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DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia.

Chen CW, Koche RP, Sinha AU, Deshpande AJ, Zhu N, Eng R, Doench JG, Xu H, Chu SH, Qi J, Wang X, Delaney C, Bernt KM, Root DE, Hahn WC, Bradner JE, Armstrong SA - Nat. Med. (2015)

Bottom Line: However, the mechanisms underlying this dependency are unclear.We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL fusion target genes after DOT1L inhibition.These results indicate that the dynamic interplay between chromatin regulators controlling the activation and repression of gene expression could provide novel opportunities for combination therapy.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

ABSTRACT
Rearrangements of MLL (encoding lysine-specific methyltransferase 2A and officially known as KMT2A; herein referred to as MLL to denote the gene associated with mixed-lineage leukemia) generate MLL fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the disruptor of telomeric silencing 1-like histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small-molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and the H3K9 methyltransferase SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced antiproliferative activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling the activation and repression of gene expression could provide novel opportunities for combination therapy.

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SIRT1 activator SRT1720 sensitizes MLL-r leukemia to DOT1L inhibitor EPZ4777. (a–e) Mouse MLL-AF9 leukemic cells were treated with DMSO (blue), SRT1720 alone (1 μM; red), EPZ4777 alone (10 μM; orange) or the combination of SRT1720 plus EPZ4777 (green) for 6 days in tissue culture. (a) RT-qPCR and (b) H3K9ac-ChIP-qPCR for Hoxa7 and Meis1 genes. (c) Kaplan-Meier survival curves of mice transplanted with pretreated cells. (d) Percentage of GFP-positive MLL-AF9 leukemic blasts in the peripheral blood of the mice described in (c) on day 15 post-transplantation. (e) Relative blast colony count of pretreated cells further cultured in methylcellulose without the small molecules. (f,g) Effect of EPZ4777 on the proliferation of (f) mouse MLL-AF9 leukemic cells, and (g) human Molm13 (MLL-AF9), MV4-11 (MLL-AF4), SEMK2 (MLL-AF4), Kasumi-1 (AML1-ETO) and HL-60 leukemic cell lines. Cells were co-treated with the indicated concentration of EPZ4777 and either DMSO (red) or SRT1720 (1 μM; green) for (f) 3, 6 and 10 days, and (g) 17 days, respectively. Data represent the observed values and mean ± s.d. of (a,b,f,g) two and (e) six replicates, and (d) ten mice per group. *P < 0.05 and #P <0.01 compared to DMSO group; **P < 0.05 and ##P < 0.01 compared to both DMSO and EPZ4777 alone groups using (a,b,d,e) Student’s t-test and (c) Mantel-Cox test.
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Figure 6: SIRT1 activator SRT1720 sensitizes MLL-r leukemia to DOT1L inhibitor EPZ4777. (a–e) Mouse MLL-AF9 leukemic cells were treated with DMSO (blue), SRT1720 alone (1 μM; red), EPZ4777 alone (10 μM; orange) or the combination of SRT1720 plus EPZ4777 (green) for 6 days in tissue culture. (a) RT-qPCR and (b) H3K9ac-ChIP-qPCR for Hoxa7 and Meis1 genes. (c) Kaplan-Meier survival curves of mice transplanted with pretreated cells. (d) Percentage of GFP-positive MLL-AF9 leukemic blasts in the peripheral blood of the mice described in (c) on day 15 post-transplantation. (e) Relative blast colony count of pretreated cells further cultured in methylcellulose without the small molecules. (f,g) Effect of EPZ4777 on the proliferation of (f) mouse MLL-AF9 leukemic cells, and (g) human Molm13 (MLL-AF9), MV4-11 (MLL-AF4), SEMK2 (MLL-AF4), Kasumi-1 (AML1-ETO) and HL-60 leukemic cell lines. Cells were co-treated with the indicated concentration of EPZ4777 and either DMSO (red) or SRT1720 (1 μM; green) for (f) 3, 6 and 10 days, and (g) 17 days, respectively. Data represent the observed values and mean ± s.d. of (a,b,f,g) two and (e) six replicates, and (d) ten mice per group. *P < 0.05 and #P <0.01 compared to DMSO group; **P < 0.05 and ##P < 0.01 compared to both DMSO and EPZ4777 alone groups using (a,b,d,e) Student’s t-test and (c) Mantel-Cox test.

Mentions: Based on the enlarged H3K9achi domains observed at HOXA and MEIS1 loci in MLL-AF9 leukemias (Fig. 4c,d), and since Sirt1 is required for deacetylation of H3K9, accumulation of H3K9 methylation and silencing of MLL-AF9 target genes, we explored whether pharmacological activation of Sirt1 enhances efficacy of DOT1L inhibitors against MLL-r leukemias. While EPZ4777 treatment alone suppressed the expression of Hoxa7 and Meis1, the combination of SRT1720 (a potent SIRT1 agonist)44,45 with EPZ4777 further diminished the expression of these two genes (Fig. 6a). Concomitantly, we observed a further reduction of the H3K9ac levels at the TSS of Hoxa7 and Meis1 loci in cells receiving the SRT1720 and EPZ4777 combination treatment (Fig. 6b).


DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia.

Chen CW, Koche RP, Sinha AU, Deshpande AJ, Zhu N, Eng R, Doench JG, Xu H, Chu SH, Qi J, Wang X, Delaney C, Bernt KM, Root DE, Hahn WC, Bradner JE, Armstrong SA - Nat. Med. (2015)

SIRT1 activator SRT1720 sensitizes MLL-r leukemia to DOT1L inhibitor EPZ4777. (a–e) Mouse MLL-AF9 leukemic cells were treated with DMSO (blue), SRT1720 alone (1 μM; red), EPZ4777 alone (10 μM; orange) or the combination of SRT1720 plus EPZ4777 (green) for 6 days in tissue culture. (a) RT-qPCR and (b) H3K9ac-ChIP-qPCR for Hoxa7 and Meis1 genes. (c) Kaplan-Meier survival curves of mice transplanted with pretreated cells. (d) Percentage of GFP-positive MLL-AF9 leukemic blasts in the peripheral blood of the mice described in (c) on day 15 post-transplantation. (e) Relative blast colony count of pretreated cells further cultured in methylcellulose without the small molecules. (f,g) Effect of EPZ4777 on the proliferation of (f) mouse MLL-AF9 leukemic cells, and (g) human Molm13 (MLL-AF9), MV4-11 (MLL-AF4), SEMK2 (MLL-AF4), Kasumi-1 (AML1-ETO) and HL-60 leukemic cell lines. Cells were co-treated with the indicated concentration of EPZ4777 and either DMSO (red) or SRT1720 (1 μM; green) for (f) 3, 6 and 10 days, and (g) 17 days, respectively. Data represent the observed values and mean ± s.d. of (a,b,f,g) two and (e) six replicates, and (d) ten mice per group. *P < 0.05 and #P <0.01 compared to DMSO group; **P < 0.05 and ##P < 0.01 compared to both DMSO and EPZ4777 alone groups using (a,b,d,e) Student’s t-test and (c) Mantel-Cox test.
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Figure 6: SIRT1 activator SRT1720 sensitizes MLL-r leukemia to DOT1L inhibitor EPZ4777. (a–e) Mouse MLL-AF9 leukemic cells were treated with DMSO (blue), SRT1720 alone (1 μM; red), EPZ4777 alone (10 μM; orange) or the combination of SRT1720 plus EPZ4777 (green) for 6 days in tissue culture. (a) RT-qPCR and (b) H3K9ac-ChIP-qPCR for Hoxa7 and Meis1 genes. (c) Kaplan-Meier survival curves of mice transplanted with pretreated cells. (d) Percentage of GFP-positive MLL-AF9 leukemic blasts in the peripheral blood of the mice described in (c) on day 15 post-transplantation. (e) Relative blast colony count of pretreated cells further cultured in methylcellulose without the small molecules. (f,g) Effect of EPZ4777 on the proliferation of (f) mouse MLL-AF9 leukemic cells, and (g) human Molm13 (MLL-AF9), MV4-11 (MLL-AF4), SEMK2 (MLL-AF4), Kasumi-1 (AML1-ETO) and HL-60 leukemic cell lines. Cells were co-treated with the indicated concentration of EPZ4777 and either DMSO (red) or SRT1720 (1 μM; green) for (f) 3, 6 and 10 days, and (g) 17 days, respectively. Data represent the observed values and mean ± s.d. of (a,b,f,g) two and (e) six replicates, and (d) ten mice per group. *P < 0.05 and #P <0.01 compared to DMSO group; **P < 0.05 and ##P < 0.01 compared to both DMSO and EPZ4777 alone groups using (a,b,d,e) Student’s t-test and (c) Mantel-Cox test.
Mentions: Based on the enlarged H3K9achi domains observed at HOXA and MEIS1 loci in MLL-AF9 leukemias (Fig. 4c,d), and since Sirt1 is required for deacetylation of H3K9, accumulation of H3K9 methylation and silencing of MLL-AF9 target genes, we explored whether pharmacological activation of Sirt1 enhances efficacy of DOT1L inhibitors against MLL-r leukemias. While EPZ4777 treatment alone suppressed the expression of Hoxa7 and Meis1, the combination of SRT1720 (a potent SIRT1 agonist)44,45 with EPZ4777 further diminished the expression of these two genes (Fig. 6a). Concomitantly, we observed a further reduction of the H3K9ac levels at the TSS of Hoxa7 and Meis1 loci in cells receiving the SRT1720 and EPZ4777 combination treatment (Fig. 6b).

Bottom Line: However, the mechanisms underlying this dependency are unclear.We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL fusion target genes after DOT1L inhibition.These results indicate that the dynamic interplay between chromatin regulators controlling the activation and repression of gene expression could provide novel opportunities for combination therapy.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

ABSTRACT
Rearrangements of MLL (encoding lysine-specific methyltransferase 2A and officially known as KMT2A; herein referred to as MLL to denote the gene associated with mixed-lineage leukemia) generate MLL fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the disruptor of telomeric silencing 1-like histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small-molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and the H3K9 methyltransferase SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced antiproliferative activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling the activation and repression of gene expression could provide novel opportunities for combination therapy.

Show MeSH
Related in: MedlinePlus