Abstract SY33-01: Mechanism of tumor suppression by the Fanconi anemia proteins

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA Interstrand crosslink (ICL) repair takes place at sites where the two strands of the DNA have become covalently linked by the byproducts of lipid peroxidation, endogenous aldehydes, and yet unknown metabolites. The repair of these lethal lesions takes place during DNA replication and requires a dual excision of the crosslinked bases and repair of the resulting double-strand breaks. This feat is accomplished in a multi-step process mediated by the Fanconi anemia (FA) pathway and factors that promote Homologous Recombination (HR), including BRCA1 and BRCA2 (reviewed in (1)). FA patients lack components of this pathway and suffer from bone marrow failure and infertility presumably due to the inability to maintain hematopoietic and germline stem cells. FA is also associated with a very high incidence of cancer, most likely due to the mutagenic nature of incompletely repaired ICLs. On the other hand, the induction of ICL is a major method of cancer treatment and often leads to excellent outcomes (e.g. cisplatin treatment of testicular cancer). Major effort has been directed into the identification of all of the components of the ICL repair pathways and the elucidation of the mechanism of such repair in the hopes of understanding how it suppresses tumorigenesis. Fanconi anemia registry (IFAR) housed at the Rockefeller University has been collecting patient samples and information since 1982. Using candidate gene sequencing of patient samples from the IFAR, we first found SLX4 as a new FA gene. SLX4 is a scaffold protein that interacts with three nucleases: XPF, MUS81, and SLX1 (2-4). We showed that this “Swiss army knife of repair” is mutated in patients with typical features of Fanconi anemia (5). Using SLX4 null patient cell lines, we explored the nuclease requirements in different DNA repair pathways during DNA replication and showed that the SLX4-bound XPF nuclease is essential for the ICL repair (6). This has been recently validated by work that identified XPF as a gene mutated in FA (7). In a separate study, we demonstrated that SLX4 is an important regulator of processing of replication intermediates including the Holliday junctions even under conditions of unperturbed DNA replication (8). This activity, although important for normal cellular growth, can be very mutagenic and thus cancer inducing. In our newest work, we have concentrated on a 12-year-old patient who was born with radial dysplasia, absent right thumb and tethered cord; the patient also exhibited enuresis and several absent permanent teeth. Marrow function is normal to date. No mutations in any of the known FA genes were identified. Whole exome sequencing identified an unexpected novel de novo mutation in RAD51. RAD51 is the mammalian homolog of RecA, mediating the homology search during HR. Although RAD51 foci formation is reduced and delayed following treatment with a range of DNA damaging agents, the patient fibroblasts do not exhibit sensitivity to ionizing radiation. In addition, the patient fibroblasts show wild type levels of sister chromatid exchanges following mitomycin C treatment, and are capable of performing HR for repairing dysfunctional GFP gene, as assessed by the DR-GFP assay. Collectively, these results suggest HR repair of double strand breaks is not impaired in the patient cells. Patient fibroblasts are hypersensitive to crosslinking agents and PARP inhibition. Following mitomycin C treatment, the cells exhibit elevated levels of both RPA foci formation and phosphorylation, indicating that RAD51 inhibits resection of nascent DNA strands, thereby stabilizing the ICL for its subsequent repair. Our study of the RAD51 patient mutation identifies the essential function of RAD51 at sites of stalled replication forks that is critical for the accurate repair of ICLs. Our results also suggest that the tumor suppressive role of RAD51-interacting proteins, including BRCA2, RAD51C, and RAD51D (all implicated in breast and ovarian cancer development) may exert their effect through this stabilizing activity of RAD51 at stalled replication forks. References 1. Kottemann MC, Smogorzewska A. Fanconi anaemia and the repair of Watson and Crick DNA crosslinks. Nature2013 Jan 17;493(7432):356-63. 2. Fekairi S, Scaglione S, Chahwan C, Taylor ER, Tissier A, Coulon S, Dong MQ, Ruse C, Yates JR, 3rd, Russell P, Fuchs RP, McGowan CH, Gaillard PH. Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases. Cell2009 Jul 10;138(1):78-89. 3. Munoz IM, Hain K, Declais AC, Gardiner M, Toh GW, Sanchez-Pulido L, Heuckmann JM, Toth R, Macartney T, Eppink B, Kanaar R, Ponting CP, Lilley DM, Rouse J. Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol Cell2009 Jul 10;35(1):116-27. 4. Svendsen JM, Smogorzewska A, Sowa ME, O'Connell BC, Gygi SP, Elledge SJ, Harper JW. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair. Cell2009 Jul 10;138(1):63-77. 5. Kim Y, Lach FP, Desetty R, Hanenberg H, Auerbach AD, Smogorzewska A. Mutations of the SLX4 gene in Fanconi anemia. Nat Genet2011 Feb;43(2):142-6. 6. Kim Y, Spitz GS, Veturi U, Lach FP, Auerbach AD, Smogorzewska A. Regulation of multiple DNA repair pathways by the Fanconi anemia protein SLX4. Blood2013 Jan 3;121(1):54-63. 7. Bogliolo M, Schuster B, Stoepker C, Derkunt B, Su Y, Raams A, Trujillo JP, Minguillon J, Ramirez MJ, Pujol R, Casado JA, Banos R, Rio P, Knies K, Zuniga S, Benitez J, Bueren JA, Jaspers NG, Scharer OD, de Winter JP, Schindler D, Surralles J. Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia. Am J Hum Genet 2013 May 2;92(5):800-6. 8. Garner E, Kim Y, Lach FP, Kottemann MC, Smogorzewska A. Human GEN1 and the SLX4-Associated Nucleases MUS81 and SLX1 Are Essential for the Resolution of Replication-Induced Holliday Junctions. Cell Rep2013 Oct 17;5(1):207-15. Citation Format: Agata Smogorzewska. Mechanism of tumor suppression by the Fanconi anemia proteins. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY33-01. doi:10.1158/1538-7445.AM2014-SY33-01