Dual Fluorescence Isogenic Synthetic Lethal Kinase Screen and High-Content Secondary Screening for MUC16/CA125-Selective Agents

Abstract

Significant strides have been made in the development of precision therapeutics for cancer. Aberrantly expressed glycoproteins represent a potential avenue for therapeutic development. The MUC16/CA125 glycoprotein serves as a biomarker of disease and a driver of malignant transformation in epithelial ovarian cancer. Previously, we demonstrated a proof-of-principle approach to selectively targeting MUC16+ cells. In this report, we performed a synthetic lethal kinase screen using a human kinome RNAi library and identified key pathways preferentially targetable in MUC16+ cells using isogenic dual-fluorescence ovarian cancer cell lines. Using a separate approach, we performed high-content small-molecule screening of six different libraries of 356,982 compounds for MUC16/CA125-selective agents and identified lead candidates that showed preferential cytotoxicity in MUC16+ cells. Compounds with differential activity were selected and tested in various other ovarian cell lines or isogenic pairs to identify lead compounds for structure-activity relationship (SAR) selection. Lead siRNA and small-molecule inhibitor candidates preferentially inhibited invasion of MUC16+ cells in vitro and in vivo, and we show that this is due to decreased activation of MAPK, and non-receptor tyrosine kinases. Taken together, we present a comprehensive screening approach to the development of a novel class of MUC16-selective targeted therapeutics and identify candidates suitable for further clinical development.

Figure 1:. siRNA screen to identify MUC16-selective candidates by cytotoxicity.

A, Schematic diagram of MUC16. B, Workflow of synthetic lethal kinase killing assay on A2780 MUC16- or A2780 MUC16+ cells. C, Heap Map showing 4 siRNAs per kinase that showed either MUC16+ or MUC16- inhibition. The siRNA kinases were validated by Qiagen.

Figure 2:. siRNA screen to identify kinases with selective inhibition of invasion and proliferation in MUC16+ cells.

A, Western blot analysis of A2780-phrGFP (MUC16-) or A2780-M4CL (MUC16+) treated with individually pooled ILK or GCK siRNAs. B, A2780-phrGFP or A2780-M4CL cells treated with pooled ILK siRNAs or negative control siRNAs and subjected to matrigel invasion assay. Results are expressed as percentages compared by paired t-test to A2780-phrGFP control after 48 hrs (Mean ± SE) (n=3). A2780-M4CL cells treated with pooled ILK siRNAs showed a statistically significant (**p=0.003) inhibition of matrigel invasion compared to negative control siRNAs.

Figure 3:. Dual fluorescence High-content screening.

A, Schematic showing high-content screening workflow. B, Details of various available compound libraries used in the study. Compounds were acquired from NCI, ChemDiv, ChemBridge, Analyticon, Biofocus, and SPECS. Parental compounds and analogs shown with nomenclature, and chemical structures.

Figure 4:. Functional validation of Primary and SAR compounds.

Primary and SAR compounds were validated in A2780 and SKOV3 isogenic pairs using matrigel invasion assays. A, A2780 isogenic cell pairs screened with parental compounds; P1– 9. ** p = 0.001; *** p = 0.0001. B, SKOV3 isogenic cell pairs screened with parental compounds. # p = 0.02; ## p = 0.001; ### p < 0.0001. Multiple comparisons of primary compounds showed statistically significant inhibition of MUC16+ cells (p < 0.0056) by Bonferroni correction for both A2780 and SKOV3 cells. C, A2780 isogenic cell pairs screened with SAR analogues derived from primary compounds; SAR 1–12. * p = 0.01; *** p = 0.008. D, SKOV3 isogenic cell pairs screened with SAR analogues. ## p = 0.001; ### p = 0.0004. Multiple comparisons of SAR analogues showed statistically significant inhibition of MUC16+ cells (p < 0.0042) by Bonferroni correction for both A2780 and SKOV3 cells. Matrigel invasion was measured in triplicate (n = 3). Results are expressed as percentages compared by paired t-test to parental phrGFP control cells after 48 hrs (mean ± SE) unless otherwise stated. Two or more biologic replicates were performed for each condition.

Figure 5:. Validation of lead candidates and SAR analogs.

Determination of relative IC50 of primary lead compounds and SAR analogs in 4 isogenic pairs and wild type ovarian cell lines by Alamar blue cytotoxic assay. The ratio of the IC50 in MUC16+ cells to MUC16- cells are plotted for each compound. Values less than 1 reflect lower IC50 in MUC16+ cells, and hence preferential potency. A, IC50 ratios for the parental P2 compound and the Analyticon Flavone scaffold (SAR 7) are shown. IC50 for OVCAR3 cells treated with P2 and SAR7 are shown separately. B, OVCAR3 cells treated with flavopiridol in concentrations between 0.25 μM- 1.0 μM for 5 days and assayed for shed CA-125. C, Spheroid cell assay with OVCAR3 cells cultured in DMSO or indicated flavopiridol concentrations for various time points. ns: not significant. *p < 0.05 (DMSO vs 0.25 μM), **p < 0.05 (DMSO vs 0.5 μM), ***p < 0.05 (DMSO vs 1.0 μM). D, Viability at 72hrs. ***p < 0.05. E, Wound healing scratch assay with OVCAR3 cells cultured in DMSO or indicated flavopiridol concentrations for various time points. Data are expressed as percentage of fold-decrease of open wound area compared with control (0 h) set as 100% and are reported as mean of three independent experiments ± S.E. ns: not significant, ***p<0.05. F, A2780 isogenic cells cultured in serum-free media with or without 10 ug/mL flavopiridol for various time points. Cell lysates were prepared and assayed by western blotting for 4H11, total and phospho EGFR, AKT, ERK, and SRC antibodies. G, Western blot analysis performed on lysates derived from flank tumor-bearing mice treated with a single dose of intravenous flavopiridol. Equal amounts of proteins were separated on 10% SDS-PAGE gels and probed for 4H11 (to detect MUC16), total or phospho AKT, ERK, and SRC antibodies.

Figure 6:. In vivo evaluation of flavopiridol.

Ten million A2780-phrGFP or A2780-M4CL tumor cells were implanted into the peritoneum of female NSG mice and treated with flavopiridol 10 mg/kg i.p three times a week. n =10, experiment performed in duplicate. **p < 0.05.