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. 2018 Apr 15;24(8):1944-1953.
doi: 10.1158/1078-0432.CCR-17-0443. Epub 2018 Feb 1.

Overcoming Resistance to DNA-Targeted Agents by Epigenetic Activation of Schlafen 11 (SLFN11) Expression with Class I Histone Deacetylase Inhibitors

Sai-Wen Tang #  1 , Anish Thomas #  1 , Junko Murai #  1 , Jane B Trepel  1 , Susan E Bates  1   2 , Vinodh N Rajapakse  1 , Yves Pommier  3
Affiliations

Overcoming Resistance to DNA-Targeted Agents by Epigenetic Activation of Schlafen 11 (SLFN11) Expression with Class I Histone Deacetylase Inhibitors

Sai-Wen Tang et al. Clin Cancer Res. .

Abstract

Purpose: Schlafen 11 (SLFN11), a putative DNA/RNA helicase is a dominant genomic determinant of response to DNA-damaging agents and is frequently not expressed in cancer cells. Whether histone deacetylase (HDAC) inhibitors can be used to release SLFN11 and sensitize SLFN11-inactivated cancers to DNA-targeted agents is tested here.Experimental Design:SLFN11 expression was examined in The Cancer Genome Atlas (TCGA), in cancer cell line databases and in patients treated with romidepsin. Isogenic cells overexpressing or genetically inactivated for SLFN11 were used to investigate the effect of HDAC inhibitors on SLFN11 expression and sensitivity to DNA-damaging agents.Results:SLFN11 expression is suppressed in a broad fraction of common cancers and cancer cell lines. In cancer cells not expressing SLFN11, transfection of SLFN11 sensitized the cells to camptothecin, topotecan, hydroxyurea, and cisplatin but not to paclitaxel. SLFN11 mRNA and protein levels were strongly induced by class I (romidepsin, entinostat), but not class II (roclinostat) HDAC inhibitors in a broad panel of cancer cells. SLFN11 expression was also enhanced in peripheral blood mononuclear cells of patients with circulating cutaneous T-cell lymphoma treated with romidepsin. Consistent with the epigenetic regulation of SLFN11, camptothecin and class I HDAC inhibitors were synergistic in many of the cell lines tested.Conclusions: This study reports the prevalent epigenetic regulation of SLFN11 and the dominant stimulatory effect of HDAC inhibitors on SLFN11 expression. Our results provide a rationale for combining class I HDAC inhibitors and DNA-damaging agents to overcome epigenetic inactivation of SLFN11-mediated resistance to DNA-targeted agents. Clin Cancer Res; 24(8); 1944-53. ©2018 AACR.

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Conflict of interest statement

Conflict of interest: The other authors declare no conflicts of interest

Figures

Fig. 1
Fig. 1
SLFN11 is both repressed and overexpressed across the various cancer types of the TCGA database and is epigenetically regulated in the 1,000 cancer cell line databases. (A) When available, for each tissue type, normal samples are shown in green and cancer samples in red. SLFN11 transcript expression was determined by RNASeq and is represented as the log2 of the RSEM-quantified transcript abundance level. The salmon color shaded area corresponds to average normal transcript levels. Abbreviations: ChRCC: Chromophobe renal cell carcinoma; CCRCC: clear cell renal cell carcinoma; PRCC: papillary renal cell carcinoma; ADC: adenocarcinoma; SCC: squamous cell carcinoma. (B) SLFN11 expression in the cancer cell lines of the CCLE collection (http://www.broadinstitute.org/ccle/) matched with promoter methylation determined from the data from the GDSC database (http://www.cancerrxgene.org/) for the individual common cell lines across the two databases (blue dots) obtained with CellMiner (http://discover.nci.nih.gov/cellminercdb/). Cell lines in groups 1 do not express SLFN11 and have promoter methylation. Cell lines in group 2 do not express SLFN11 presumably due to chromatin epigenetic drivers such as histone deacetylation. Cell lines used in this study are marked in red (see also Supplementary Fig. S1). (C) Scheme for the rationale of the study. Recent studies have shown that SLFN11 can be derepressed by inhibiting DNA methylation and by EZH2 inhibitors (9,13). The present study explores the relevance of epigenetic acetylation by using HDAC inhibitors.
Fig. 2
Fig. 2
Forcing SLFN11 expression sensitizes K562 cells to DNA-damaging drugs. (A) Protein levels of SLFN11 in K562/Vector and K562/SLFN11 cells determined by Western blotting using antibodies against SLFN11 (98 kDa). Actin (42 kDa) was used as loading control. (B) K562/Vector (empty circles) and K562/SLFN11 (solid circles) cells were treated with camptothecin, topotecan, hydroxyurea, cisplatin or paclitaxel for 2 days before assessing cell viability. Representative results in triplicate from three independent experiments are shown as mean ± SD.
Fig. 3
Fig. 3
Class I HDAC inhibitors induce SLFN11 expression. K562 (A) and HT1080 (B) cells were treated with romidepsin, entinostat or rocilinostat for 16 h. Upper panels: SLFN11 mRNA levels were measured by quantitative real-time PCR. The y-axis represents relative SLFN11 expression normalized to untreated cells (0). Representative results in triplicate from three independent experiments are shown as mean ± SD. *, p < 0.05; **, p < 0.001 by t test. Lower panels: SLFN11 protein levels were determined by western blotting using antibodies against SLFN11 (98 kDa). Actin (42 kDa) was used as loading control.
Fig. 4
Fig. 4
SLFN11 expression is induced in response to romidepsin in patient samples. Microarray analysis of total RNA isolated from PBMCs of seven patients with significant CTCL blood involvement. Samples were obtained pre-romidepsin infusion and at 4 h and 24 h after the initiation of romidepsin therapy (at romidepsin infusion end). The y-axis represents relative SLFN11 expression normalized to the samples before romidepsin infusion (open bars).
Fig. 5
Fig. 5
Class I HDAC inhibitors show synergistic effects with camptothecin but not with paclitaxel. K562 (A) and HT1080 (B) cells were pretreated with romidepsin (left), entinostat (middle) or rocilinostat (right) for 16 h, washed and then treated with the indicated concentrations of camptothecin for another 2 days before assessing cell viability (upper panel). Representative results in triplicate from three independent experiments are shown as mean ± SD. Lower panels: graphs of combination index versus Fa (fraction affected) for data points of romidepsin (left), entinostat (middle) or rocilinostat (right) in combination with camptothecin. (C) K562 cells were pretreated with romidepsin (left) or entinostat (right) for 16 h, washed and then treated with camptothecin for another 2 days before assessing cell viability (upper panels). Representative results in triplicate from three independent experiments are shown as mean ± SD. Lower panels: graphs of combination index versus Fa (fraction affected) for data points of romidepsin (left) or entinostat (right) in combination with camptothecin. Shading represents the levels of synergism. Combination index between 0.7 and 0.3, combination index between 0.3 and 0.1, and combination index less than 0.1 indicate synergy, strong synergy, and very strong synergy, respectively.
Fig. 6
Fig. 6
SLFN11 induction is crucial for the synergy of class I HDAC inhibitors with camptothecin. (A) Protein levels of SLFN11 and acetyl histone H3 (K9) in K562 WT and K562/SLFN11-knockout cells determined by Western blotting using antibodies against SLFN11 (98 kDa) and acetyl histone H3 (17 kDa). Actin (42 kDa) was used as loading control. (B) K562 WT (left) and K562/SLFN11-knockout (right) cells were pretreated with entinostat for 16 h, washed and then treated with the indicated concentrations of camptothecin for another 2 days before assessing cell viability (upper panels). Representative results in triplicate from three independent experiments are shown as mean ± SD. Lower panels: graphs of combination index versus Fa (fraction affected) for data points of entinostat in combination with camptothecin.
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