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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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Methylation of H3K9 by Suv39h1 is involved in Sirt1-mediated silencing of the MLL-AF9 leukemic program upon suppression of Dot1L. (a) Scatterplots and boxplots showing changes in ChIP-seq signals for H3K9ac (x-axis; both panels), H3K9me2 (y-axis; left panel) and H3K27me3 (y-axis; right panel) at TSS ± 2 kb regions of genome (gray; 18,240 genes) and MLL-AF9 targets (129 genes) in sh-LUC (red) or sh-Sirt1 (blue) transduced mouse MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. (b) Venn diagram showing the overlap genes in “SIRT1-interacting proteins” and “candidate antagonists of Dot1L”. (c) Effect of EPZ4777 on the proliferation of MLL-AF9 leukemic cells transduced with sh-LUC (green), sh-Sirt1 (red) or sh-Suv39h1 (blue). (d) Immunoblot of Suv39h1, Sirt1, H3K9me2 and histone H3 in MLL-AF9 leukemic cells transduced with sh-LUC or sh-Suv39h1. (e,g,h) ChIP-qPCR of (e) H3K9me2, (g) Sirt1, and (h) Suv39h1 for Hoxa7 and Meis1 gene TSS regions in MLL-AF9 leukemic cells transduced with sh-LUC, sh-Sirt1, or sh-Suv39h1. (f) RT-qPCR of Hoxa7 and Meis1 in sh-LUC or sh-Suv39h1 transduced MLL-AF9 leukemic cells. (i) Bar-graph and boxplot showing changes in ATAC-seq signals at TSS ± 2 kb regions of MLL-AF9 targets (129 genes) in sh-LUC (green), sh-Sirt1 (red) and sh-Suv39h1 (blue) transduced MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. Individual MLL-AF9 target genes in bar-graph are ranked according to ATAC-seq (EPZ/DMSO) ratio in sh-LUC cells from high (left) to low (right). Cells were cultured in the presence of EPZ4777 or DMSO for (a,e–i) 6 days and (c) 9 days, respectively. Data represent the observed values and mean ± s.d. of (c,e) two and (g,h) four replicates, and (f) three independent experiments. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 using (a,i) Welch’s t-test and (c, e–h) Student’s t-test.
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Figure 5: Methylation of H3K9 by Suv39h1 is involved in Sirt1-mediated silencing of the MLL-AF9 leukemic program upon suppression of Dot1L. (a) Scatterplots and boxplots showing changes in ChIP-seq signals for H3K9ac (x-axis; both panels), H3K9me2 (y-axis; left panel) and H3K27me3 (y-axis; right panel) at TSS ± 2 kb regions of genome (gray; 18,240 genes) and MLL-AF9 targets (129 genes) in sh-LUC (red) or sh-Sirt1 (blue) transduced mouse MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. (b) Venn diagram showing the overlap genes in “SIRT1-interacting proteins” and “candidate antagonists of Dot1L”. (c) Effect of EPZ4777 on the proliferation of MLL-AF9 leukemic cells transduced with sh-LUC (green), sh-Sirt1 (red) or sh-Suv39h1 (blue). (d) Immunoblot of Suv39h1, Sirt1, H3K9me2 and histone H3 in MLL-AF9 leukemic cells transduced with sh-LUC or sh-Suv39h1. (e,g,h) ChIP-qPCR of (e) H3K9me2, (g) Sirt1, and (h) Suv39h1 for Hoxa7 and Meis1 gene TSS regions in MLL-AF9 leukemic cells transduced with sh-LUC, sh-Sirt1, or sh-Suv39h1. (f) RT-qPCR of Hoxa7 and Meis1 in sh-LUC or sh-Suv39h1 transduced MLL-AF9 leukemic cells. (i) Bar-graph and boxplot showing changes in ATAC-seq signals at TSS ± 2 kb regions of MLL-AF9 targets (129 genes) in sh-LUC (green), sh-Sirt1 (red) and sh-Suv39h1 (blue) transduced MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. Individual MLL-AF9 target genes in bar-graph are ranked according to ATAC-seq (EPZ/DMSO) ratio in sh-LUC cells from high (left) to low (right). Cells were cultured in the presence of EPZ4777 or DMSO for (a,e–i) 6 days and (c) 9 days, respectively. Data represent the observed values and mean ± s.d. of (c,e) two and (g,h) four replicates, and (f) three independent experiments. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 using (a,i) Welch’s t-test and (c, e–h) Student’s t-test.

Mentions: It has been reported that mammalian SIRT1 physically interacts with other proteins that modify histones and is found in complexes that mediate chromatin silencing. We therefore investigated dimethylation of histone 3 lysine 9 (H3K9me2) and trimethylation of histone 3 lysine 27 (H3K27me3), both of which have been connected to Sirt1-associated gene silencing40–42. Concomitant with the reduction of H3K9ac, inhibition of Dot1L increased the amount of both H3K9me2 and H3K27me3 around the TSS of MLL-AF9 bound genes, suggesting potential involvement of multiple epigenetic repressive mechanisms in silencing the MLL-fusion target loci (Fig. 5a). Interestingly, depletion of Sirt1 completely blocked the removal of H3K9ac and selectively inhibited the accumulation of H3K9me2 at MLL-AF9 targets after Dot1L inhibition, whereas the gain of H3K27me3 at those loci remained largely unaffected (Fig. 5a). Thus Sirt1 function is more critical for H3K9 methylation than for H3K27 methylation subsequent to Dot1L inhibition at the MLL-AF9 targets.


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)

Methylation of H3K9 by Suv39h1 is involved in Sirt1-mediated silencing of the MLL-AF9 leukemic program upon suppression of Dot1L. (a) Scatterplots and boxplots showing changes in ChIP-seq signals for H3K9ac (x-axis; both panels), H3K9me2 (y-axis; left panel) and H3K27me3 (y-axis; right panel) at TSS ± 2 kb regions of genome (gray; 18,240 genes) and MLL-AF9 targets (129 genes) in sh-LUC (red) or sh-Sirt1 (blue) transduced mouse MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. (b) Venn diagram showing the overlap genes in “SIRT1-interacting proteins” and “candidate antagonists of Dot1L”. (c) Effect of EPZ4777 on the proliferation of MLL-AF9 leukemic cells transduced with sh-LUC (green), sh-Sirt1 (red) or sh-Suv39h1 (blue). (d) Immunoblot of Suv39h1, Sirt1, H3K9me2 and histone H3 in MLL-AF9 leukemic cells transduced with sh-LUC or sh-Suv39h1. (e,g,h) ChIP-qPCR of (e) H3K9me2, (g) Sirt1, and (h) Suv39h1 for Hoxa7 and Meis1 gene TSS regions in MLL-AF9 leukemic cells transduced with sh-LUC, sh-Sirt1, or sh-Suv39h1. (f) RT-qPCR of Hoxa7 and Meis1 in sh-LUC or sh-Suv39h1 transduced MLL-AF9 leukemic cells. (i) Bar-graph and boxplot showing changes in ATAC-seq signals at TSS ± 2 kb regions of MLL-AF9 targets (129 genes) in sh-LUC (green), sh-Sirt1 (red) and sh-Suv39h1 (blue) transduced MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. Individual MLL-AF9 target genes in bar-graph are ranked according to ATAC-seq (EPZ/DMSO) ratio in sh-LUC cells from high (left) to low (right). Cells were cultured in the presence of EPZ4777 or DMSO for (a,e–i) 6 days and (c) 9 days, respectively. Data represent the observed values and mean ± s.d. of (c,e) two and (g,h) four replicates, and (f) three independent experiments. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 using (a,i) Welch’s t-test and (c, e–h) Student’s t-test.
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Figure 5: Methylation of H3K9 by Suv39h1 is involved in Sirt1-mediated silencing of the MLL-AF9 leukemic program upon suppression of Dot1L. (a) Scatterplots and boxplots showing changes in ChIP-seq signals for H3K9ac (x-axis; both panels), H3K9me2 (y-axis; left panel) and H3K27me3 (y-axis; right panel) at TSS ± 2 kb regions of genome (gray; 18,240 genes) and MLL-AF9 targets (129 genes) in sh-LUC (red) or sh-Sirt1 (blue) transduced mouse MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. (b) Venn diagram showing the overlap genes in “SIRT1-interacting proteins” and “candidate antagonists of Dot1L”. (c) Effect of EPZ4777 on the proliferation of MLL-AF9 leukemic cells transduced with sh-LUC (green), sh-Sirt1 (red) or sh-Suv39h1 (blue). (d) Immunoblot of Suv39h1, Sirt1, H3K9me2 and histone H3 in MLL-AF9 leukemic cells transduced with sh-LUC or sh-Suv39h1. (e,g,h) ChIP-qPCR of (e) H3K9me2, (g) Sirt1, and (h) Suv39h1 for Hoxa7 and Meis1 gene TSS regions in MLL-AF9 leukemic cells transduced with sh-LUC, sh-Sirt1, or sh-Suv39h1. (f) RT-qPCR of Hoxa7 and Meis1 in sh-LUC or sh-Suv39h1 transduced MLL-AF9 leukemic cells. (i) Bar-graph and boxplot showing changes in ATAC-seq signals at TSS ± 2 kb regions of MLL-AF9 targets (129 genes) in sh-LUC (green), sh-Sirt1 (red) and sh-Suv39h1 (blue) transduced MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. Individual MLL-AF9 target genes in bar-graph are ranked according to ATAC-seq (EPZ/DMSO) ratio in sh-LUC cells from high (left) to low (right). Cells were cultured in the presence of EPZ4777 or DMSO for (a,e–i) 6 days and (c) 9 days, respectively. Data represent the observed values and mean ± s.d. of (c,e) two and (g,h) four replicates, and (f) three independent experiments. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 using (a,i) Welch’s t-test and (c, e–h) Student’s t-test.
Mentions: It has been reported that mammalian SIRT1 physically interacts with other proteins that modify histones and is found in complexes that mediate chromatin silencing. We therefore investigated dimethylation of histone 3 lysine 9 (H3K9me2) and trimethylation of histone 3 lysine 27 (H3K27me3), both of which have been connected to Sirt1-associated gene silencing40–42. Concomitant with the reduction of H3K9ac, inhibition of Dot1L increased the amount of both H3K9me2 and H3K27me3 around the TSS of MLL-AF9 bound genes, suggesting potential involvement of multiple epigenetic repressive mechanisms in silencing the MLL-fusion target loci (Fig. 5a). Interestingly, depletion of Sirt1 completely blocked the removal of H3K9ac and selectively inhibited the accumulation of H3K9me2 at MLL-AF9 targets after Dot1L inhibition, whereas the gain of H3K27me3 at those loci remained largely unaffected (Fig. 5a). Thus Sirt1 function is more critical for H3K9 methylation than for H3K27 methylation subsequent to Dot1L inhibition at the MLL-AF9 targets.

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