Targeting methyltransferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia
Yanli Jin, Jingfeng Zhou, Fang Xu, Bei Jin, Lijing Cui, Yun Wang, Xin Du, Juan Li, Peng Li, Ruibao Ren, Jingxuan Pan
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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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.