CRISPRi screens to identify combination therapies for the improved treatment of ovarian cancer

Abstract

Ovarian cancer is the fifth leading cause of cancer death for women in the United States, with only modest improvements in patient survival in the past few decades. Standard-of-care consists of surgical debulking followed by a combination of platinum and taxane agents, but relapse and resistance frequently occur. To identify genes that confer sensitivity or resistance in tumor cells treated with platinum chemotherapeutics, I performed genome-wide screens combining cisplatin or oxaliplatin with CRISPRi pooled gene knockdowns. Screens were analyzed at 9-days to mimic patient care, and at 48-hours to isolate the short-term DNA damage response. Genes whose knockdown caused sensitivity to the platinum chemotherapeutics were identified through a multi-objective optimization approach to account for knockdown efficiencies and variances in sequencing depth.

To filter the noise in the genome-wide screen and more confidently identify 'hits,' a smaller pooled CRISPRi screen of four hundred targets was designed, and a few 'hits' were validated. Interestingly, knockdown of FAAP24, a component of the FA core complex, was found to sensitize multiple ovarian cancer cells to platinum compounds, and thus may be a promising candidate for a combination treatment with oxaliplatin and cisplatin. Chapter 5 details an implementation of a combination therapy with cisplatin using peptide nanoparticles. Peptide nanoparticles are a promising therapeutic for the delivery of siRNA and allow for targeting of specific proteins that are difficult to inhibit with small molecular inhibitors; specifically, nanoplexes allowed for the targeting of the REV3 protein, the catalytic component of the translesion synthesis polymerase.

Interfering with REV3 expression through siRNA has a synergistic effect with cisplatin treatment in both human and mouse models of lung cancer, indicating that REV3 is an excellent target to combine with cisplatin therapies. This REV3 knock-down sensitivity was also extended to human ovarian cancer cell lines, indicating the potential of the combination treatment for both lung and ovarian cancers.