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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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Anti-leukemic effect of EZH2 inhibition is specific for MLL fusion leukemia. (a) Cell proliferation analysis of several types of leukemia cell lines. Leukemia cell lines are cultured for 3 days in medium containing indicated concentration of DZNep. Cell counts are plotted as percentage treating counts of DZNep non-containing cultures as 100%. (b) Leukemic fusion gene transduced cells or c-Kit positive BM cells (cKit-BM) were transduced with pSIREN-RetroQ-shEZH2 or control vector. 1 × 104 of cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Replating was performed for three times. Colony counts for each round are shown. N = 2 for each. (c) Leukemic fusion gene transduced cells or cKit-BM cells were transduced with pSIREN-RetroQ control or shp16, and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The cells were transduced with pSIREN-ZsGreen-shEZH2 or control vector. 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. N = 2 for each. (d) Mice were injected intravenously with 1 × 105 or 1 × 106 of TPAE leukemic cells. From 7 days after injection, mice were treated intraperitoneally with DZNep, 2 mg/kg 3 days per week, until death. Survival of the mice in both groups is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 5 for 1 × 105 and N = 8 for 1 × 106 cohort. (e) Total BM cells of TPAE leukemic mice (injected with 1 × 106 cells) treated with DDW or DZNep were collected and lysed at 15 days after transplantation. Immunoblot analysis was performed for H3K27Me3.
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fig04: Anti-leukemic effect of EZH2 inhibition is specific for MLL fusion leukemia. (a) Cell proliferation analysis of several types of leukemia cell lines. Leukemia cell lines are cultured for 3 days in medium containing indicated concentration of DZNep. Cell counts are plotted as percentage treating counts of DZNep non-containing cultures as 100%. (b) Leukemic fusion gene transduced cells or c-Kit positive BM cells (cKit-BM) were transduced with pSIREN-RetroQ-shEZH2 or control vector. 1 × 104 of cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Replating was performed for three times. Colony counts for each round are shown. N = 2 for each. (c) Leukemic fusion gene transduced cells or cKit-BM cells were transduced with pSIREN-RetroQ control or shp16, and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The cells were transduced with pSIREN-ZsGreen-shEZH2 or control vector. 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. N = 2 for each. (d) Mice were injected intravenously with 1 × 105 or 1 × 106 of TPAE leukemic cells. From 7 days after injection, mice were treated intraperitoneally with DZNep, 2 mg/kg 3 days per week, until death. Survival of the mice in both groups is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 5 for 1 × 105 and N = 8 for 1 × 106 cohort. (e) Total BM cells of TPAE leukemic mice (injected with 1 × 106 cells) treated with DDW or DZNep were collected and lysed at 15 days after transplantation. Immunoblot analysis was performed for H3K27Me3.

Mentions: Next, we studied whether anti-leukemic effect for MLL fusion leukemia by EZH2 inhibition can be applied to various kinds of leukemia. We exposed several human leukemia cell lines to DZNep. K562, HEL, Kasumi-1 and ME-1 cells, all of which do not harbor MLL fusion genes were not sensitive to low concentration of DZNep while both of MLL leukemia cell lines Mv4-11 and MOLM13 were sensitive to 50 nM of DZNep (Fig.4a). Knockdown of EZH2 led to minimal effect on E2A/HLF transduced BM cells or c-Kit positive BM cells as we previously reported,32 while colony forming capacity of MLL/ENL and Hoxa9/Meis1 transduced cells were strongly suppressed (Fig.4b). Knockdown of p16 rescued colony forming capacity of MLL/ENL or Hoxa9/Meis1 transduced cells by EZH2 inhibition (Fig.4c). Expression levels of p16 were not influenced by knockdown of EZH2 in the BM cells transduced with leukemia fusion genes other than MLL/ENL (Fig. S5). In addition, survival of mice transplanted with leukemia cells induced by TPAE is not prolonged by DZNep administration (Fig.4d), although H3K27 were globally hypomethylated (Fig.4e). These data imply that anti-leukemic effect of EZH2 inhibition through p16 upregulation is specific for MLL fusion leukemia.


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)

Anti-leukemic effect of EZH2 inhibition is specific for MLL fusion leukemia. (a) Cell proliferation analysis of several types of leukemia cell lines. Leukemia cell lines are cultured for 3 days in medium containing indicated concentration of DZNep. Cell counts are plotted as percentage treating counts of DZNep non-containing cultures as 100%. (b) Leukemic fusion gene transduced cells or c-Kit positive BM cells (cKit-BM) were transduced with pSIREN-RetroQ-shEZH2 or control vector. 1 × 104 of cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Replating was performed for three times. Colony counts for each round are shown. N = 2 for each. (c) Leukemic fusion gene transduced cells or cKit-BM cells were transduced with pSIREN-RetroQ control or shp16, and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The cells were transduced with pSIREN-ZsGreen-shEZH2 or control vector. 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. N = 2 for each. (d) Mice were injected intravenously with 1 × 105 or 1 × 106 of TPAE leukemic cells. From 7 days after injection, mice were treated intraperitoneally with DZNep, 2 mg/kg 3 days per week, until death. Survival of the mice in both groups is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 5 for 1 × 105 and N = 8 for 1 × 106 cohort. (e) Total BM cells of TPAE leukemic mice (injected with 1 × 106 cells) treated with DDW or DZNep were collected and lysed at 15 days after transplantation. Immunoblot analysis was performed for H3K27Me3.
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fig04: Anti-leukemic effect of EZH2 inhibition is specific for MLL fusion leukemia. (a) Cell proliferation analysis of several types of leukemia cell lines. Leukemia cell lines are cultured for 3 days in medium containing indicated concentration of DZNep. Cell counts are plotted as percentage treating counts of DZNep non-containing cultures as 100%. (b) Leukemic fusion gene transduced cells or c-Kit positive BM cells (cKit-BM) were transduced with pSIREN-RetroQ-shEZH2 or control vector. 1 × 104 of cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Replating was performed for three times. Colony counts for each round are shown. N = 2 for each. (c) Leukemic fusion gene transduced cells or cKit-BM cells were transduced with pSIREN-RetroQ control or shp16, and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The cells were transduced with pSIREN-ZsGreen-shEZH2 or control vector. 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. N = 2 for each. (d) Mice were injected intravenously with 1 × 105 or 1 × 106 of TPAE leukemic cells. From 7 days after injection, mice were treated intraperitoneally with DZNep, 2 mg/kg 3 days per week, until death. Survival of the mice in both groups is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 5 for 1 × 105 and N = 8 for 1 × 106 cohort. (e) Total BM cells of TPAE leukemic mice (injected with 1 × 106 cells) treated with DDW or DZNep were collected and lysed at 15 days after transplantation. Immunoblot analysis was performed for H3K27Me3.
Mentions: Next, we studied whether anti-leukemic effect for MLL fusion leukemia by EZH2 inhibition can be applied to various kinds of leukemia. We exposed several human leukemia cell lines to DZNep. K562, HEL, Kasumi-1 and ME-1 cells, all of which do not harbor MLL fusion genes were not sensitive to low concentration of DZNep while both of MLL leukemia cell lines Mv4-11 and MOLM13 were sensitive to 50 nM of DZNep (Fig.4a). Knockdown of EZH2 led to minimal effect on E2A/HLF transduced BM cells or c-Kit positive BM cells as we previously reported,32 while colony forming capacity of MLL/ENL and Hoxa9/Meis1 transduced cells were strongly suppressed (Fig.4b). Knockdown of p16 rescued colony forming capacity of MLL/ENL or Hoxa9/Meis1 transduced cells by EZH2 inhibition (Fig.4c). Expression levels of p16 were not influenced by knockdown of EZH2 in the BM cells transduced with leukemia fusion genes other than MLL/ENL (Fig. S5). In addition, survival of mice transplanted with leukemia cells induced by TPAE is not prolonged by DZNep administration (Fig.4d), although H3K27 were globally hypomethylated (Fig.4e). These data imply that anti-leukemic effect of EZH2 inhibition through p16 upregulation is specific for MLL fusion leukemia.

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