Ezh2 loss propagates hypermethylation at T cell differentiation–regulating genes to promote leukemic transformation
Changshan Wang, … , Atsushi Iwama, Goro Sashida
Changshan Wang, … , Atsushi Iwama, Goro Sashida
Published August 6, 2018
Citation Information: J Clin Invest. 2018;128(9):3872-3886. https://doi.org/10.1172/JCI94645.
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Research Article Hematology Oncology

Ezh2 loss propagates hypermethylation at T cell differentiation–regulating genes to promote leukemic transformation

Abstract

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a new pathological entity with poor outcomes in T cell ALL (T-ALL) that is characterized by a high incidence of loss-of-function mutations in polycomb repressive complex 2 (PRC2) genes. We generated a mouse model of ETP-ALL by deleting Ezh2, one of the PRC2 genes, in p53-null hematopoietic cells. The loss of Ezh2 in p53-null hematopoietic cells impeded the differentiation of ETPs and eventually induced ETP-ALL–like disease in mice, indicating that PRC2 functions as a bona fide tumor suppressor in ETPs. A large portion of PRC2 target genes acquired DNA hypermethylation of their promoters following reductions in H3K27me3 levels upon the loss of Ezh2, which included pivotal T cell differentiation–regulating genes. The reactivation of a set of regulators by a DNA-demethylating agent, but not the transduction of single regulator genes, effectively induced the differentiation of ETP-ALL cells. Thus, PRC2 protects key T cell developmental regulators from DNA hypermethylation in order to keep them primed for activation upon subsequent differentiation phases, while its insufficiency predisposes ETPs to leukemic transformation. These results revealed a previously unrecognized epigenetic switch in response to PRC2 dysfunction and provide the basis for specific rational epigenetic therapy for ETP-ALL with PRC2 insufficiency.

Authors

Changshan Wang, Motohiko Oshima, Daisuke Sato, Hirotaka Matsui, Sho Kubota, Kazumasa Aoyama, Yaeko Nakajima-Takagi, Shuhei Koide, Jun Matsubayashi, Makiko Mochizuki-Kashio, Takako Nakano-Yokomizo, Jie Bai, Toshitaka Nagao, Akinori Kanai, Atsushi Iwama, Goro Sashida

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Figure 2

Ezh2 loss promoted the development of ETP-ALL in the absence of p53.

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Ezh2 loss promoted the development of ETP-ALL in the absence of p53. (A)...
(A) Proportions of CD4–CD8– DN cells among CD45.2+ cells in the thymus of WT, Ezh2Δ/Δ, p53Δ/Δ, and Ezh2Δ/Δp53Δ/Δ (n = 4–7) mice 3 months after transplantation. Data are shown as mean ± SEM. (B) Representative flow cytometric profiles of CD4 and CD8 expression on donor-derived CD45.2+ cells in the thymus of WT, Ezh2Δ/Δ, p53Δ/Δ, and Ezh2Δ/Δp53Δ/Δ mice. (C) Proportions of CD44+CD25– DN1, CD44+CD25+ DN2, CD44–CD25+ DN3, and CD44–CD25– DN4 cells among donor-derived CD45.2+CD4–CD8– DN cells in the thymus (n = 4–7). Data are shown as mean ± SEM. (D) Representative flow cytometric profiles of CD44 and CD25 expression on CD45.2+ DN cells in the thymus of WT and Ezh2Δ/Δp53Δ/Δ mice. (E) Thymus weight of WT (n = 10), Ezh2Δ/Δ (n = 10), p53Δ/Δ (n = 10), and Ezh2Δ/Δp53Δ/Δ (n = 9) mice 3 months after transplantation and moribund Ezh2Δ/Δp53Δ/Δ ETP-ALL mice (n = 8) at the time of sacrifice. Data are shown as mean ± SEM. *P < 0.05; ***P < 0.001, Student’s t test (WT vs. Ezh2Δ/Δ) or Mann-Whitney U test (WT vs. ETP-ALL and Ezh2Δ/Δp53Δ/Δ vs. ETP-ALL). (F) Spleen weight of WT (n = 10), Ezh2Δ/Δ (n = 10), p53Δ/Δ (n = 10), and Ezh2Δ/Δp53Δ/Δ (n = 9) mice 3 months after transplantation and moribund Ezh2Δ/Δp53Δ/Δ ETP-ALL mice (n = 8) at the time of sacrifice. Data are shown as mean ± SEM. **P < 0.01, Mann-Whitney U test. (G) Immature leukemic cells in the thymus of Ezh2Δ/Δp53Δ/Δ ETP-ALL mice (mouse nos. 4 and 6) observed by May-Grünwald Giemsa staining. Original magnification, ×400. (H) Pie chart illustrating the frequencies of the disease phenotypes of Ezh2Δ/Δp53Δ/Δ mice (n = 18). ETP-ALL with DN1, DN2, and DN1/2 type and thymic lymphoma were identified. (I) Representative flow cytometric profiles of CD4/CD8 expression in donor-derived CD45.2+ cells, and c-Kit, CD44, and CD25 expression in CD45.2+Lin– cells in the thymus of each subtype of ETP-ALL shown in H. (J) Representative histology of the thymus, spleen, and liver of an Ezh2Δ/Δp53Δ/Δ ETP-ALL mouse observed by H&E staining and CD3 staining. Original magnification, ×400 (left panels); ×100 (right panels). Scale bars: 100 μm. (K) Rearrangement of the TCR-β gene in DN2 cells from WT and Ezh2Δ/Δp53Δ/Δ mice 3 months after transplantation and ETP-ALL mice (mouse nos. 13 and 41) assessed by Dβ1-Jβ1 amplifications by PCR. Germline bands were observed in WT Gr1+Mac1+ neutrophils and B220+ B cells.