ABSTRACT
Double-stranded DNA breaks (DSB)s are repaired by two major mechanistically distinct pathways: DNA homology-directed repair (HDR) and non-homologous end joining (NHEJ). A decisive factor in the choice between HDR and NHEJ is the competition between DNA end protection (necessary for NHEJ) and DNA end resection (necessary for HDR). DSB end resection is appropriately restricted to S/G2 phases of the cell cycle, as HDR requires the presence of an intact sister chromatid. Depletion of NHEJ-promoting factors such as 53BP1 allows DNA end resection in the G1 phase, thereby impairing DSB repair and causing genomic instability. Conversely, loss of the HDR protein BRCA1 (critical for initiating end resection) allows the error-prone NHEJ pathway to dominate throughout the cell cycle, potentially leading to tumorigenesis. BRCA1 deficient tumors are exquisitely sensitive to inhibitors of the DNA repair protein, poly (ADP-ribose) polymerase (PARP). Surprisingly, loss of 53BP1 or associated factors (Shieldin complex, CST complex, etc) in these tumors render them insensitive to PARP inhibitors (PARPi) as DNA end resection and the subsequent steps of the HDR pathway are restored. Loss of DYNLL1, a factor that is constitutively bound to 53BP1, also results in enhanced end resection and HDR. DYNLL1 directly binds to and inhibits MRE11, thereby blocking the initiation of DNA end resection. Phosphorylation of DYNLL1 on serine 88 enhances the formation of the DYNLL1/MRE11 complex, albeit reducing DYNLL1’s interaction with 53BP1. Our preliminary studies suggest that DNA-PKcs is responsible for the phosphorylation of DYNLL1 and may be critical for inhibiting MRE11 and blocking HDR in BRCA1-deficient cells. BRCA1 impedes the activation of DNA-PKcs, indirectly preventing DYNLL1 phosphorylation and the inhibition of MRE11. In BRCA1 proficient cells, DYNLL1 promotes BRCA1/BARD1 mediated ubiquitination of MRE11, thereby facilitating end resection at DSBs. In AIM1, we propose to investigate the dynamics between BRCA1 and DNA-PKcs and how they regulate the DYNLL1 and MRE11 activity during the cell cycle. Like DYNLL1, the Shieldin (SHLD1–SHLD2–SHLD3) and CST (CTC, STN1, and TEN1) complexes are recruited to DSBs in a 53BP1- dependent manner, and loss of any of the subunits is also associated with increased end resection and HDR. Intriguingly, SHLD1 is recruited to short-resected ssDNA in the G1 phase of the cell cycle. This brings up one key issue: how the Shieldin complex, which primarily functions in G1, influences PARPi sensitivity in BRCA1- mutant tumors. PARPi sensitivity has now been closely tied to replication fork stability and ssDNA gap formation. Loss of REV7 and the CST complex has been shown to destabilize the fork, which should cause PARPi sensitivity. This is in contrast to PARPi resistance in BRCA1-mutant cells. Together, they suggest that the Shieldin complex may have a differential function in the absence of BRCA1. We speculate that in BRCA1-mutant cells, components of the Shieldin complex cannot be removed from DNA lesions, thereby disrupting replication fork dynamics and causing PARPi sensitivity. Therefore, in AIM2, we will test the hypothesis that (i) BRCA1 regulates Shieldin localization and function at DSBs, and (ii) Shieldin is recruited to specific DNA “scars” where the loss of Shieldin confers PARPi resistance. Investigating the dynamics of these end resecting factors and their regulation in DSB repair bears significant clinical relevance in combating PARPi resistance in BRCA1-mutant tumors. Therefore, in AIM3, we determine how alterations in 53BP1-dependent mechanisms of attenuated DNA end resection may drive the development of PARPi resistance in high-grade serous ovarian cancer using patient-derived xenografts and primary tumors.
