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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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Genome-scale RNAi screen for “antagonists of Dot1L” in MLL-AF9 leukemia. (a) Schematic outline of a genome-scale shRNA library screen coupled with high-throughput sequencing (HiSeq) in mouse MLL-AF9 leukemia cells harboring Dot1lfl/fl alleles and tamoxifen-inducible Cre recombinase (CreER). (b) Genotyping PCR for Dot1L engineered allele and immunoblot for histone H3 modifications in MLL-AF9-Dot1lfl/fl-CreER leukemia cells after tamoxifen-induced Dot1L-excision. (c) Wright-Giemsa stain of MLL-AF9-Dot1lfl/fl-CreER leukemia cells before and after tamoxifen treatment. Scale bar, 20 μm. (d) Cell number expansion of Dot1lfl/fl-CreER (red) and Dot1lwt/wt-CreER (blue) MLL-AF9 leukemia cells cultured in tamoxifen. (e) Volcano plot depicts the changes in representation (x-axis) and significance (y-axis) of each shRNA construct in the screen before versus after tamoxifen-induced Dot1L deletion. Total library (gray; 92,425 shRNA), enriched shRNA (red; more than 4-fold increase and p value ≤ 0.05 in the six replicates; 934 shRNA) and sh-Sirt1 (blue; five shRNA) are highlighted. (f) Relative blast colony number from sh-LUC or sh-Sirt1 transduced MLL-AF9-Dot1lfl/fl-CreER leukemic cells cultured in ethanol (green) or tamoxifen (red). The numbers of blast colonies were normalized to the total colony count in control cultures transduced with the same shRNA construct. Data represent the observed values and mean ± s.d. of (d) three independent experiments and (f) four replicates. *P < 0.01 using Student’s t-test.
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Figure 1: Genome-scale RNAi screen for “antagonists of Dot1L” in MLL-AF9 leukemia. (a) Schematic outline of a genome-scale shRNA library screen coupled with high-throughput sequencing (HiSeq) in mouse MLL-AF9 leukemia cells harboring Dot1lfl/fl alleles and tamoxifen-inducible Cre recombinase (CreER). (b) Genotyping PCR for Dot1L engineered allele and immunoblot for histone H3 modifications in MLL-AF9-Dot1lfl/fl-CreER leukemia cells after tamoxifen-induced Dot1L-excision. (c) Wright-Giemsa stain of MLL-AF9-Dot1lfl/fl-CreER leukemia cells before and after tamoxifen treatment. Scale bar, 20 μm. (d) Cell number expansion of Dot1lfl/fl-CreER (red) and Dot1lwt/wt-CreER (blue) MLL-AF9 leukemia cells cultured in tamoxifen. (e) Volcano plot depicts the changes in representation (x-axis) and significance (y-axis) of each shRNA construct in the screen before versus after tamoxifen-induced Dot1L deletion. Total library (gray; 92,425 shRNA), enriched shRNA (red; more than 4-fold increase and p value ≤ 0.05 in the six replicates; 934 shRNA) and sh-Sirt1 (blue; five shRNA) are highlighted. (f) Relative blast colony number from sh-LUC or sh-Sirt1 transduced MLL-AF9-Dot1lfl/fl-CreER leukemic cells cultured in ethanol (green) or tamoxifen (red). The numbers of blast colonies were normalized to the total colony count in control cultures transduced with the same shRNA construct. Data represent the observed values and mean ± s.d. of (d) three independent experiments and (f) four replicates. *P < 0.01 using Student’s t-test.

Mentions: We hypothesized that there are other effectors required for the decrease in MLL-fusion driven gene expression that occurs upon Dot1L inhibition, and that genetic suppression of these effectors would reverse the antiproliferative effect of DOT1L inactivation in MLL-fusion leukemias. To identify these negative regulators of the DOT1L pathway, or “antagonists of Dot1L,” we conducted a pooled screen by introducing a mouse genome-scale shRNA library (containing 92,425 hairpins targeting 16,924 mouse genes)37,38 into Dot1Lfl/fl-MLL-AF9 leukemic cells8 harboring tamoxifen-inducible Cre recombinase (CreER) (Fig. 1a). We confirmed the bi-allelic excision of Dot1L and loss of H3K79me2 in these cells following induction of Cre recombinase activity by tamoxifen treatment (Fig. 1b). We then assessed the relative frequencies of each integrated shRNA sequence before and after Dot1L gene excision by massively parallel sequencing (Hi-seq). Since inactivation of Dot1L induced myeloid differentiation and severely inhibited proliferation of MLL-AF9 leukemic cells (Fig. 1c,d), shRNA constructs that rendered a growth or survival advantage to these cells were expected to be enriched in the screen after tamoxifen-induced Dot1L deletion. Analyses that compared hairpin frequency on day 9 and day 0 identified 934 significantly enriched shRNA constructs (more than 4-fold increase; p ≤ 0.05) after Dot1L deletion (Fig. 1e and Supplementary Table 3). Remarkably, we found three shRNAs targeting Sirtuin 1 (Sirt1) in this top 1% of enriched hairpins, making Sirt1 our leading candidate “antagonist of Dot1L” in MLL-AF9 leukemia (additional candidates are shown in Supplementary Fig. 1).


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)

Genome-scale RNAi screen for “antagonists of Dot1L” in MLL-AF9 leukemia. (a) Schematic outline of a genome-scale shRNA library screen coupled with high-throughput sequencing (HiSeq) in mouse MLL-AF9 leukemia cells harboring Dot1lfl/fl alleles and tamoxifen-inducible Cre recombinase (CreER). (b) Genotyping PCR for Dot1L engineered allele and immunoblot for histone H3 modifications in MLL-AF9-Dot1lfl/fl-CreER leukemia cells after tamoxifen-induced Dot1L-excision. (c) Wright-Giemsa stain of MLL-AF9-Dot1lfl/fl-CreER leukemia cells before and after tamoxifen treatment. Scale bar, 20 μm. (d) Cell number expansion of Dot1lfl/fl-CreER (red) and Dot1lwt/wt-CreER (blue) MLL-AF9 leukemia cells cultured in tamoxifen. (e) Volcano plot depicts the changes in representation (x-axis) and significance (y-axis) of each shRNA construct in the screen before versus after tamoxifen-induced Dot1L deletion. Total library (gray; 92,425 shRNA), enriched shRNA (red; more than 4-fold increase and p value ≤ 0.05 in the six replicates; 934 shRNA) and sh-Sirt1 (blue; five shRNA) are highlighted. (f) Relative blast colony number from sh-LUC or sh-Sirt1 transduced MLL-AF9-Dot1lfl/fl-CreER leukemic cells cultured in ethanol (green) or tamoxifen (red). The numbers of blast colonies were normalized to the total colony count in control cultures transduced with the same shRNA construct. Data represent the observed values and mean ± s.d. of (d) three independent experiments and (f) four replicates. *P < 0.01 using Student’s t-test.
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Related In: Results  -  Collection

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Figure 1: Genome-scale RNAi screen for “antagonists of Dot1L” in MLL-AF9 leukemia. (a) Schematic outline of a genome-scale shRNA library screen coupled with high-throughput sequencing (HiSeq) in mouse MLL-AF9 leukemia cells harboring Dot1lfl/fl alleles and tamoxifen-inducible Cre recombinase (CreER). (b) Genotyping PCR for Dot1L engineered allele and immunoblot for histone H3 modifications in MLL-AF9-Dot1lfl/fl-CreER leukemia cells after tamoxifen-induced Dot1L-excision. (c) Wright-Giemsa stain of MLL-AF9-Dot1lfl/fl-CreER leukemia cells before and after tamoxifen treatment. Scale bar, 20 μm. (d) Cell number expansion of Dot1lfl/fl-CreER (red) and Dot1lwt/wt-CreER (blue) MLL-AF9 leukemia cells cultured in tamoxifen. (e) Volcano plot depicts the changes in representation (x-axis) and significance (y-axis) of each shRNA construct in the screen before versus after tamoxifen-induced Dot1L deletion. Total library (gray; 92,425 shRNA), enriched shRNA (red; more than 4-fold increase and p value ≤ 0.05 in the six replicates; 934 shRNA) and sh-Sirt1 (blue; five shRNA) are highlighted. (f) Relative blast colony number from sh-LUC or sh-Sirt1 transduced MLL-AF9-Dot1lfl/fl-CreER leukemic cells cultured in ethanol (green) or tamoxifen (red). The numbers of blast colonies were normalized to the total colony count in control cultures transduced with the same shRNA construct. Data represent the observed values and mean ± s.d. of (d) three independent experiments and (f) four replicates. *P < 0.01 using Student’s t-test.
Mentions: We hypothesized that there are other effectors required for the decrease in MLL-fusion driven gene expression that occurs upon Dot1L inhibition, and that genetic suppression of these effectors would reverse the antiproliferative effect of DOT1L inactivation in MLL-fusion leukemias. To identify these negative regulators of the DOT1L pathway, or “antagonists of Dot1L,” we conducted a pooled screen by introducing a mouse genome-scale shRNA library (containing 92,425 hairpins targeting 16,924 mouse genes)37,38 into Dot1Lfl/fl-MLL-AF9 leukemic cells8 harboring tamoxifen-inducible Cre recombinase (CreER) (Fig. 1a). We confirmed the bi-allelic excision of Dot1L and loss of H3K79me2 in these cells following induction of Cre recombinase activity by tamoxifen treatment (Fig. 1b). We then assessed the relative frequencies of each integrated shRNA sequence before and after Dot1L gene excision by massively parallel sequencing (Hi-seq). Since inactivation of Dot1L induced myeloid differentiation and severely inhibited proliferation of MLL-AF9 leukemic cells (Fig. 1c,d), shRNA constructs that rendered a growth or survival advantage to these cells were expected to be enriched in the screen after tamoxifen-induced Dot1L deletion. Analyses that compared hairpin frequency on day 9 and day 0 identified 934 significantly enriched shRNA constructs (more than 4-fold increase; p ≤ 0.05) after Dot1L deletion (Fig. 1e and Supplementary Table 3). Remarkably, we found three shRNAs targeting Sirtuin 1 (Sirt1) in this top 1% of enriched hairpins, making Sirt1 our leading candidate “antagonist of Dot1L” in MLL-AF9 leukemia (additional candidates are shown in Supplementary Fig. 1).

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