Michael Longo

Introduction. The breast cancer susceptibility genes BRCA1 and BRCA2 were originally identified as the genetic source of familial breast cancer ^1,2 and have since been used to identify one’s own risk of breast cancer development. Carriers of BRCA1 mutations have a 72% chance of developing breast cancer; 69% chance for BRCA2 carriers. Subsequently, BRCA1 & 2 protein:protein interactions with DNA repair factors were identified and characterized, thus associating these mutations with DNA repair deficits ^3,4. An overlap of DNA lesions produced in Poly ADP Ribose Polymerase (PARP)-deficient cells and similar unrepaired lesions in BRCA-mutant cells ^5-7 lead to the development of PARP inhibitors (PARPi) as BRCA therapies^8,9. Initial success lead to rapid clinical approval¹⁰, though instances of PARPi resistance emerged ¹¹. Studies have now further analyzed the molecular mechanisms of PARPi’s role in targeting BRCA mutations and in the development of resistance^12-15. Methods In further elucidating the mechanism of resistance development, 3 approaches were used to identify molecular “networks” involved: an siRNA library, CRISPR-Cas9 knock-out library, and a proteomics/pulldown strategy ^12,15-19. Results Using a siRNA-knock-down library, inhibiting expression of REV1 was found to diminish PARPi sensitivity in BRCA1 cells ^12,17. Lack of REV1 was found to restore the initiating step (DNA end resection) of DNA repair via homologous recombination. A CRISPR-Cas9 knock-out library was used to further expand on the REV1 network, finding it to be part of a larger SHIELDIN complex which appears to protect (“shield”) DNA ends from resection ^18,19. Independently, a proteomics approach involving isolating proteins-of-interest and identifying co-purified interacting proteins, also identified the SHIELDIN complex as involved in DNA-end processing ¹⁶. With respect to BRCA2-PARPi resistance, a CRISPR-Cas9 knock-out library found that knock-out of RNaseH re-introduced PARPi sensitivity¹⁵. Further investigation revealed that RNaseH2 and Topoisomerase 1 (TopI) both remove aberrant incorporation of ribonucleotides throughout the genome, but TopI is dependent on PARP1 activity. Thus, knocking-out RNaseH2 leaves the removal of these misincorporated ribonucleotides dependent on a PARP1-susceptible pathway. Conclusions The susceptibility of BRCA1 mutations to PARPi can be overcome by subsequent acquired mutation in factors the protect broken DNA ends from nucleolytic processing. The Shieldin complex was identified as a molecular component protecting DNA ends, likely from aberrant processing. Also, PARP inhibitors function not only in blocking the sealing of single strand breaks/nicks (which lead to double strand breaks when encountered by replication forks), but also by producing persistent TOP1-DNA adducts (also leading to dsDNA breaks).

1. Wooster, R. et al. Identification of the breast cancer susceptibility gene BRCA2. Nature 378, 789-792, doi:10.1038/378789a0 (1995).
2. Hall, J. M. et al. Linkage of early-onset familial breast cancer to chromosome 17q21. Science 250, 1684-1689, doi:10.1126/science.2270482 (1990).
3. Pellegrini, L. et al. Insights into DNA recombination from the structure of a RAD51-BRCA2 complex. Nature 420, 287-293, doi:10.1038/nature01230 (2002).
4. Sharan, S. K. et al. Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature 386, 804-810, doi:10.1038/386804a0 (1997).
5. Saleh-Gohari, N. et al. Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks. Mol Cell Biol 25, 7158-7169, doi:10.1128/MCB.25.16.7158-7169.2005 (2005).
6. Saleh-Gohari, N. & Helleday, T. Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells. Oncogene 23, 9136-9141, doi:10.1038/sj.onc.1208178 (2004).
7. Saleh-Gohari, N. & Helleday, T. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res 32, 3683-3688, doi:10.1093/nar/gkh703 (2004).
8. Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917-921, doi:10.1038/nature03445 (2005).
9. Bryant, H. E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913-917, doi:10.1038/nature03443 (2005).
10. Fong, P. C. et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 361, 123-134, doi:10.1056/NEJMoa0900212 (2009).
11. Edwards, S. L. et al. Resistance to therapy caused by intragenic deletion in BRCA2. Nature 451, 1111-1115, doi:10.1038/nature06548 (2008).
12. Boersma, V. et al. MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end resection. Nature 521, 537-540, doi:10.1038/nature14216 (2015).
13. Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243-254, doi:10.1016/j.cell.2010.03.012 (2010).
14. Mirman, Z. et al. 53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polalpha-dependent fill-in. Nature 560, 112-116, doi:10.1038/s41586-018-0324-7 (2018).
15. Zimmermann, M. et al. CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions. Nature 559, 285-289, doi:10.1038/s41586-018-0291-z (2018).
16. Gupta, R. et al. DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity. Cell 173, 972-988 e923, doi:10.1016/j.cell.2018.03.050 (2018).
17. Xu, G. et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature 521, 541-544, doi:10.1038/nature14328 (2015).
18. Noordermeer, S. M. et al. The shieldin complex mediates 53BP1-dependent DNA repair. Nature 560, 117-121, doi:10.1038/s41586-018-0340-7 (2018).
19. Dev, H. et al. Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells. Nat Cell Biol 20, 954-965, doi:10.1038/s41556-018-0140-1 (2018).