ARID1A-mutated ovarian cancers depend on HDAC6 activity

Abstract

ARID1A, encoding a subunit of the SWI/SNF chromatin-remodelling complex, is the most frequently mutated epigenetic regulator across all human cancers. ARID1A and TP53 mutations are typically mutually exclusive. Therapeutic approaches that correlate with this genetic characteristic remain to be explored. Here, we show that HDAC6 activity is essential in ARID1A-mutated ovarian cancers. Inhibition of HDAC6 activity using a clinically applicable small-molecule inhibitor significantly improved the survival of mice bearing ARID1A-mutated tumours. This correlated with the suppression of growth and dissemination of ARID1A-mutated, but not wild-type, tumours. The dependence on HDAC6 activity in ARID1A-mutated cells correlated with a direct transcriptional repression of HDAC6 by ARID1A. HDAC6 inhibition selectively promoted apoptosis of ARID1A-mutated cells. HDAC6 directly deacetylates Lys120 of p53, a pro-apoptotic post-translational modification. Thus, ARID1A mutation inactivates the apoptosis-promoting function of p53 by upregulating HDAC6. Together, these results indicate that pharmacological inhibition of HDAC6 is a therapeutic strategy for ARID1A-mutated cancers.

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Figure 1: ARID1A-inactivated cells are selectively sensitive to HDAC6 knockdown.
Figure 2: The selectivity against ARID1A mutation depends on the enzymatic activity of HDAC6.
Figure 3: HDAC6 inhibition induces apoptosis in ARID1A-inactivated cells.
Figure 4: ARID1A represses HDAC6 expression.
Figure 5: The selectivity of HDAC6 inhibition against ARID1A inactivation depends on p53 and HDAC6 deacetylase Lys120 residues on p53.
Figure 6: Apoptosis induced by HDAC6 inhibition in ARID1A-mutated cells correlates with mitochondrial localization of p53K120Ac.
Figure 7: Apoptosis induced by HDAC6 inhibition in ARID1A-mutated cells correlates with a decrease in mitochondrial membrane potential.
Figure 8: HDAC6 inhibition improves the survival of mice bearing ARID1A-mutated ovarian tumours.

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Acknowledgements

We thank C. Kadoch for the ARID1A CRISPR plasmid and K. Payne and T. Fukumoto for technical assistance. This work was supported by US National Institutes of Health grants (R01CA160331, R01CA163377 and R01CA202919 to R.Z., K99CA194318 to B.G.B., K99CA194309 to K.M.A. and R01GM49758 to D.W.C.), US Department of Defense (OC140632P1 and OC150446 to R.Z.), an Ovarian Cancer Research Fund (OCRF) program project (to R.Z.) and The Jayne Koskinas & Ted Giovanis Breast Cancer Research Consortium at Wistar (to R.Z.). Support of Core Facilities was provided by Cancer Centre Support Grant (CCSG) CA010815 to The Wistar Institute.

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B.G.B., S.W., P.H.P., Y.H., K.M.A., Y.W. and A.V.-A. performed the experiments, and analysed data. B.G.B. and R.Z. designed the experiments. A.V.K. performed the bioinformatics analysis. F.J.R.III, J.R.C.-G., W.Z. and D.W.S. participated in the experimental design; K.R.C. and Y.Z. contributed key reagents. D.G.H., D.W.C. and R.Z. supervised studies. B.G.B., K.M.A., Y.W., K.R.C., D.W.C. and R.Z. wrote the manuscript. R.Z. conceived the study.

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Correspondence to Rugang Zhang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 ARID1A-inactivated cells are more sensitive to HDAC6 inhibition.

(a) ARID1A wild-type RMG1 cells with or without ARID1A knockdown were transduced with lentivirus encoding shRNA to each of the 11 individual HDACs. RNA was isolated from the indicated cells and subjected to qRT-PCR for the indicated HDACs. n = 3 independent experiments. (b) Confirmation of HDAC6 knockdown by qRT-PCR in OVCA429 cells. n = 6 independent experiments. (c) HDAC6 knockdown is selective against ARID1A-mutated clear cell or endometrioid ovarian cancer cell lines based on the Project Achilles database. The whiskers of the boxplot represent minima to maxima of the relative growth of cell lines with (n = 5 cell lines) or without ARID1A mutation (n = 11 cell lines). The box represents median bar with the first and the third quartiles. (d) Expression of HDAC6 determined by immunoblot in a panel of ovarian clear cell carcinoma cell lines with known ARID1A mutational status without or with HDAC6 knockdown using two individual shHDAC6s. GAPDH expression was used as a loading control. Error bars represent mean with S.E.M. P-value calculated via two-tailed t-test. Statistical source data are provided in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.

Supplementary Figure 2 BRG knockdown does not affect sensitivity to HDAC6 inhibition.

(a,b) Expression of HDAC6, FLAG and loading control β-actin in ARID1A-mutated OVISE cells with knocking down of endogenous HDAC6 expression using a shRNA that targets the 3’ UTR region of the human HDAC6 gene and concurrent expression of FLAG-tagged shRNA resistant wildtype HDAC6 or a catalytically inactivated H216/611A mutant (a). The indicated cells were subjected to colony formation assay and integrated density was measured with NIH Image J software as a surrogate for cell growth (b). n = 4 independent experiments. (c) ARID1A wildtype RMG1 cells with or without ARID1A knockdown were determined for Vorinostat dose responsive curves in a 12-day colony formation assay, n = 3 independent experiments. (df) ARID1A wildtype RMG1 cells with or without BRG1 knockdown were examined for expression of BRG1, HDAC6 or a loading control GAPDH by immunoblot (d); examined for HDAC6 mRNA expression by qRT-PCR (e), n = 3 independent experiments; or determined for ACY1215 dose responsive curves in a 12-day colony formation assay (f), n = 4 independent experiments. (gi) BRM compensates for the knockdown of BRG1 at the HDAC6 gene promoter. ARID1A wildtype RMG1 cells were infected with the indicated shBRG1 or shControl. Expression of BRG1, BRM1, ARID1A and a loading control β-actin was determined by immunoblot (g). The indicated cells were subjected to ChIP analysis for the HDAC6 gene promoter using antibodies against BRG1 (h) or BRM (i). An isotype matched IgG was used as a control. n = 4 independent experiments. Error bars represent mean with S.E.M. P-value calculated via two-tailed t-test. Statistical source data are provided in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.

Supplementary Figure 3 Caspase 3, Caspase 8 and Caspase 9 knockdown in TOV21G cells.

(a) The gating strategy used for determining apoptosis based on AnnexinV-FITC and propidium iodide staining. Note that total apoptotic cells are calculated based on both early and late apoptotic fractions. (b,c) Expression of CASP3 (encodes caspase 3) mRNA (b), n = 4 independent experiments; and CASP9 (encodes caspase 9) mRNA (c), n = 3 independent experiments; determined by qRT-PCR in ARID1A-mutated TOV21G cells with or without Caspase 3 (b) or Caspase 9 knockdown (c). (d,e). Caspase 8 inhibition does not affect apoptosis induced by HDAC6 inhibition. ARID1A-mutated TOV21G cells were infected with the indicated shCaspase 8 or shControl. Relative expression of CASP8 (encodes caspase 8) was determined by qRT-PCR (d). n = 4 independent experiments. The indicated cells were treated with HDAC6 inhibitor ACY1215 (1.25 μM) or vehicle DMSO control for 72 hours and the percentages of apoptotic cells were quantified by Annexin V staining (e). n = 3 independent experiments. Error bars represent mean with S.E.M. P-value calculated via two-tailed t-test. Statistical source data are provided in Supplementary Table 6.

Supplementary Figure 4 ARID1A regulates HDAC6 expression.

(a) Expression of HDAC6, BRG1 and a loading control β-actin in ARID1A-mutated TOV21G cells with or without wildtype ARID1A restoration and with or without concurrent BRG1 knockdown. ARID1A restoration the same as Fig. 3f. (b) Relative expression of the indicated HDACs mRNA determined by qRT-PCR in ARID1A-mutated TOV21G cells with wildtype ARID1A restoration or ARID1A wildtype RMG1 cells with ARID1A knockdown. n = 3 independent experiments. P < 0.01. P-value calculated via two-tailed t-test. (c) Tumour cells derived from the indicated genetic mouse endometrioid tumours were treated with ACY1215 (1.25 μM). The percentage of surviving cells was determined by colony formation. n = 3 independent experiments. (d) Relative mRNA expression of the indicated class II HDACs in ARID1A wildtype (n = 12) and mutated (n = 7) human ovarian clear cell carcinoma specimens. Note that none of them are significant. (e) ARID1A expression negatively correlates with HDAC6 expression at the mRNA level in clear cell and endometrioid ovarian cancer cells in the cell lines encyclopedia1. Pearson correlation was used for calculating P value. (f) ARID1A expression negatively correlates with HDAC6 expression at the mRNA level in a published dataset of laser capture microdissected (LCM) clear cell ovarian carcinomas and normal human ovarian surface epithelial cells2. Pearson correlation was used for calculating P value. (g) Schematic of primers’ positions for ChIP-PCR in the human HDAC6 gene locus. (h,i) ARID1A wildtype RMG1 cells with or without ARID1A knockdown were subjected to ChIP analysis using antibodies against ARID1A (h), anti-Pol II (i) or an IgG control. ChIP products were subjected to qPCR analysis using primers as indicated in (g) for the human HDAC6 gene locus. n = 4 independent experiments. (j) ARID1A wildtype RMG1 cells with or without ARID1A knockdown were subjected to ChIP analysis using an antibody against acetylated histone H3 (H3Ac) or an IgG control for the human HDAC6 gene locus. n = 4 independent experiments. Error bars represent mean with S.E.M. P-value calculated via two-tailed t-test. Statistical source data are provided in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.

Supplementary Figure 5 p53 is required for the observed selectivity against ARID1A mutation by HDAC6 inhibitor.

(ac) ARID1A-mutated OVISE cells with or without p53 knockdown were examined for TP53 mRNA expression by qRT-PCR (a), n = 4 independent experiments; or examined for p53 protein expression by immunoblot (b). GAPDH expression was used as a loading control; or determined for dose response curves with the indicated concentration of ACY1215 for 12 days in a colony formation assay (c). Growth inhibition was calculated based on integrated density as measured in NIH ImageJ, and values were normalized to vehicle control. n = 4 independent experiments. (d) ARID1A-mutated TOV21G cells with or without p53 knockdown were treated with the indicated concentration of CAY10603 to generate dose response curves. ARID1A wildtype RMG1 and OVCA429 were used as controls for comparison. n = 4 independent experiments. (e) ARID1A-mutated TOV21G treated with vehicle DMSO control or the HDAC6 inhibitor ACY1215 (1.25 μM). Expression of the indicated proteins was determined by immunoblot. GAPDH expression was used as a loading control. Error bars represent mean with S.E.M. P-value calculated via two-tailed t-test. Statistical source data are provided in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.

Supplementary Figure 6 TIP60 inhibition impaired apoptosis induced by HDAC6 inhibition in ARID1A-mutated cells.

(a) ARID1A-mutated TOV21G cells were treated with vehicle control, shControl, shHDAC6, ACY1215 (1.25 μM) or CAY10603 (312 nM). RNA was extracted and utilized for next generation sequencing (RNA-seq). Expression of p53 target genes known to regulate apoptosis such as BAX, PUMA and NOXA, and known to regulate cell cycle arrest such as CDKN1A were not altered by HDAC6 inhibition in RNA-seq analysis. (b,c) ARID1A-mutated TOV21G cells were treated with the HDAC6 inhibitor ACY1215 (1.25 μM), or TIP60 inhibitor NU9056 (10 μM) or a combination. Expression of p53K120Ac and a loading control β-actin was determined by immunoblot (b). Percent apoptosis was quantified by Annexin V staining in the indicated cells (c). n = 3 independent experiments. Error bars represent mean with S.E.M. P-value calculated via two-tailed t-test. (d) The gating strategy in determining mitochondria membrane potential (MMP) by FACS analysis. Examples of maintenance of MMP and loss of MMP are shown. Statistical source data are provided in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.

Supplementary Figure 7 A model for the mechanism by which HDAC6 inhibition promotes apoptosis in ARID1A-mutated cells.

Supplementary Figure 8 HDAC6 inhibition significantly inhibits tumour growth ARID1A-mutated, but not wildtype, tumours.

(a,b) Luciferase-expressing ARID1A-mutated TOV21G cells were orthotopically transplanted into the ovarian bursa sac of SCID/nude female mice. Tumours were allowed to establish for 14 days before randomized into two groups (n = 6 mice/group). Mice were treated with vehicle control or HDAC6 inhibitor (ACY1215, 50mg/kg) daily for 21 days. Representative images of control and ACY1215 treated mice at the end of treatment (a). Total flux (photons/sec) is graphed at the indicated time points (b). P = 0.0313. Error bars represent S.E.M. P-value calculated via two-tailed t-test. (c) 6-10-weeks-old Pik3caH1047R/Arid1aflox/flox female mice were intrabursally injected with adenovirus-Cre to induce clear cell ovarian carcinomas. Mice were randomized and treated with vehicle control (n = 5 mice) or ACY1215 (50 mg/kg, n = 4 mice) daily for 21 days. The changes in volumes of tumours formed on the injected ovary were calculated against the contrary side non-injected ovary from the same mice. (di) Luciferase-expressing ARID1A-wildtype RMG1 cells were orthotopically transplanted into the ovarian bursa sac of SCID/nude female mouse. Tumours were allowed to establish for 14 days before randomized into two groups (n = 6 mice/group). Mice were treated with vehicle control or HDAC6 inhibitor (ACY1215, 50 mg/kg) daily for 21 days. Representative images of control and ACY1215 treated mice at the end of treatment (d). At the indicated time interval during treatment, mice were imaged for luciferase expression to monitor tumour growth. Total flux (photons/sec) is graphed (e). The weight of tumours dissected from control and ACY1215 treated mice was measured at the end of treatment as a surrogate for tumour burden (f). The number of disseminated tumour nodules was counted in the indicated treatment groups (g). The serial sections of tumours dissected were subjected to immunohistochemical staining for HDAC6, Ki67, cleaved caspase 3 and p53K120Ac (h). Scale bar = 100 μm. Histological score (H-score) was calculated for 5 separate fields from 6 tumours from 6 individual mice from each of the indicated groups (i). Error bars represent mean with S.E.M. P-value calculated via two-tailed t-test. Statistical source data are provided in Supplementary Table 6.

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Bitler, B., Wu, S., Park, P. et al. ARID1A-mutated ovarian cancers depend on HDAC6 activity. Nat Cell Biol 19, 962–973 (2017). https://doi.org/10.1038/ncb3582

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