Exploiting DNA Repair Targets in Breast Cancer
October 3, 2013 | eNews
Normal cells acquire various types of DNA damage throughout their lifetime. To prevent mutations arising in the cell’s DNA, several different DNA repair mechanisms must collaborate to reverse the damage or minimize its deleterious effects on the cell. To repair double strand breaks (DSBs), one of the most dangerous forms of DNA damage, cells use two major pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). Although each pathway can operate throughout the cell cycle, HR is largely dedicated to repairing breaks that arise during DNA replication.
Homologous recombination is mediated by the BRCA gene pathway, which is impaired in cancers caused by germline BRCA mutations and in some non-BRCA-linked triple-negative breast cancers or ovarian cancers. These HR-deficient cancer cells may become vulnerable to inhibition of other DNA repair mechanisms, which may be why inhibition of the poly(ADP-ribose) polymerase (PARP) enzyme system can sensitize BRCA mutant cells to killing. One idea to explain how PARP inhibitors work to kill BRCA mutant cells is that they prevent the sealing of DNA “nicks” that commonly arise during the repair of damaged DNA bases, explains Ralph Scully, MBBS, PhD (BIDMC). Replication across these nicks will force the formation of new DSBs during replication, and might particularly stress the BRCA gene-regulated HR system. However, this appealing idea is complicated by the fact that PARPs are activated by a number of different types of DNA damage, including DSBs themselves.
“The precise mechanism by which PARP inhibition kills BRCA mutant cells is not well understood. If we knew what that mechanism was, this might lead us to other molecular targets and to the discovery of new therapeutics,” says Scully. Then, the additional stress on the cell’s DNA repair function from additional DNA repair targeting might enhance the therapeutic effect of PARP inhibition in BRCA-linked cancer.
To look for new DNA repair targets, Scully, in collaboration with Jagesh Shah, PhD (BIDMC), subjected cells to DNA damage, and then followed the time course of fluorescently labeled DNA repair proteins to the sites of DNA breakage. They saw that one of the first proteins to arrive at the break is Ku, a protein that controls the NHEJ pathway of DSB repair. Ku proteins bind directly to the broken DNA ends and recruit other NHEJ enzymes to stitch the ends back together. Provocatively, Scully and Shah have recently found that there is a connection between NHEJ- and PARP-mediated DNA repair pathways.
Scully thinks it might be possible to gain a therapeutic advantage by manipulating NHEJ in tandem with PARP inhibition, striking out a third DNA repair mechanism in BRCA-deficient cells. “The process is conceptually simple but biochemically complex. We know some of the biochemical players but we don’t know all of them. So we are taking a functional genome-wide approach to identify genes that regulate non-homologous end joining in an unbiased way.”
For that effort, postdoctoral fellow Emilie Rass, PhD, developed a miniaturized high throughput screen that allows her to inactivate one gene at a time and quantify the efficiency of NHEJ, using a fluorescent measure of re-ligation. If a deletion greatly reduces NHEJ function, she can then use the screen to measure the efficiency of inhibitory molecules.
“We’re still some distance away from having additional new targeted agents that kill BRCA-linked breast cancers, but we now have a quick way to test and screen individual candidates,” says Scully. “When we do have a good candidate, the translational research can happen quickly here with the help of the DF/HCC Breast Cancer Program.”
Research detailed in this article was funded in part by NIH grants, including CA065164 and CA145867, and a Susan G. Komen for the Cure Postdoctoral Fellowship PDF12230760.
— Cathryn Delude