FANCN Hypomorphic Mutation Retains BRCA1 Binding Domain

Introduction: Fanconi anemia (FA) is a recessive genetic disease that can result in bone marrow failure, morphological defects and cancer predisposition. Patients with mutations in the FA gene, FANCN/PALB2 (partner and localizer of BRCA2) typically have more severe disease with earlier onset of cancer and bone marrow failure. FANCN is known to promote homologous recombination (HR) and contains an N-terminal BRCA1 binding domain, and a C-terminal BRCA2 binding domain. To further investigate the function of FANCN, we used recombinant adeno-associated virus (rAAV)-mediated gene targeting techniques to make a conditional null FANCN cell line. We targeted early exons 2 and 3 and found that removal of the conditional allele resulted in spontaneous chromosomal breaks and rearrangements that ultimately lead to chromosomal catastrophe within 48 hours. Due to the essential function of FANCN in genome stability, we were curious how FANCN patients can survive with biallelic loss of function mutations in FANCN. Methods: A hypomorphic FANCN cell line was created using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9)-mediated gene targeting of FANCN in exon 5. In addition, we complemented the FANCN conditional null cell line with four different FANCN cDNAs containing patient mutations with premature stop codons that completely eliminate or disrupt the N-terminal BRCA2 binding domain. These FANCN mutant cell lines have been investigated for expression of truncated protein and will be tested in their ability to bind and form foci with BRCA1 and BRCA2. Finally, these FANCN mutant cell lines will be evaluated in their sensitivity to mitomycin C (MMC), PARP inhibitors, replication stress, and in their ability to repair DNA. Results: We have determined that FANCN is essential for maintaining genomic stability and cellular viability. However, contrary to the lethality of deleting exons 2 and 3, the biallelic frameshift mutations in exon 5 resulted in a viable FANCN mutant cell line. These hypomorphic FANCN exon 5 frameshift mutations truncate the C-terminal BRCA2 binding domain, and this cell line has a growth defect and MMC sensitivity. Furthermore, we have demonstrated that the patient mutation complemented cell lines are expressing truncated FANCN proteins that will be functionally characterized. Conclusions: We are beginning to unveil important and separate functional domains of FANCN. For example, the FANCN N-terminus with the BRCA1 binding domain serves an essential function in viability and genomic stability, whereas the FANCN C-terminus with the BRCA2 binding domain servers an essential role in cell proliferation and MMC resistance. This research is expected to continue to advance our mechanistic understanding of FANCN and how these FANCN patient mutations result in disease. In addition, these novel FANCN cell lines provide a means for screening potential new therapeutics for the treatment of Fanconi anemia. Disclosures Hendrickson: Horizon Discovery , Humanetics and Intellia Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: licensing fees, Research Funding.