Cyclin E1/CDK2 activation defines a key vulnerability to WEE1 kinase inhibition in gynecological cancers

Abstract

Upregulation of Cyclin E1 and subsequent activation of CDK2 accelerates cell cycle progression from G1 to S phase and is a common oncogenic driver in gynecological malignancies. WEE1 kinase counteracts the effects of Cyclin E1/CDK2 activation by regulating multiple cell cycle checkpoints. Here we characterized the relationship between Cyclin E1/CDK2 activation and sensitivity to the selective WEE1 inhibitor azenosertib. We found that ovarian cancer cell lines with high levels of endogenous Cyclin E1 expression or forced overexpression were exquisitely sensitive to azenosertib and these results extended to in vivo models of ovarian and uterine serous carcinoma. Models with high Cyclin E1 expression showed higher baseline levels of replication stress and enhanced cellular responses to azenosertib treatment. We found azenosertib synergized with different classes of chemotherapy and described distinct underlying mechanisms. Finally, we provided early evidence from an ongoing phase I study demonstrating the clinical activity of monotherapy azenosertib in patients with Cyclin E1/CDK2-activated ovarian and uterine serous carcinomas.

Fig. 1. Cyclin E1^high HGSOC cell lines are more sensitive to azenosertib in vitro.

a Jess analysis showing cyclin E1 expression level in nine high-grade serous ovarian cancer (HGSOC) cell lines. b Quantification of Cyclin E1 level normalized by Vinculin using densitometry. c CellTiter-Glo (CTG) assay to evaluate the growth rate inhibition (GR) performed on nine HGSOC cell lines grown in 2D culture conditions in a 3-day assay. GR and GR_max were determined by measurement of cell viability before and after 72 hours treatment of azenosertib and the GR calculator (http://www.grcalculator.org). (left) The GR values were generated in at least two independent experiments. The representative GR curve is shown. (right) The GR plot in combination with Cyclin E1 expression level showing the correlation of the level of Cyclin E1 and cytotoxic effect (lowering GR_max). (bottom) Summary of the average GR_max and the copy number of nine HGSOC cell lines used in the study.

Fig. 2. Cyclin E1 overexpression sensitizes HGSOC cell lines to azenosertib, resulting in greater growth inhibition and replication stress.

a Jess analysis of Cyclin E1^high OVCAR3, OVCAR4, and Cyclin E1^low KURAMOCHI cell lines treated with different doses of azenosertib for 16 hours. Biomarkers of target engagement (p-CDK1^Y15), cell cycle (Cyclin E1, A2, B1, pHH3^S10), DNA damage response (pCHK1^S345, γH2AX^S139), and apoptosis (Cleaved caspase-3 and 7) were analyzed. b The plot showing the decrease of IC50 and GR50 of OV90 stably expressing Cyclin E1 relative to that of vector control cells determined by measurement of cell viability. c Jess analysis of isogenic pair cell lines of OV90 stably expressing Cyclin E1 treated with different doses of azenosertib for 16 hours. Biomarkers of target engagement, DNA damage response, and mitosis were analyzed. d DNA fiber length analysis of isogenic pair of OV90 control cells (OV90/Vector) and overexpression cells (OV90/Cyclin E1) treated with 200 nM of azenosertib and 0.5 mM of hydroxyurea (HU) for 24 hours. Statistical significance was calculated using two-way ANOVA. * P < 0.05, ****P < 0.0001.

Fig. 3. Sensitivity of cyclin E1^high HGSOC cells to azenosertib is dependent on CDK2/Cyclin E1.

a Jess analysis showing Cyclin E1 expression in OVCAR3 and OVCAR4 cell lines transfected with two different CCNE1 siRNAs. b Dose response of OVCAR3 (top) and OVCAR4 (bottom) cell lines transfected with CCNE1 siRNAs to azenosertib for 72 hours. Statistical significance was calculated using two-way ANOVA. ****P < 0.0001. c Jess analysis showing CDK1 and CDK2 expression in OVCAR4 cell line transfected with two different CDK2 siRNAs. d Dose response of OVCAR4 cell line transfected with CDK2 siRNAs to azenosertib for 72 hours. Statistical significance was calculated using two-way ANOVA. ****P < 0.0001. e Jess analysis of indicated antibodies with OVCAR4 cell lines transfected with two different CDK2 siRNAs and treated with different doses of azenosertib for 16 hours.

Fig. 4. Greater anti-tumor effects of azenosertib in a Cyclin E1^high tumor model are associated with increased replication stress.

a Azenosertib was administered via daily oral gavage at the doses indicated to NOD/SCID mice bearing established cyclin E1^low SKOV3 (left) and cyclin E1^high OVCAR3 (right) xenografts (n = 10/group). No animals were excluded from the analysis. Dosing was initiated when tumors were approximately 200 mm³. Azenosertib was administered to mice daily until day 28. Data are shown as mean tumor volume ± SEM. Statistical significance was calculated using two-way ANOVA followed by Bonferroni post-test. *P < 0.05, **P < 0.01. b, c Immunohistochemistry (IHC) analysis of pCDK1^Y15 (b) and γH2AX^S139 (c) from the SKOV3 and OVCAR3 xenograft tumors treated with azenosertib for 5 days and collected at 12 hours after the last dose. The Y axis presents the average H-score of 2-3 tumors. d Representative images of immunofluorescence (IF) staining of pRPA32^S4/S8 and γH2AX^S139 for MCF10A cell line samples treated with gemcitabine for 24 hours and xenograft tumor samples of SKOV3 and OVCAR3 treated with azenosertib for 5 days and collected at 4, 12, and 24 hours after the last dose. MCF10A cells treated with gemcitabine were used as a positive control as gemcitabine is known to induce replication stress. e Quantification of γH2AX foci-positive cells in (d) was shown in top plot. Ratio of dual positivity of pRPA32^S4/S8 and γH2AX^S139 was shown in bottom plot.

Fig. 5. Increased synergy between azenosertib and chemotherapy was observed in Cyclin E1^high HGSOC cells.

a The drug synergistic effect based on Loewe model in Cyclin E1^high OVCAR3 (left), Cyclin E1^low OV90 (center), and Cyclin E1^low TYK-nu (right) cell lines of the combination of azenosertib and oxaliplatin (top), paclitaxel (middle), and gemcitabine (bottom). Loewe values > 10 indicate synergistic effects of the drugs, Loewe values from −10 to 10 indicate additive effects, and Loewe values <-10 indicate antagonism. b Jess analysis of OVCAR3 and OV90 cell lines treated with azenosertib in combination with different doses of paclitaxel for 48 hours with the indicated antibodies. c Jess analysis of OVCAR3 and OV90 cell lines treated with azenosertib in combination with different doses of carboplatin (Carbo) for 48 hours with the indicated antibodies. d Jess analysis of OVCAR3 and OV90 cell lines treated with azenosertib in combination with gemcitabine (Gem) for 48 hours with the indicated antibodies. 123 or 370 nM of azenosertib and 22 or 5 nM of gemcitabine were treated in OVCAR3 and OV90 cell lines, respectively. e The indicated doses of azenosertib, paclitaxel, or a combination were administered to mice bearing the OVCAR3 cell line xenografts (n = 8/group). Statistical significance was calculated on day 20 using Mann–Whitney U test. **P < 0.01, ***P < 0.001 compared to Vehicle treatment, ⁺⁺⁺P < 0.001 compared to azenosertib or paclitaxel single treatment. f The indicated doses of azenosertib, carboplatin, or a combination were administered to mice bearing the OVCAR3 cell line xenografts (n = 8/group). Statistical significance was calculated on day 28 using two-way ANOVA followed by Bonferroni post-test. *P < 0.05 **P < 0.01 compared to Vehicle treatment, ⁺⁺P < 0.01 compared to azenosertib or carboplatin single treatment. g The indicated doses of azenosertib, paclitaxel, or a combination were administered to mice bearing the A2780 cell line xenografts (n = 8/group). Statistical significance was calculated on day 10 using Dunnett T3 test. ⁺P < 0.05 compared to azenosertib single treatment. h The indicated doses of azenosertib, carboplatin, or a combination were administered to mice bearing the SKOV3 cell line xenografts (n = 8/group). Tumor growth curves were shown in e–h. TGI, Tumor Growth Inhibition. No animals were excluded from the analysis.

Fig. 6. Azenosertib monotherapy in patients with Cyclin E1^high cancers.

a Each symbol represents an individual subject from azenosertib phase I clinical trial and shows the percentage of pCDK1^Y15 change in surrogate skin tissue (% change from baseline) as quantified by H-score. Green-filled circles indicated subjects that showed partial response to azenosertib. The dotted horizontal line represents the threshold used to define target engagement, defined as ≥50% of pCDK1^Y15 on-treatment as compared to baseline. b IHC analysis of Cyclin E1 and H&E staining of archival tumor samples from 3 representative cases. c Azenosertib monotherapy efficacy in patients with Cyclin E1^high cancers. Patient 0173-008, HGSOC, week 12 scan indicated a 57% reduction in target lesions. Patient 0171-010, HGSOC, week 12 scan indicated a 42% reduction in target lesion. Patient 0171-019, Uterine Serous Carcinoma, week 12 scan indicated 30% reduction in target lesion.