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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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Suppression of p16 restores anti-leukemic effect of EZH2 inhibition. (a) MLL/ENL leukemia cells were transduced with pSIREN-RetroQ-shp16 (shp16) or control vector and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The total RNAs were isolated and analyzed for p16 by qRT-PCR. N = 2 for each. (b) shp16 transduced leukemia cells were transduced with pSIREN-ZsGreen control or shEZH2. 1 × 104 of sorted ZsGreen positive cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 2 for each. (c) Mice were injected intravenously with 1 × 104 of control or shp16 transduced MLL/ENL leukemic cells. Mice were treated with DZNep or DDW as previously shown in Figure1a. N = 7 for each group. (d) Retrovirus encoding p16-IRES-GFP was infected into MLL/ENL leukemia cells. GFP positive cells were sorted and 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 = 5 for each. (e) Mice were intravenously injected with 1 × 104 of p16-IRES-GFP or mock transduced MLL/ENL leukemic cells. N = 7 for each group.
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fig03: Suppression of p16 restores anti-leukemic effect of EZH2 inhibition. (a) MLL/ENL leukemia cells were transduced with pSIREN-RetroQ-shp16 (shp16) or control vector and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The total RNAs were isolated and analyzed for p16 by qRT-PCR. N = 2 for each. (b) shp16 transduced leukemia cells were transduced with pSIREN-ZsGreen control or shEZH2. 1 × 104 of sorted ZsGreen positive cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 2 for each. (c) Mice were injected intravenously with 1 × 104 of control or shp16 transduced MLL/ENL leukemic cells. Mice were treated with DZNep or DDW as previously shown in Figure1a. N = 7 for each group. (d) Retrovirus encoding p16-IRES-GFP was infected into MLL/ENL leukemia cells. GFP positive cells were sorted and 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 = 5 for each. (e) Mice were intravenously injected with 1 × 104 of p16-IRES-GFP or mock transduced MLL/ENL leukemic cells. N = 7 for each group.

Mentions: To validate whether upregulation of p16 is responsible for anti-leukemic effect of EZH2 inhibition, we tested shRNA for p16 (shp16) (Fig.3a). Colony forming capacity of MLL/ENL leukemia cells attenuated by shEZH2 was markedly restored by p16 knockdown (Fig.3b). Furthermore, similar results were obtained using MLL/ENL leukemia mice treated with shp16 and DZNep (Fig.3c). In contrast, overexpression of p16 diminished colony number of MLL/ENL leukemia cells (Fig.3d) and showed decreased leukemia initiating capacity in transplantation assay (Fig.3e). These data support the idea that p16 is one of the main targets of 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)

Suppression of p16 restores anti-leukemic effect of EZH2 inhibition. (a) MLL/ENL leukemia cells were transduced with pSIREN-RetroQ-shp16 (shp16) or control vector and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The total RNAs were isolated and analyzed for p16 by qRT-PCR. N = 2 for each. (b) shp16 transduced leukemia cells were transduced with pSIREN-ZsGreen control or shEZH2. 1 × 104 of sorted ZsGreen positive cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 2 for each. (c) Mice were injected intravenously with 1 × 104 of control or shp16 transduced MLL/ENL leukemic cells. Mice were treated with DZNep or DDW as previously shown in Figure1a. N = 7 for each group. (d) Retrovirus encoding p16-IRES-GFP was infected into MLL/ENL leukemia cells. GFP positive cells were sorted and 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 = 5 for each. (e) Mice were intravenously injected with 1 × 104 of p16-IRES-GFP or mock transduced MLL/ENL leukemic cells. N = 7 for each group.
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Related In: Results  -  Collection

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fig03: Suppression of p16 restores anti-leukemic effect of EZH2 inhibition. (a) MLL/ENL leukemia cells were transduced with pSIREN-RetroQ-shp16 (shp16) or control vector and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The total RNAs were isolated and analyzed for p16 by qRT-PCR. N = 2 for each. (b) shp16 transduced leukemia cells were transduced with pSIREN-ZsGreen control or shEZH2. 1 × 104 of sorted ZsGreen positive cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 2 for each. (c) Mice were injected intravenously with 1 × 104 of control or shp16 transduced MLL/ENL leukemic cells. Mice were treated with DZNep or DDW as previously shown in Figure1a. N = 7 for each group. (d) Retrovirus encoding p16-IRES-GFP was infected into MLL/ENL leukemia cells. GFP positive cells were sorted and 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 = 5 for each. (e) Mice were intravenously injected with 1 × 104 of p16-IRES-GFP or mock transduced MLL/ENL leukemic cells. N = 7 for each group.
Mentions: To validate whether upregulation of p16 is responsible for anti-leukemic effect of EZH2 inhibition, we tested shRNA for p16 (shp16) (Fig.3a). Colony forming capacity of MLL/ENL leukemia cells attenuated by shEZH2 was markedly restored by p16 knockdown (Fig.3b). Furthermore, similar results were obtained using MLL/ENL leukemia mice treated with shp16 and DZNep (Fig.3c). In contrast, overexpression of p16 diminished colony number of MLL/ENL leukemia cells (Fig.3d) and showed decreased leukemia initiating capacity in transplantation assay (Fig.3e). These data support the idea that p16 is one of the main targets of 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