Identification of therapeutic targets applicable to clinical strategies in ovarian cancer

Marianne K. Kim, Natasha Caplen, Sirisha Chakka, Lidia Hernandez, Carrie House, Georgios Pongas, Elizabeth Jordan, Christina M. Annunziata

shRNA-mediated lethality screening is a useful tool to identify essential targets in cancer biology. Ovarian cancer (OC) is extremely heterogeneous and most cases are advanced stages at diagnosis. OC has a high response rate initially, but becomes resistant to standard chemotherapy. We previously employed high throughput global shRNA sensitization screens to identify NF-kB related pathways. Here, we re-analyzed our previous shRNA screens in an unbiased manner to identify clinically applicable molecular targets. We proceeded with siRNA lethality screening using the top 55 genes in an expanded set of 6 OC cell lines. We investigated clinical relevance of candidate targets in The Cancer Genome Atlas OC dataset. To move these findings towards the clinic, we chose four pharmacological inhibitors to recapitulate the top siRNA effects: Oxozeaenol (for MAP3K7/TAK1), BI6727 (PLK1), MK1775 (WEE1), and Lapatinib (ERBB2). Cytotoxic effects were measured by cellular viability assay, as single agents and in 2-way combinations. Co-treatments were evaluated in either sequential or simultaneous exposure to drug for short term and extended periods to simulate different treatment strategies. Loss-of-function shRNA screens followed by short-term siRNA validation screens identified therapeutic targets in OC cells. Candidate genes were dysregulated in a subset of TCGA OCs although the alterations of these genes showed no statistical significance to overall survival. Pharmacological inhibitors such as Oxozeaenol, BI6727, and MK1775 showed cytotoxic effects in OC cells regardless of cisplatin responsiveness, while all OC cells tested were cytostatic to Lapatinib. Co-treatment with BI6727 and MK1775 at sub-lethal concentrations was equally potent to BI6727 alone at lethal concentrations without cellular re-growth after the drugs were washed off, suggesting the co-inhibition at reduced dosages may be more efficacious than maximal single-agent cytotoxic concentrations. Loss-of-function screen followed by in vitro target validation using chemical inhibitors identified clinically relevant targets. This approach has the potential to systematically refine therapeutic strategies in OC. These molecular target-driven strategies may provide additional therapeutic options for women whose tumors have become refractory to standard chemotherapy.