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Inhibition of histone methyltransferase EZH2 depletes leukemia stem cell of mixed lineage leukemia fusion leukemia through upregulation of p16.

Ueda K, Yoshimi A, Kagoya Y, Nishikawa S, Marquez VE, Nakagawa M, Kurokawa M - Cancer Sci. (2014)

Bottom Line: Epigenetic regulators are associated with many cellular processes including maintenance of stem cells.Expression analysis suggested that p16 upregulation was responsible for LICs reduction.Knockdown of p16 canceled the survival advantage of mice treated with DZNep.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

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Knockdown of EZH2 reproduces the effect of EZH2 inhibitor for MLL fusion leukemic mice. (a) MLL/ENL leukemia cells were transduced with pSIREN-ZsGreen control vector or pSIREN-ZsGreen-shEZH2 (shEZH2) and cultured for 3 days. The total RNAs were isolated and analyzed for EZH2 by qRT-PCR. N = 2 for each. (b) Mice were injected intravenously with 1 × 104 ZsGreen (shRNA targeting EZH2 or control vector) positive leukemia cells. Survival of the mice in both groups is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 6 for each group. (c) Immunoblot analysis of H3K27Me3 and total H3. Values indicate relative density scale. (d) 1 × 104 of sorted ZsGreen positive BM cells were placed in 1 ml of methocult M3434 and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 3 for each. (e) ZsGreen positive cells were sorted and analyzed by qRT-PCR. Relative expression of EZH2 and its target genes are shown. P-values were calculated by T test. N = 3 for each group.
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fig02: Knockdown of EZH2 reproduces the effect of EZH2 inhibitor for MLL fusion leukemic mice. (a) MLL/ENL leukemia cells were transduced with pSIREN-ZsGreen control vector or pSIREN-ZsGreen-shEZH2 (shEZH2) and cultured for 3 days. The total RNAs were isolated and analyzed for EZH2 by qRT-PCR. N = 2 for each. (b) Mice were injected intravenously with 1 × 104 ZsGreen (shRNA targeting EZH2 or control vector) positive leukemia cells. Survival of the mice in both groups is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 6 for each group. (c) Immunoblot analysis of H3K27Me3 and total H3. Values indicate relative density scale. (d) 1 × 104 of sorted ZsGreen positive BM cells were placed in 1 ml of methocult M3434 and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 3 for each. (e) ZsGreen positive cells were sorted and analyzed by qRT-PCR. Relative expression of EZH2 and its target genes are shown. P-values were calculated by T test. N = 3 for each group.

Mentions: To confirm whether the effect of DZNep against MLL related leukemia is due to EZH2 inhibition, we transduced shRNAs for EZH2 (shEZH2) into MLL/ENL leukemic cells (Fig.2a). The transduced cells were FACS sorted and transplanted to sublethally irradiated mice. Knockdown of EZH2 prolonged the survival of MLL/ENL leukemic mice (Fig.2b). The BM cells transduced with shEZH2 were globally hypomethylated (Fig.2c), and had diminished colony forming capacity with elevated p16 expression (Fig.2d,e). The ratios of L-GMP in mice transplanted with shEZH2 transduced cells were markedly reduced (Fig. S4). These data indicate that therapeutic efficacy of DZNep is due to EZH2 inhibition.


Inhibition of histone methyltransferase EZH2 depletes leukemia stem cell of mixed lineage leukemia fusion leukemia through upregulation of p16.

Ueda K, Yoshimi A, Kagoya Y, Nishikawa S, Marquez VE, Nakagawa M, Kurokawa M - Cancer Sci. (2014)

Knockdown of EZH2 reproduces the effect of EZH2 inhibitor for MLL fusion leukemic mice. (a) MLL/ENL leukemia cells were transduced with pSIREN-ZsGreen control vector or pSIREN-ZsGreen-shEZH2 (shEZH2) and cultured for 3 days. The total RNAs were isolated and analyzed for EZH2 by qRT-PCR. N = 2 for each. (b) Mice were injected intravenously with 1 × 104 ZsGreen (shRNA targeting EZH2 or control vector) positive leukemia cells. Survival of the mice in both groups is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 6 for each group. (c) Immunoblot analysis of H3K27Me3 and total H3. Values indicate relative density scale. (d) 1 × 104 of sorted ZsGreen positive BM cells were placed in 1 ml of methocult M3434 and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 3 for each. (e) ZsGreen positive cells were sorted and analyzed by qRT-PCR. Relative expression of EZH2 and its target genes are shown. P-values were calculated by T test. N = 3 for each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: Knockdown of EZH2 reproduces the effect of EZH2 inhibitor for MLL fusion leukemic mice. (a) MLL/ENL leukemia cells were transduced with pSIREN-ZsGreen control vector or pSIREN-ZsGreen-shEZH2 (shEZH2) and cultured for 3 days. The total RNAs were isolated and analyzed for EZH2 by qRT-PCR. N = 2 for each. (b) Mice were injected intravenously with 1 × 104 ZsGreen (shRNA targeting EZH2 or control vector) positive leukemia cells. Survival of the mice in both groups is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 6 for each group. (c) Immunoblot analysis of H3K27Me3 and total H3. Values indicate relative density scale. (d) 1 × 104 of sorted ZsGreen positive BM cells were placed in 1 ml of methocult M3434 and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 3 for each. (e) ZsGreen positive cells were sorted and analyzed by qRT-PCR. Relative expression of EZH2 and its target genes are shown. P-values were calculated by T test. N = 3 for each group.
Mentions: To confirm whether the effect of DZNep against MLL related leukemia is due to EZH2 inhibition, we transduced shRNAs for EZH2 (shEZH2) into MLL/ENL leukemic cells (Fig.2a). The transduced cells were FACS sorted and transplanted to sublethally irradiated mice. Knockdown of EZH2 prolonged the survival of MLL/ENL leukemic mice (Fig.2b). The BM cells transduced with shEZH2 were globally hypomethylated (Fig.2c), and had diminished colony forming capacity with elevated p16 expression (Fig.2d,e). The ratios of L-GMP in mice transplanted with shEZH2 transduced cells were markedly reduced (Fig. S4). These data indicate that therapeutic efficacy of DZNep is due to EZH2 inhibition.

Bottom Line: Epigenetic regulators are associated with many cellular processes including maintenance of stem cells.Expression analysis suggested that p16 upregulation was responsible for LICs reduction.Knockdown of p16 canceled the survival advantage of mice treated with DZNep.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus