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. 2023 Apr 22;16(1):80.
doi: 10.1186/s13048-023-01160-y.

Treatment for ovarian clear cell carcinoma with combined inhibition of WEE1 and ATR

Wenwen Chien  1 , Jeffrey W Tyner  2 , Sigal Gery  3 , Yueyuan Zheng  4 , Li-Yan Li  5 , Mohan Shankar Gopinatha Pillai  3 , Chehyun Nam  6 , Neil A Bhowmick  3 , De-Chen Lin  6 , H Phillip Koeffler  3   7
Affiliations

Treatment for ovarian clear cell carcinoma with combined inhibition of WEE1 and ATR

Wenwen Chien et al. J Ovarian Res. .

Abstract

Background: Standard platinum-based therapy for ovarian cancer is inefficient against ovarian clear cell carcinoma (OCCC). OCCC is a distinct subtype of epithelial ovarian cancer. OCCC constitutes 25% of ovarian cancers in East Asia (Japan, Korea, China, Singapore) and 6-10% in Europe and North America. The cancer is characterized by frequent inactivation of ARID1A and 10% of cases of endometriosis progression to OCCC. The aim of this study was to identify drugs that are either FDA-approved or in clinical trials for the treatment of OCCC.

Results: High throughput screening of 166 compounds that are either FDA-approved, in clinical trials or are in pre-clinical studies identified several cytotoxic compounds against OCCC. ARID1A knockdown cells were more sensitive to inhibitors of either mTOR (PP242), dual mTOR/PI3K (GDC0941), ATR (AZD6738) or MDM2 (RG7388) compared to control cells. Also, compounds targeting BH3 domain (AZD4320) and SRC (AZD0530) displayed preferential cytotoxicity against ARID1A mutant cell lines. In addition, WEE1 inhibitor (AZD1775) showed broad cytotoxicity toward OCCC cell lines, irrespective of ARID1A status.

Conclusions: In a selection of 166 compounds we showed that inhibitors of ATR and WEE1 were cytotoxic against a panel of OCCC cell lines. These two drugs are already in other clinical trials, making them ideal candidates for treatment of OCCC.

Keywords: ATR; Ovarian clear cell carcinoma; WEE1.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Sensitivity of small molecule inhibitors of OCCC cell lines with silencing of ARID1A. ARID1A isogenic OVCA429 OCCC cell lines were treated with drugs for 72 h with 2 replicates (Rep 1, Rep 2) each from KD (ARID1A shRNA knockdown) versus WTC (wildtype control). (A) The 38 small molecule inhibitors showed significantly different cytotoxicity when comparing KD to WTC. IC50 values (0–10,000 nM, Supplementary Table 2) for each inhibitor are expressed in a clustered heatmap. (B) IC50 of 18 compounds with cytotoxicity toward both KD and WTC are presented in dot plot fashion. (C) Significant differential response to 9 different combination treatment (KD vs. WTC). Red, higher IC50; Blue, lower IC50.
Fig. 2
Fig. 2
IC50s in OCCC cell lines. (A) Summary of IC50 values in 3 sets of ARID1A isogenic cell lines (JHOC5, ES2, OVCA429; KD: ARID1A shRNA knockdown; WTC: wildtype control). Solid shapes, WTC; Open shapes, KD. (B) Summary of IC50 values in OCCC cell lines (WT: JHOC5, ES2, OVCA429, RMG1; Mut: OVTOKO, KOC7C). Values of IC50 were calculated from dose curves (Supplementary. Figure 2). Circles, WT; Triangles, Mut; *: p < 0.05
Fig. 3
Fig. 3
Combination treatment with AZD1775 and AZD6738 in OCCC cell lines. (A) OCCC cell lines (WT: ARID1A wild type, ES2; Mut: ARID1A mutant, KOC7C) were treated with AZD1775 or AZD6738 alone and combination of both at 0, 0.25, 0.5, 1, 2 µM (< IC50) for 72 h. Dose-response matrices and CI (combination index) heatmap between the interaction of two drugs are shown. Dose-response matrices: Red; 100% growth inhibition; White; 50%; Blue, 0%. CI’s were analyzed by CompuSyn as described in Methods. CI values: < 1, synergistic; =1, additive; > 1, antagonistic. CI < 0.3, strong synergy. (B) ARID1A isogenic ES2 OCCC cell lines (WTC : wildtype control; KD: ARID1A shRNA knockdown). Also see Supplementary Fig. 4
Fig. 4
Fig. 4
AZD1775 and AZD6738 inhibited cell growth in OCCC cell lines. OCCC cell lines were cultured on plastic or soft agar and treated with drugs for 14 days. Colony formation was fixed, stained, and number of colonies was counted. (A) WT (ES2) and Mut (KOC7C) cell lines. (B) ARID1A isogenic cell lines (ES2 WTC, ES2 KD). (Two-tailed t test of combination to single agent: *, p < 0.05; *, p < 0.01, n = 2)
Fig. 5
Fig. 5
Analysis of cell cycle and apoptosis. Analysis of OCCC cell lines treated with AZD1775 and AZD6738 (WT: ES2; Mut: KOC7C; ARID1A isogenic: ES2 WTC, ES2 KD) treated with AZD1775, AZD6738 or combination of both at IC50 for 24 h. (A) Cell cycle analysis. Stacked bar graphs show the fractions of cells at Pre-G1, G1, S or G2/M phase. (B) Apoptosis analysis. Bar graphs show the fractions of annexin V positive cells. (Two-tailed t test: *, p < 0.05 compared to control; #, p < 0.05 combination compared to single agent, n = 2). Also see Supplementary Fig. 6
Fig. 6
Fig. 6
Effect of AZD1775 and AZD6738 on expression levels of protein related to apoptosis and DNA damage. A & B. Two OCCC cell lines (WT: ES; Mut: KOC7C) were treated with AZD1775, AZD6738, or combination of both and protein expression levels were examined by Western blots. C. Western blot analysis of ARID1A isogenic pair ES2 WTC, ES2 KD after drug treatment. GAPDH was used as loading control. p-CHK1, phosphorylated CHK1; Cas3, caspase 3; p-CDC25C, phosphorylated CDC25C. Bar graphs show quantification of Western blots using ImageJ as described in Methods. (Two-tailed t test: *, p < 0.05, **, p < 0.01, ****, p < 0.0001, compared to control; #, p < 0.05, ##, p < 0.01, ###, p < 0.001, combination compared to single agent; n = 2)
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