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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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Administration of EZH2 inhibitor is therapeutically effective in MLL fusion leukemic mouse models. (a) Mice were injected intravenously with 1 × 104 of leukemia 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 (DDW as vehicle control and DZNep) is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 7 for each group. (b) Total BM cells were collected and lysed at 21 days after transplantation. Immunoblot analysis was performed for H3K27Me3. The levels of total H3 served as the loading control. Values indicate relative density scale. (c) GFP positive BM cells were sorted and analyzed by qRT-PCR at 21 days after transplantation. Relative expression of EZH2 and its target genes are shown. P-values were calculated by unpaired T test. N = 6 for DDW group and N = 5 for DZNep group. (d) Immunoblot analysis was performed for p16. The levels of β-actin served as the loading control. (e) The BM cells of MLL/AF9 leukemic mice treated with DDW, DZNep or AraC (100 mg/kg intraperitoneally from day 14–18) were collected and analyzed by flow cytometry at 21 days after transplantation. The percentages of L-GMP in GFP positive cells are shown (N = 6, 4 and 5). P-values were calculated by unpaired T test. (f) GFP positive BM cells were sorted and limiting dilution assay was performed. Poisson distribution of LIC frequency is shown. P-value was calculated by chi-square test. (g) 1 × 104 of sorted GFP 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 = 4 for each.
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fig01: Administration of EZH2 inhibitor is therapeutically effective in MLL fusion leukemic mouse models. (a) Mice were injected intravenously with 1 × 104 of leukemia 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 (DDW as vehicle control and DZNep) is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 7 for each group. (b) Total BM cells were collected and lysed at 21 days after transplantation. Immunoblot analysis was performed for H3K27Me3. The levels of total H3 served as the loading control. Values indicate relative density scale. (c) GFP positive BM cells were sorted and analyzed by qRT-PCR at 21 days after transplantation. Relative expression of EZH2 and its target genes are shown. P-values were calculated by unpaired T test. N = 6 for DDW group and N = 5 for DZNep group. (d) Immunoblot analysis was performed for p16. The levels of β-actin served as the loading control. (e) The BM cells of MLL/AF9 leukemic mice treated with DDW, DZNep or AraC (100 mg/kg intraperitoneally from day 14–18) were collected and analyzed by flow cytometry at 21 days after transplantation. The percentages of L-GMP in GFP positive cells are shown (N = 6, 4 and 5). P-values were calculated by unpaired T test. (f) GFP positive BM cells were sorted and limiting dilution assay was performed. Poisson distribution of LIC frequency is shown. P-value was calculated by chi-square test. (g) 1 × 104 of sorted GFP 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 = 4 for each.

Mentions: To clarify whether in vivo administration of EZH2 inhibitor has therapeutically effective against MLL fusion leukemic mice, we employed DZNep, an inhibitor for H3K27 methyltransferase which is feasible for in vivo administration.26 First, we administered DZNep intraperitoneally to MLL fusion leukemia mouse models and analyzed epigenetic and transcriptional changes in the BM cells. In vivo administration of DZNep prolonged survival of MLL/ENL and MLL/AF9 leukemic mice (Fig.1a). The BM cells of mice treated with DZNep were globally hypomethylated at H3K27 (Fig.1b). Next, we investigated the expression of specific genes concerning apoptosis and cellular senescence that are known to be targets of EZH2.26,27,33 As previously reported,34 DZNep did not suppress EZH2 transcript level (Fig. S1a) but decreased protein level (Fig. S1b). Most of the genes tested showed a tendency to upregulation by DZNep treatment in vitro (Fig. S1a), among which only p16 was significantly upregulated by in vivo administration of DZNep (Fig.1c,d). p16 is known to be a tumor suppressor which is typified by association with oncogene-induced senescence (OIS) and is epigenetically regulated by PRC2 and PRC1.35–37 Loss of epigenetic suppression of p16 is reported to induce depletion of stem cells.38 Thus, we investigated whether LIC ratio in MLL/AF9 leukemic mice is influenced by administration of DZNep. The LICs in MLL/AF9 leukemic mice are known to be enriched in L-GMP fraction.31 In FACS analysis, the ratios of L-GMP in mice treated with DZNep were significantly decreased compared with control mice, while administration of typical cytotoxic agent did not reduce them (Fig.1e, Fig. S2). Moreover, the BM cells from MLL/AF9 leukemic mice treated with DZNep had impaired capacity to reconstitute leukemia in vivo and attenuated colony forming capacity in vitro (Fig.1f,g, Fig. S3). These data suggest that LIC are decreased by DZNep administration in MLL/AF9 leukemic mice.


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)

Administration of EZH2 inhibitor is therapeutically effective in MLL fusion leukemic mouse models. (a) Mice were injected intravenously with 1 × 104 of leukemia 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 (DDW as vehicle control and DZNep) is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 7 for each group. (b) Total BM cells were collected and lysed at 21 days after transplantation. Immunoblot analysis was performed for H3K27Me3. The levels of total H3 served as the loading control. Values indicate relative density scale. (c) GFP positive BM cells were sorted and analyzed by qRT-PCR at 21 days after transplantation. Relative expression of EZH2 and its target genes are shown. P-values were calculated by unpaired T test. N = 6 for DDW group and N = 5 for DZNep group. (d) Immunoblot analysis was performed for p16. The levels of β-actin served as the loading control. (e) The BM cells of MLL/AF9 leukemic mice treated with DDW, DZNep or AraC (100 mg/kg intraperitoneally from day 14–18) were collected and analyzed by flow cytometry at 21 days after transplantation. The percentages of L-GMP in GFP positive cells are shown (N = 6, 4 and 5). P-values were calculated by unpaired T test. (f) GFP positive BM cells were sorted and limiting dilution assay was performed. Poisson distribution of LIC frequency is shown. P-value was calculated by chi-square test. (g) 1 × 104 of sorted GFP 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 = 4 for each.
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Related In: Results  -  Collection

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fig01: Administration of EZH2 inhibitor is therapeutically effective in MLL fusion leukemic mouse models. (a) Mice were injected intravenously with 1 × 104 of leukemia 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 (DDW as vehicle control and DZNep) is represented by Kaplan–Meier plot. P-values were calculated by log-rank test. N = 7 for each group. (b) Total BM cells were collected and lysed at 21 days after transplantation. Immunoblot analysis was performed for H3K27Me3. The levels of total H3 served as the loading control. Values indicate relative density scale. (c) GFP positive BM cells were sorted and analyzed by qRT-PCR at 21 days after transplantation. Relative expression of EZH2 and its target genes are shown. P-values were calculated by unpaired T test. N = 6 for DDW group and N = 5 for DZNep group. (d) Immunoblot analysis was performed for p16. The levels of β-actin served as the loading control. (e) The BM cells of MLL/AF9 leukemic mice treated with DDW, DZNep or AraC (100 mg/kg intraperitoneally from day 14–18) were collected and analyzed by flow cytometry at 21 days after transplantation. The percentages of L-GMP in GFP positive cells are shown (N = 6, 4 and 5). P-values were calculated by unpaired T test. (f) GFP positive BM cells were sorted and limiting dilution assay was performed. Poisson distribution of LIC frequency is shown. P-value was calculated by chi-square test. (g) 1 × 104 of sorted GFP 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 = 4 for each.
Mentions: To clarify whether in vivo administration of EZH2 inhibitor has therapeutically effective against MLL fusion leukemic mice, we employed DZNep, an inhibitor for H3K27 methyltransferase which is feasible for in vivo administration.26 First, we administered DZNep intraperitoneally to MLL fusion leukemia mouse models and analyzed epigenetic and transcriptional changes in the BM cells. In vivo administration of DZNep prolonged survival of MLL/ENL and MLL/AF9 leukemic mice (Fig.1a). The BM cells of mice treated with DZNep were globally hypomethylated at H3K27 (Fig.1b). Next, we investigated the expression of specific genes concerning apoptosis and cellular senescence that are known to be targets of EZH2.26,27,33 As previously reported,34 DZNep did not suppress EZH2 transcript level (Fig. S1a) but decreased protein level (Fig. S1b). Most of the genes tested showed a tendency to upregulation by DZNep treatment in vitro (Fig. S1a), among which only p16 was significantly upregulated by in vivo administration of DZNep (Fig.1c,d). p16 is known to be a tumor suppressor which is typified by association with oncogene-induced senescence (OIS) and is epigenetically regulated by PRC2 and PRC1.35–37 Loss of epigenetic suppression of p16 is reported to induce depletion of stem cells.38 Thus, we investigated whether LIC ratio in MLL/AF9 leukemic mice is influenced by administration of DZNep. The LICs in MLL/AF9 leukemic mice are known to be enriched in L-GMP fraction.31 In FACS analysis, the ratios of L-GMP in mice treated with DZNep were significantly decreased compared with control mice, while administration of typical cytotoxic agent did not reduce them (Fig.1e, Fig. S2). Moreover, the BM cells from MLL/AF9 leukemic mice treated with DZNep had impaired capacity to reconstitute leukemia in vivo and attenuated colony forming capacity in vitro (Fig.1f,g, Fig. S3). These data suggest that LIC are decreased by DZNep administration in MLL/AF9 leukemic mice.

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