***P < 0.0001.