Limits...
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.

Show MeSH

Related in: MedlinePlus

Unique H3K9 epigenomic signature at MLL-AF9 bound gene loci in MLL-fusion leukemia. (a,b) Scatterplots showing ChIP-seq signals for H3K79me2 (x-axis) and H3K9ac (y-axis) in mouse (a) MLL-AF9 leukemic cells, and (b) LSK cells sorted from normal mouse bone marrow. Hoxa cluster genes and Meis1 are highlighted in black circles. (c,h) Screen shots showing ChIP-seq profiles of MLL-AF9 fusion protein (black), H3K79me2 (blue) and H3K9ac (red) at select MLL-AF9 bound target (HOXA cluster and MEIS1), active gene (GAPDH) and silent gene (HBB/OLFR) loci in (c) mouse and (h) human MLL-AF9 leukemic cell lines. The core occupied regions for MLL-AF9 fusion protein in mouse MLL-AF9 leukemia are highlighted (c; green-dashed box). (d–g) Boxplots showing ChIP-seq signal of H3K9ac in (d) mouse and (e–g) human MLL-AF9 leukemic cells including (e) Molm13, (f) Nomo1 and (g) MonoMac6 cells. (a,b,d–g) Data showing ChIP-seq signals of H3K79me2 or H3K9ac at TSS ± 2 kb regions of genome (gray; 18,240 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes). (d–g) Data represent mean ± s.d. *P < 0.001 to MLL-AF9 targets using Welch’s t-test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4390532&req=5

Figure 4: Unique H3K9 epigenomic signature at MLL-AF9 bound gene loci in MLL-fusion leukemia. (a,b) Scatterplots showing ChIP-seq signals for H3K79me2 (x-axis) and H3K9ac (y-axis) in mouse (a) MLL-AF9 leukemic cells, and (b) LSK cells sorted from normal mouse bone marrow. Hoxa cluster genes and Meis1 are highlighted in black circles. (c,h) Screen shots showing ChIP-seq profiles of MLL-AF9 fusion protein (black), H3K79me2 (blue) and H3K9ac (red) at select MLL-AF9 bound target (HOXA cluster and MEIS1), active gene (GAPDH) and silent gene (HBB/OLFR) loci in (c) mouse and (h) human MLL-AF9 leukemic cell lines. The core occupied regions for MLL-AF9 fusion protein in mouse MLL-AF9 leukemia are highlighted (c; green-dashed box). (d–g) Boxplots showing ChIP-seq signal of H3K9ac in (d) mouse and (e–g) human MLL-AF9 leukemic cells including (e) Molm13, (f) Nomo1 and (g) MonoMac6 cells. (a,b,d–g) Data showing ChIP-seq signals of H3K79me2 or H3K9ac at TSS ± 2 kb regions of genome (gray; 18,240 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes). (d–g) Data represent mean ± s.d. *P < 0.001 to MLL-AF9 targets using Welch’s t-test.

Mentions: Our data identified a significantly stronger loss of H3K9ac at the MLL-AF9 target genes compared to other active gene loci after Dot1L inhibition. Interestingly, the majority of MLL-AF9 target genes in these leukemic cells possessed not only the highest H3K79me2 levels in the genome as previously reported8, but they also showed elevated H3K9ac levels relative to other expressed loci (Fig. 4a,c,d and Supplementary Figs. 7). We also note that H4K16ac (another known Sirt1 substrate) showed the same elevated pattern as H3K9ac (Supplementary Fig. 8). On the contrary, the MLL-AF9 target gene set exhibited a scattered distribution of these two histone marks among expressed genes in normal Lin−Sca1+cKit+ (LSK) hematopoietic stem/progenitor cells (Fig. 4b). Because the leukemic cells used in this study were established through transformation of mouse LSK cells by MLL-AF9, the current analyses suggest that the unique H3K9 epigenomic signature observed at the MLL-AF9 targets in these leukemias is attributable to the presence of MLL-fusion oncoprotein at those loci. Indeed, we found that the genomic regions showing exaggerated H3K9achi status overlapped remarkably with the previously defined MLL-AF9 occupied peaks (Fig. 4c)8. Similarly, the increased H3K9ac level at MLL-AF9 target loci was confirmed in human MLL-AF9 cell line models (Fig. 4e–g). In fact, the two most well-studied MLL-fusion target loci, HOXA cluster and MEIS1, represent two of the most enlarged H3K9achi domains in these MLL-r leukemias (Fig. 4h).


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)

Unique H3K9 epigenomic signature at MLL-AF9 bound gene loci in MLL-fusion leukemia. (a,b) Scatterplots showing ChIP-seq signals for H3K79me2 (x-axis) and H3K9ac (y-axis) in mouse (a) MLL-AF9 leukemic cells, and (b) LSK cells sorted from normal mouse bone marrow. Hoxa cluster genes and Meis1 are highlighted in black circles. (c,h) Screen shots showing ChIP-seq profiles of MLL-AF9 fusion protein (black), H3K79me2 (blue) and H3K9ac (red) at select MLL-AF9 bound target (HOXA cluster and MEIS1), active gene (GAPDH) and silent gene (HBB/OLFR) loci in (c) mouse and (h) human MLL-AF9 leukemic cell lines. The core occupied regions for MLL-AF9 fusion protein in mouse MLL-AF9 leukemia are highlighted (c; green-dashed box). (d–g) Boxplots showing ChIP-seq signal of H3K9ac in (d) mouse and (e–g) human MLL-AF9 leukemic cells including (e) Molm13, (f) Nomo1 and (g) MonoMac6 cells. (a,b,d–g) Data showing ChIP-seq signals of H3K79me2 or H3K9ac at TSS ± 2 kb regions of genome (gray; 18,240 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes). (d–g) Data represent mean ± s.d. *P < 0.001 to MLL-AF9 targets using Welch’s t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4390532&req=5

Figure 4: Unique H3K9 epigenomic signature at MLL-AF9 bound gene loci in MLL-fusion leukemia. (a,b) Scatterplots showing ChIP-seq signals for H3K79me2 (x-axis) and H3K9ac (y-axis) in mouse (a) MLL-AF9 leukemic cells, and (b) LSK cells sorted from normal mouse bone marrow. Hoxa cluster genes and Meis1 are highlighted in black circles. (c,h) Screen shots showing ChIP-seq profiles of MLL-AF9 fusion protein (black), H3K79me2 (blue) and H3K9ac (red) at select MLL-AF9 bound target (HOXA cluster and MEIS1), active gene (GAPDH) and silent gene (HBB/OLFR) loci in (c) mouse and (h) human MLL-AF9 leukemic cell lines. The core occupied regions for MLL-AF9 fusion protein in mouse MLL-AF9 leukemia are highlighted (c; green-dashed box). (d–g) Boxplots showing ChIP-seq signal of H3K9ac in (d) mouse and (e–g) human MLL-AF9 leukemic cells including (e) Molm13, (f) Nomo1 and (g) MonoMac6 cells. (a,b,d–g) Data showing ChIP-seq signals of H3K79me2 or H3K9ac at TSS ± 2 kb regions of genome (gray; 18,240 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes). (d–g) Data represent mean ± s.d. *P < 0.001 to MLL-AF9 targets using Welch’s t-test.
Mentions: Our data identified a significantly stronger loss of H3K9ac at the MLL-AF9 target genes compared to other active gene loci after Dot1L inhibition. Interestingly, the majority of MLL-AF9 target genes in these leukemic cells possessed not only the highest H3K79me2 levels in the genome as previously reported8, but they also showed elevated H3K9ac levels relative to other expressed loci (Fig. 4a,c,d and Supplementary Figs. 7). We also note that H4K16ac (another known Sirt1 substrate) showed the same elevated pattern as H3K9ac (Supplementary Fig. 8). On the contrary, the MLL-AF9 target gene set exhibited a scattered distribution of these two histone marks among expressed genes in normal Lin−Sca1+cKit+ (LSK) hematopoietic stem/progenitor cells (Fig. 4b). Because the leukemic cells used in this study were established through transformation of mouse LSK cells by MLL-AF9, the current analyses suggest that the unique H3K9 epigenomic signature observed at the MLL-AF9 targets in these leukemias is attributable to the presence of MLL-fusion oncoprotein at those loci. Indeed, we found that the genomic regions showing exaggerated H3K9achi status overlapped remarkably with the previously defined MLL-AF9 occupied peaks (Fig. 4c)8. Similarly, the increased H3K9ac level at MLL-AF9 target loci was confirmed in human MLL-AF9 cell line models (Fig. 4e–g). In fact, the two most well-studied MLL-fusion target loci, HOXA cluster and MEIS1, represent two of the most enlarged H3K9achi domains in these MLL-r leukemias (Fig. 4h).

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