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What is wrong with Fanconi anemia cells?

Fanconi anemia (FA) patient cells are very sensitive to agents that interfere with DNA replication, such as DNA interstrand crosslinks (ICLs).  Even in the absence of exogenous genotoxic stress, FA cells have abnormally elevated DNA damage response (DDR). In patients, this constitutively active DDR is most prominent in rapidly dividing tissue and correlates with bone marrow failure. Understanding the underlying cause of the overactive DDR in FA cells could provide clues for therapeutic intervention, to mitigate bone marrow failure or prevent malignancy in FA patients. Given that the DDR is also abnormal in tumors that evolve from loss of FA proteins including the hereditary breast cancer genes, these tumors are very sensitive to crosslinking chemotherapy agents, such as platinum agents. Chemotherapies that further exacerbate the DDR could also selectively cripple BRCA-associated tumors.

Pathways linked to the aberrant DDR. Clues to what causes the DDR in FA cells has come from the identification of proteins or pathways whose inactivation reduces the DDR. In particular, the nonhomologous end-joining (NHEJ) pathway contributes to an exacerbated DDR, genomic instability and DNA crosslink hypersensitivity in FA cells. Loss of NHEJ factor, Ku in FANCC mutant cells enhanced ICL resistance and reduced genomic instability 1. Inhibition of the NHEJ factor, DNA-PKcs enhanced ICL resistance in cells deficient in FANCD2, FANCA or FANCC 2. Loss of 53BP1 restored homologous recombination (HR) and rescues the lethality in Brca1-/- mice 3-6. However, loss of NHEJ does not uniformly rescue FA cells.  For example, ICL resistance is not improved in cells that are deficient for the BRCA1-interacting helicase FANCJ (BACH1/BRIP1) 2,7.  Moreover, in Fancd2-null mouse cells inactivation of NHEJ exacerbated ICL sensitivity and mice had more severe developmental defects 8,9. Thus, we sought to identify other repair pathways that are causative in FA defects.

MSH2 is linked to aberrant DDR. We considered that proteins of the mismatch repair (MMR) pathway could be problematic in FA cells given that the FANCJ interaction with the MMR protein, MLH1 is essential for ICL repair 10. Indeed, we found that depletion of the MMR protein, MSH2 suppresses defects found in cells deficient for the FANCJ-MLH1 interaction, including ICL-induced sensitivity, chromosomal aberrations, abnormal G2/M accumulation, and as well as an over-active NHEJ pathway. Furthermore, we found that MSH2 loss also suppresses defects in cells deficient for BRCA1 or FANCD2, and this rescue was confirmed in Fancd2-null mouse cells. Given that MSH2 depletion did not suppress ICL sensitivity in FANCA deficient cells 7 or FANCM-null chicken cells 11, regulation of MMR function in ICL processing or at stalled forks may be restricted to components of the FA pathway that crosstalk with MMR proteins. Normally, the interaction between FANCJ and MLH1 could serve to coordinate the DDR and to prevent unproductive MMR processing that impedes the recovery of cells following replication stress. Indeed, our data indicate that at stalled replication forks, MSH2 interferes with replication restart in cells lacking the FANCJ-MLH1 interaction 7

Clinical Implication. It is still unclear how to best reduce the DDR in FA to mitigate bone marrow failure. MMR-deficiency is linked to hematologic malignancy in mice 12,13, conversion of anemia to cancer in myelodysplastic syndrome 14,15, and chemoresistance in patients 16,17. Thus, targeting MSH2 could reduce risk of bone marrow failure but at the same time enhance the risk of leukemia in FA patients.  A more effective approach could be to find therapies that eliminate or reduce the DNA structures that MSH2 or other repair proteins bind. We envision that the BRCA-FA pathway is a “comb” for the genome” that eliminates “knots” that are enriched upon replication stress (Figure 1).  Current efforts include strategies to identify these knots/DNA structures that attract MSH2 associated complexes.  If these MMR bound knots also contribute to the elevated DDR in tumors that evolve from loss of BRCA-FA proteins, further activation of MMR in the course of cisplatin could generate insurmountable replication stress.  This hypothesis is currently being tested in the lab. 

 Figure 1: BRCA-FA pathway; a comb for the genome.

Figure 1: BRCA-FA pathway; a comb for the genome.

1        Pace, P. et al. Ku70 corrupts DNA repair in the absence of the Fanconi anemia pathway. Science 329, 219-223, doi:10.1126/science.1192277 (2010).

2        Adamo, A. et al. Preventing nonhomologous end joining suppresses DNA repair defects of Fanconi anemia. Molecular cell 39, 25-35, doi:10.1016/j.molcel.2010.06.026 (2010).

3        Cao, L. et al. A selective requirement for 53BP1 in the biological response to genomic instability induced by Brca1 deficiency. Molecular cell 35, 534-541, doi:10.1016/j.molcel.2009.06.037 (2009).

4        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).

5        Bunting, S. F. & Nussenzweig, A. Dangerous liaisons: Fanconi anemia and toxic nonhomologous end joining in DNA crosslink repair. Molecular cell 39, 164-166, doi:10.1016/j.molcel.2010.07.016 (2010).

6        Bouwman, P. et al. 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nature structural & molecular biology 17, 688-695, doi:10.1038/nsmb.1831 (2010).

7        Peng, M., Xie, J., Ucher, A., Stavnezer, J. & Cantor, S. B. Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses. The EMBO journal, doi:10.15252/embj.201387530 (2014).

8        Houghtaling, S. et al. Fancd2 functions in a double strand break repair pathway that is distinct from non-homologous end joining. Hum Mol Genet 14, 3027-3033 (2005).

9        Bunting, S. F. et al. BRCA1 Functions Independently of Homologous Recombination in DNA Interstrand Crosslink Repair. Molecular cell 46, 125-135 (2012).

10      Peng, M. et al. The FANCJ/MutLalpha interaction is required for correction of the cross-link response in FA-J cells. Embo J 26, 3238-3249 (2007).

11       Huang, M. et al. Human MutS and FANCM complexes function as redundant DNA damage sensors in the Fanconi Anemia pathway. DNA repair 10, 1203-1212 (2011).

12      Reitmair, A. H. et al. MSH2 deficient mice are viable and susceptible to lymphoid tumours. Nature genetics 11, 64-70 (1995).

13      Campbell, M. R., Nation, P. N. & Andrew, S. E. A lack of DNA mismatch repair on an athymic murine background predisposes to hematologic malignancy. Cancer research 65, 2626-2635 (2005).

14      Casorelli, I. et al. Drug treatment in the development of mismatch repair defective acute leukemia and myelodysplastic syndrome. DNA repair 2, 547-559 (2003).

15      Offman, J. et al. Defective DNA mismatch repair in acute myeloid leukemia/myelodysplastic syndrome after organ transplantation. Blood 104, 822-828 (2004).

16      Jiricny, J. The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 7, 335-346 (2006).

17      Karran, P. Mechanisms of tolerance to DNA damaging therapeutic drugs. Carcinogenesis 22, 1931-1937 (2001).