Person:

Nagel, Zachary

Repair of DNA damage is a critical survival mechanism that impacts susceptibility to a variety of human diseases and represents a key target for cancer therapy. A major barrier to applying this knowledge in research and clinical translation has been the lack of efficient, quantitative functional assays for measuring DNA repair capacity in living primary cells. To overcome this barrier, we recently developed a technology termed fluorescence multiplex host cell reactivation (FM-HCR). We describe a method for using standard molecular biology techniques to generate large quantities of FM-HCR reporter plasmids containing site-specific DNA lesions and using these reporters to assess DNA repair capacity in at least six major DNA repair pathways in live cells. We improve upon previous methodologies by i) providing a universal workflow for generating reporter plasmids, ii) improving yield and purity to enable large-scale studies that demand milligram quantities, and iii) reducing preparation time more than 10-fold.

Synthetic lethality, an interaction whereby the co-occurrence of two genetic events leads to cell death but one event alone does not, can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets since many cancers exhibit an impaired DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors in homologous recombination-deficient cancers highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair (dMMR). Here we analyzed data from large-scale CRISPR/Cas9 knockout and RNA interference (RNAi) silencing screens and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in microsatellite stable (MSS) models. WRN depletion induced double-strand DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity, but not the exonuclease activity of WRN. These findings expose WRN as a synthetic lethal vulnerability and promising drug target for MSI cancers.