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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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EZH2 is enriched at the TSS of p16 in MLL fusion leukemia. (a) Relative expression of EZH2 and p16 in immortalized cells harboring several types of leukemic fusion genes. N = 4 for all fusion genes. (b) Relative expression of EZH2 and p16 in BM cells of wild type mice (Wt BM) or mice harboring several kinds of leukemic fusion genes. N = 4 for Wt BM, N = 3 for MLL/ENL and N = 2 for TPAE. (c) 5 × 105 of MLL/ENL, Hoxa9/Meis1 or E2A/HLF transduced cells were harvested from methocult M3434 for ChIP assays using indicated antibodies. N = 3 for each. (d) Relative expression of p16 and Hoxa9 in MLL/ENL and Hoxa9/Meis1 immortalized cells. N = 4 for each.
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fig05: EZH2 is enriched at the TSS of p16 in MLL fusion leukemia. (a) Relative expression of EZH2 and p16 in immortalized cells harboring several types of leukemic fusion genes. N = 4 for all fusion genes. (b) Relative expression of EZH2 and p16 in BM cells of wild type mice (Wt BM) or mice harboring several kinds of leukemic fusion genes. N = 4 for Wt BM, N = 3 for MLL/ENL and N = 2 for TPAE. (c) 5 × 105 of MLL/ENL, Hoxa9/Meis1 or E2A/HLF transduced cells were harvested from methocult M3434 for ChIP assays using indicated antibodies. N = 3 for each. (d) Relative expression of p16 and Hoxa9 in MLL/ENL and Hoxa9/Meis1 immortalized cells. N = 4 for each.

Mentions: To clarify the molecular mechanism of p16 regulation by EZH2 in MLL fusion leukemia, we investigated p16 transcription levels and epigenetic status of p16 promoter in MLL/ENL, Hoxa9/Meis1 and E2A/HLF transduced cells. Among several types of immortalized cells and leukemia cells, the transcript level of p16 is remarkably low in MLL/ENL immortalized or leukemia cells (Fig.5a,b). This suggests existence of specific mechanism of p16 suppression in MLL fusion leukemia and we speculated that difference in epigenetic status between MLL fusion leukemia and other types of leukemia is a possible cause. The promoter of p16 is suggested to locate around the transcription-start-site (TSS), and a previous study using mouse embryonic fibroblasts revealed that H3K27 methylation status around this region is altered by OIS.37 As expected, our ChIP assay revealed that EZH2 is enriched at the p16 TSS together with Bmi1 and H3K27Me3 in MLL/ENL or Hoxa9/Meis1 transduced cells, while they are not enriched at the p16 TSS of E2A/HLF transduced cells (Fig.5c, Fig. S6). In contrast, H3K4Me3 was enriched at the TSS of p16, not only in MLL/ENL and Hoxa9/Meis1 cells, but also in E2A/HLF cells (Fig. S6), which is consistent with the report that H3K4Me3 in the p16 TSS is an initial event after transduction of oncogene leading to p16 upregulation.37,39 Our data suggest that enriched EZH2 at the p16 TSS maintains H3K27Me3 methylation status against p16 upregulation, leading to suppressed expression of p16 compared with other types of leukemia. Recently, Hoxa9 is reported to be important to recruit EZH2 to the TSS of p16.40 In fact, Hoxa9/Meis1 transduced cells which mimic downstream signaling of MLL fusion leukemia displayed similar behavior of EZH2 and H3K27Me3 on the p16 gene (Fig.5c). At this point, our data are compatible to the previous report showing a role of Hoxa9 to recruit EZH2 to p16 gene. However, despite the higher expression of Hoxa9 in Hoxa9/Meis1 transduced cells compared with MLL/ENL transduced cells, the expression of p16 in Hoxa9/Meis1 transduced cells is higher than that of MLL/ENL transduced cells (Fig.5d). These data suggest that there may be some specific mechanisms other than Hoxa9-mediated recruitment of EZH2 to suppress p16 in MLL/ENL transduced cells.


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)

EZH2 is enriched at the TSS of p16 in MLL fusion leukemia. (a) Relative expression of EZH2 and p16 in immortalized cells harboring several types of leukemic fusion genes. N = 4 for all fusion genes. (b) Relative expression of EZH2 and p16 in BM cells of wild type mice (Wt BM) or mice harboring several kinds of leukemic fusion genes. N = 4 for Wt BM, N = 3 for MLL/ENL and N = 2 for TPAE. (c) 5 × 105 of MLL/ENL, Hoxa9/Meis1 or E2A/HLF transduced cells were harvested from methocult M3434 for ChIP assays using indicated antibodies. N = 3 for each. (d) Relative expression of p16 and Hoxa9 in MLL/ENL and Hoxa9/Meis1 immortalized cells. N = 4 for each.
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fig05: EZH2 is enriched at the TSS of p16 in MLL fusion leukemia. (a) Relative expression of EZH2 and p16 in immortalized cells harboring several types of leukemic fusion genes. N = 4 for all fusion genes. (b) Relative expression of EZH2 and p16 in BM cells of wild type mice (Wt BM) or mice harboring several kinds of leukemic fusion genes. N = 4 for Wt BM, N = 3 for MLL/ENL and N = 2 for TPAE. (c) 5 × 105 of MLL/ENL, Hoxa9/Meis1 or E2A/HLF transduced cells were harvested from methocult M3434 for ChIP assays using indicated antibodies. N = 3 for each. (d) Relative expression of p16 and Hoxa9 in MLL/ENL and Hoxa9/Meis1 immortalized cells. N = 4 for each.
Mentions: To clarify the molecular mechanism of p16 regulation by EZH2 in MLL fusion leukemia, we investigated p16 transcription levels and epigenetic status of p16 promoter in MLL/ENL, Hoxa9/Meis1 and E2A/HLF transduced cells. Among several types of immortalized cells and leukemia cells, the transcript level of p16 is remarkably low in MLL/ENL immortalized or leukemia cells (Fig.5a,b). This suggests existence of specific mechanism of p16 suppression in MLL fusion leukemia and we speculated that difference in epigenetic status between MLL fusion leukemia and other types of leukemia is a possible cause. The promoter of p16 is suggested to locate around the transcription-start-site (TSS), and a previous study using mouse embryonic fibroblasts revealed that H3K27 methylation status around this region is altered by OIS.37 As expected, our ChIP assay revealed that EZH2 is enriched at the p16 TSS together with Bmi1 and H3K27Me3 in MLL/ENL or Hoxa9/Meis1 transduced cells, while they are not enriched at the p16 TSS of E2A/HLF transduced cells (Fig.5c, Fig. S6). In contrast, H3K4Me3 was enriched at the TSS of p16, not only in MLL/ENL and Hoxa9/Meis1 cells, but also in E2A/HLF cells (Fig. S6), which is consistent with the report that H3K4Me3 in the p16 TSS is an initial event after transduction of oncogene leading to p16 upregulation.37,39 Our data suggest that enriched EZH2 at the p16 TSS maintains H3K27Me3 methylation status against p16 upregulation, leading to suppressed expression of p16 compared with other types of leukemia. Recently, Hoxa9 is reported to be important to recruit EZH2 to the TSS of p16.40 In fact, Hoxa9/Meis1 transduced cells which mimic downstream signaling of MLL fusion leukemia displayed similar behavior of EZH2 and H3K27Me3 on the p16 gene (Fig.5c). At this point, our data are compatible to the previous report showing a role of Hoxa9 to recruit EZH2 to p16 gene. However, despite the higher expression of Hoxa9 in Hoxa9/Meis1 transduced cells compared with MLL/ENL transduced cells, the expression of p16 in Hoxa9/Meis1 transduced cells is higher than that of MLL/ENL transduced cells (Fig.5d). These data suggest that there may be some specific mechanisms other than Hoxa9-mediated recruitment of EZH2 to suppress p16 in MLL/ENL transduced cells.

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