Figure 3.
HDAC8 inhibits p53 activity to promote leukemia maintenance and TKI resistance. (A-C) MV4-11 cells were treated with dimethyl sulfoxide or 22d (10 μM) for 18 hours and then subjected to RNA-sequencing analysis. Differentially expressed genes were identified (P < .001). (A) Cellular functions were predicted on the basis of the overlap of differentially expressed genes with Ingenuity Pathway Analysis (IPA), ranked based on the calculated Z score. Red represents predicted activation, and blue represents predicted inhibition of the respective cellular function category in 22d vs vehicle condition. (B) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed genes. (C) Activation and inhibition of upstream regulators predicted with IPA. The upstream regulators were ranked based on Z score. Red represents predicted activation, and blue represents predicted inhibition of the upstream regulator. (D) p53 shRNA (shp53-1 and shp53-2) or control shRNA (shCtrl) was expressed in MV4-11 and MOLM-13 cells. Viability of cells exposed to different concentrations of 22d for 48 hours was analyzed by a Cell Counting Kit-8 (CCK-8) assay. (E) MV4-11 and MOLM-13 cells were treated with vehicle or AC220 (5 nM) for 24 hours. Protein lysates were immunoprecipitated with p53 antibody and then immunoblotted for HDAC8 and p53. (F) MV4-11 and MOLM-13 cells were treated with AC220 (5 nM), 22d (10 μM), or both for 24 hours and then subjected to western blot to detect the indicated proteins. (G) Primary FLT3-ITD+ AML blasts were treated with AC220 (20 nM), 22d (10 μM), or both for 24 hours and then subjected to western blot to detect the indicated proteins. (H) MV4-11 and MOLM-13 cells were treated with AC220 (5 nM), 22d (10 μM), or both for 24 hours. The relative expression of the indicated p53 target genes was analyzed and compared with AC220 treatment alone. (I) MV4-11 and MOLM-13 cells with p53 knockdown were transduced with vectors expressing WT p53 (p53-WT) or acetylation defect mutant p53 (a p53 mutant with 8 potential acetylation sites mutated; p53-8KR). Viability of cells exposed to 22d for 48 hours was analyzed using a CCK-8 assay. Data are mean ± standard error of the mean. **P < .01, ***P < .001, ****P < .0001 vs AC220;  ^^P < .01,  ^^^P < .001 vs 22d. IC50, half maximum inhibitory concentraton; OE, overexpression.

HDAC8 inhibits p53 activity to promote leukemia maintenance and TKI resistance. (A-C) MV4-11 cells were treated with dimethyl sulfoxide or 22d (10 μM) for 18 hours and then subjected to RNA-sequencing analysis. Differentially expressed genes were identified (P < .001). (A) Cellular functions were predicted on the basis of the overlap of differentially expressed genes with Ingenuity Pathway Analysis (IPA), ranked based on the calculated Z score. Red represents predicted activation, and blue represents predicted inhibition of the respective cellular function category in 22d vs vehicle condition. (B) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed genes. (C) Activation and inhibition of upstream regulators predicted with IPA. The upstream regulators were ranked based on Z score. Red represents predicted activation, and blue represents predicted inhibition of the upstream regulator. (D) p53 shRNA (shp53-1 and shp53-2) or control shRNA (shCtrl) was expressed in MV4-11 and MOLM-13 cells. Viability of cells exposed to different concentrations of 22d for 48 hours was analyzed by a Cell Counting Kit-8 (CCK-8) assay. (E) MV4-11 and MOLM-13 cells were treated with vehicle or AC220 (5 nM) for 24 hours. Protein lysates were immunoprecipitated with p53 antibody and then immunoblotted for HDAC8 and p53. (F) MV4-11 and MOLM-13 cells were treated with AC220 (5 nM), 22d (10 μM), or both for 24 hours and then subjected to western blot to detect the indicated proteins. (G) Primary FLT3-ITD+ AML blasts were treated with AC220 (20 nM), 22d (10 μM), or both for 24 hours and then subjected to western blot to detect the indicated proteins. (H) MV4-11 and MOLM-13 cells were treated with AC220 (5 nM), 22d (10 μM), or both for 24 hours. The relative expression of the indicated p53 target genes was analyzed and compared with AC220 treatment alone. (I) MV4-11 and MOLM-13 cells with p53 knockdown were transduced with vectors expressing WT p53 (p53-WT) or acetylation defect mutant p53 (a p53 mutant with 8 potential acetylation sites mutated; p53-8KR). Viability of cells exposed to 22d for 48 hours was analyzed using a CCK-8 assay. Data are mean ± standard error of the mean. **P < .01, ***P < .001, ****P < .0001 vs AC220;  ^^P < .01,  ^^^P < .001 vs 22d. IC50, half maximum inhibitory concentraton; OE, overexpression.

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