Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies well new disease models are urgently needed. We used CRISPR/Cas9 editing tools in order interrupt the human FANCA gene by generation insertions/deletions (indels) exon 4 two cancer lines from sporadic HNSCC having no mutation FA-genes: CAL27 CAL33 cells. Our approach allowed efficient editing, subsequent purification single-cell clones, Sanger sequencing validation at edited locus. Clones frameshift indels homozygosis did not express protein were selected for further analysis. When compared with parental CAL33, FANCA-mutant clones displayed a FA-phenotype they (i) highly sensitive DNA interstrand crosslink (ICL) agents such mitomycin C (MMC) or cisplatin, (ii) do monoubiquitinate FANCD2 upon MMC treatment therefore (iii) form nuclear foci, (iv) increased chromosome fragility G2 arrest after diepoxybutane (DEB) treatment. These similar growth rates their Interestingly, mutant cells acquire phenotypes associated more aggressive disease, migration wound healing assays. mutations phenocopies FA-HNSCC