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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 mediates the response of MLL-AF9 leukemia cells to DOT1L inhibitor EPZ4777. (a,c,h,i) Effect of EPZ4777 on the proliferation of mouse MLL-AF9 leukemia cells transduced with (a) sh-Sirt1 (red) or sh-LUC (green), (h) MSCV-puro-Meis1 (red), Hoxa7 (blue), or empty vector (green), and (i) MSCV-puro-Meis1 plus MSCV-ires-Tomato-Hoxa7 (red) or dual empty vectors (green), as well as (c) co-treated with Ex527 (red), suramin (blue) or DMSO (green). (b,d) Immunoblot of (b) Sirt1, H3K79me2, histone H3 and tubulin in MLL-AF9 leukemic cells transduced with sh-LUC or sh-Sirt1 and cultured in EPZ4777, and (d) Sirt1 and tubulin in sh-Sirt1 transduced MLL-AF9 cells further infected with MSCV-ires-Tomato empty vector (MIT-Vec) or Sirt1 (MIT-Sirt1) virus. (e) Relative number of the cells described in (d) cultured in EPZ4777 (red) or DMSO (green). (f) Microarray and GSEA analyses showing changes in expression of “EPZ4777_down gene set” (978 genes; Supplementary Table 3) in sh-LUC transduced MLL-AF9 cells cultured in EPZ4777 versus DMSO (left panel), as well as sh-Sirt1 versus sh-LUC transduced MLL-AF9 cells cultured in EPZ4777 (right panel). Heatmaps showing genes comprising the early leading edge (top 50 genes) of the GSEA plots. (g) RT-qPCR of Hoxa7 and Meis1 in sh-LUC or sh-Sirt1 transduced MLL-AF9 leukemic cells cultured in EPZ4777 (red) or DMSO (green). Cells were cultured in the presence of EPZ4777 or DMSO for (f,g) 6 days and (a,c,e,h,i) 9 days, respectively. Data represent the observed values and mean ± s.d. of (a,c,e,h,i) three replicates, and (g) three independent experiments. *P < 0.05; **P < 0.01 to control group using Student’s t-test.
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Figure 2: Sirt1 mediates the response of MLL-AF9 leukemia cells to DOT1L inhibitor EPZ4777. (a,c,h,i) Effect of EPZ4777 on the proliferation of mouse MLL-AF9 leukemia cells transduced with (a) sh-Sirt1 (red) or sh-LUC (green), (h) MSCV-puro-Meis1 (red), Hoxa7 (blue), or empty vector (green), and (i) MSCV-puro-Meis1 plus MSCV-ires-Tomato-Hoxa7 (red) or dual empty vectors (green), as well as (c) co-treated with Ex527 (red), suramin (blue) or DMSO (green). (b,d) Immunoblot of (b) Sirt1, H3K79me2, histone H3 and tubulin in MLL-AF9 leukemic cells transduced with sh-LUC or sh-Sirt1 and cultured in EPZ4777, and (d) Sirt1 and tubulin in sh-Sirt1 transduced MLL-AF9 cells further infected with MSCV-ires-Tomato empty vector (MIT-Vec) or Sirt1 (MIT-Sirt1) virus. (e) Relative number of the cells described in (d) cultured in EPZ4777 (red) or DMSO (green). (f) Microarray and GSEA analyses showing changes in expression of “EPZ4777_down gene set” (978 genes; Supplementary Table 3) in sh-LUC transduced MLL-AF9 cells cultured in EPZ4777 versus DMSO (left panel), as well as sh-Sirt1 versus sh-LUC transduced MLL-AF9 cells cultured in EPZ4777 (right panel). Heatmaps showing genes comprising the early leading edge (top 50 genes) of the GSEA plots. (g) RT-qPCR of Hoxa7 and Meis1 in sh-LUC or sh-Sirt1 transduced MLL-AF9 leukemic cells cultured in EPZ4777 (red) or DMSO (green). Cells were cultured in the presence of EPZ4777 or DMSO for (f,g) 6 days and (a,c,e,h,i) 9 days, respectively. Data represent the observed values and mean ± s.d. of (a,c,e,h,i) three replicates, and (g) three independent experiments. *P < 0.05; **P < 0.01 to control group using Student’s t-test.

Mentions: To validate our genome-scale shRNA library screen results, we assessed whether the Sirt1 shRNAs that were selected for in the screen also suppressed Sirt1 expression. We also performed colony-forming assays. We found that the three shRNAs selected for in the screen suppressed Sirt1 expression and depletion of Sirt1 by these individual shRNAs (sh-Sirt1) maintained more MLL-AF9 driven blast-like colonies after Dot1L deletion, as compared to the control cultures transduced with sh-LUC (Fig. 1f and Supplementary Fig. 1c, d). Of note, depletion of Sirt1 alone did not influence the proliferation and blast-like colony potential of these leukemic cells. Additionally, we subjected the MLL-AF9 leukemia cells to EPZ4777, a selective small molecular DOT1L inhibitor29, and found that suppression of Sirt1 in MLL-AF9 leukemic cells reduced their sensitivity to DOT1L inhibition (Fig. 2a,b and Supplementary Fig. 2). Similarly, small molecule inhibitors of SIRT1 including Ex527 and suramin39 desensitized MLL-AF9 leukemic cells to Dot1L inhibition, suggesting that Sirt1’s enzymatic activity is important for the suppression of MLL-AF9 leukemic cells caused by DOT1L inhibition (Fig. 2c). On the other hand, forced expression of Sirt1 by retroviral transduction re-sensitized the Sirt1 knockdown cells to EPZ4777 treatment (Fig. 2d,e).


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 mediates the response of MLL-AF9 leukemia cells to DOT1L inhibitor EPZ4777. (a,c,h,i) Effect of EPZ4777 on the proliferation of mouse MLL-AF9 leukemia cells transduced with (a) sh-Sirt1 (red) or sh-LUC (green), (h) MSCV-puro-Meis1 (red), Hoxa7 (blue), or empty vector (green), and (i) MSCV-puro-Meis1 plus MSCV-ires-Tomato-Hoxa7 (red) or dual empty vectors (green), as well as (c) co-treated with Ex527 (red), suramin (blue) or DMSO (green). (b,d) Immunoblot of (b) Sirt1, H3K79me2, histone H3 and tubulin in MLL-AF9 leukemic cells transduced with sh-LUC or sh-Sirt1 and cultured in EPZ4777, and (d) Sirt1 and tubulin in sh-Sirt1 transduced MLL-AF9 cells further infected with MSCV-ires-Tomato empty vector (MIT-Vec) or Sirt1 (MIT-Sirt1) virus. (e) Relative number of the cells described in (d) cultured in EPZ4777 (red) or DMSO (green). (f) Microarray and GSEA analyses showing changes in expression of “EPZ4777_down gene set” (978 genes; Supplementary Table 3) in sh-LUC transduced MLL-AF9 cells cultured in EPZ4777 versus DMSO (left panel), as well as sh-Sirt1 versus sh-LUC transduced MLL-AF9 cells cultured in EPZ4777 (right panel). Heatmaps showing genes comprising the early leading edge (top 50 genes) of the GSEA plots. (g) RT-qPCR of Hoxa7 and Meis1 in sh-LUC or sh-Sirt1 transduced MLL-AF9 leukemic cells cultured in EPZ4777 (red) or DMSO (green). Cells were cultured in the presence of EPZ4777 or DMSO for (f,g) 6 days and (a,c,e,h,i) 9 days, respectively. Data represent the observed values and mean ± s.d. of (a,c,e,h,i) three replicates, and (g) three independent experiments. *P < 0.05; **P < 0.01 to control group using Student’s t-test.
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Related In: Results  -  Collection

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Figure 2: Sirt1 mediates the response of MLL-AF9 leukemia cells to DOT1L inhibitor EPZ4777. (a,c,h,i) Effect of EPZ4777 on the proliferation of mouse MLL-AF9 leukemia cells transduced with (a) sh-Sirt1 (red) or sh-LUC (green), (h) MSCV-puro-Meis1 (red), Hoxa7 (blue), or empty vector (green), and (i) MSCV-puro-Meis1 plus MSCV-ires-Tomato-Hoxa7 (red) or dual empty vectors (green), as well as (c) co-treated with Ex527 (red), suramin (blue) or DMSO (green). (b,d) Immunoblot of (b) Sirt1, H3K79me2, histone H3 and tubulin in MLL-AF9 leukemic cells transduced with sh-LUC or sh-Sirt1 and cultured in EPZ4777, and (d) Sirt1 and tubulin in sh-Sirt1 transduced MLL-AF9 cells further infected with MSCV-ires-Tomato empty vector (MIT-Vec) or Sirt1 (MIT-Sirt1) virus. (e) Relative number of the cells described in (d) cultured in EPZ4777 (red) or DMSO (green). (f) Microarray and GSEA analyses showing changes in expression of “EPZ4777_down gene set” (978 genes; Supplementary Table 3) in sh-LUC transduced MLL-AF9 cells cultured in EPZ4777 versus DMSO (left panel), as well as sh-Sirt1 versus sh-LUC transduced MLL-AF9 cells cultured in EPZ4777 (right panel). Heatmaps showing genes comprising the early leading edge (top 50 genes) of the GSEA plots. (g) RT-qPCR of Hoxa7 and Meis1 in sh-LUC or sh-Sirt1 transduced MLL-AF9 leukemic cells cultured in EPZ4777 (red) or DMSO (green). Cells were cultured in the presence of EPZ4777 or DMSO for (f,g) 6 days and (a,c,e,h,i) 9 days, respectively. Data represent the observed values and mean ± s.d. of (a,c,e,h,i) three replicates, and (g) three independent experiments. *P < 0.05; **P < 0.01 to control group using Student’s t-test.
Mentions: To validate our genome-scale shRNA library screen results, we assessed whether the Sirt1 shRNAs that were selected for in the screen also suppressed Sirt1 expression. We also performed colony-forming assays. We found that the three shRNAs selected for in the screen suppressed Sirt1 expression and depletion of Sirt1 by these individual shRNAs (sh-Sirt1) maintained more MLL-AF9 driven blast-like colonies after Dot1L deletion, as compared to the control cultures transduced with sh-LUC (Fig. 1f and Supplementary Fig. 1c, d). Of note, depletion of Sirt1 alone did not influence the proliferation and blast-like colony potential of these leukemic cells. Additionally, we subjected the MLL-AF9 leukemia cells to EPZ4777, a selective small molecular DOT1L inhibitor29, and found that suppression of Sirt1 in MLL-AF9 leukemic cells reduced their sensitivity to DOT1L inhibition (Fig. 2a,b and Supplementary Fig. 2). Similarly, small molecule inhibitors of SIRT1 including Ex527 and suramin39 desensitized MLL-AF9 leukemic cells to Dot1L inhibition, suggesting that Sirt1’s enzymatic activity is important for the suppression of MLL-AF9 leukemic cells caused by DOT1L inhibition (Fig. 2c). On the other hand, forced expression of Sirt1 by retroviral transduction re-sensitized the Sirt1 knockdown cells to EPZ4777 treatment (Fig. 2d,e).

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