Chasing the backup pathway for therapeutic opportunities in cancer

Abstract

BRCA mutations are usually associated with the most aggressive subtypes of cancers such as triple‐negative breast cancer and high‐grade serous ovarian cancer. Finding a drug that can address the unique molecular features in these subtypes of cancer continues to be the best hope for treatment. In tumors with impaired BRCA‐pathway, RAD52 is a candidate for targeted inhibition as it is known to take a leading role in homologous recombination repair (HRR) of DNA double‐strand breaks (DSBs) in BRCA‐deficient cancer cells [1]. Additionally, RAD52 is the main player in single‐strand annealing (SSA), which has been reported to increase activity in a number of cancers. When RAD52 joins the DSB repair complex, RPA has already bound ssDNA and the resulting protein‐protein interaction (PPI) becomes instrumental for repair to proceed. Past work showed that conformational changes offered by RPA interaction modulate RAD52 activity [2]. A combined understanding of these bimolecular studies and the value of RAD52 in tumorigenesis were motivating factors for the development of a Fluorescence‐based protein‐protein Interaction Assay (FluorIA) [3]. FluorIA provided a tool to assess domains on RPA and RAD52 important for the PPI (Figure 1). The binding affinities and thermodynamics of those interacting domains were then evaluated by methods such as surface plasmon resonance and isothermal titration calorimetry to characterize RPA:RAD52 interaction as a vital therapeutic target.FluorIA was also used as a high throughput assay to screen over 100,000 potential small molecule inhibitors (SMIs). Eleven hits were identified, three of which are FDA approved drugs (Fig. 2). Characterization experiments thus far suggest a potential impact for at least one of these three drugs on SSA repair of DSBs. Our findings may offer molecular insights into a new mechanism of action for these drugs given their reported varied sensitivity in patients carrying BRCA deficiencies. The ultimate goal is to therapeutically target the addiction of cancer cells for DNA repair. However, we anticipate that a broader understanding of the mechanisms of action for the existing cancer drugs we screened would support the effort towards personalized medicine.Support or Funding Information1) SBMB Fellowship from U.S. Department of Education GAANN (P200A120231) 2) NASA Nebraska Space Grant: Graduate fellowship 3) Nebraska Department of Health and Human services (DHHS) LB506 4) Nebraska Research Initiative 5) Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727) pilot project fundingCompetitive binding in FluorIA. A) Domain map of RAD52. B) SDS‐PAGE gel shown full‐length RAD52(1‐418) to the right of the molecular weight marker, RAD52(1‐303), and RAD52(1‐212). C) FluorIA reaction in which 20 pmol of EGFP tagged full‐length RAD52 is incubated with RPA alone or with a 100 pmol of unlabeled RAD52(1‐303) or (1‐212) to compete with EGFP‐RAD52 for RPA binding. Reduction in RFU signal signifies inhibition of RPA:RAD52 complex formation by the unlabeled RAD52 construct.Figure 1Inhibiti of RPA:RAD52 complex by the SMI leads. Hits are designated with the letters P or C according to the first letter of the source library name. The best IC50 are P1, 110 μM; P2, 12 μM; P3, 15 μM; C2, 120 μM and C8, 100 μM.Figure 2