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

Abstract

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

Fanconi anemia-isogenic head and neck cancer cell line pairs - a basic and translational science resource

“…We observed clear cell line- and growth medium-specific differences among FANC -isogenic cell line pairs, though these differences did not clearly segregate with FANC genotype. Comparable results have been reported by Errazquin et al is FANCA-mutant CAL-SCC-27 and CAL-SCC-33 cells (21). Figure 4A provides a representative example of the proliferation of two FANC -isogenic patient-derived cell line pairs in different growth media at 37°C in ambient (20%) oxygen.…”

“…FANCA- mutant sporadic sublines were generated by Cas9 dual guide RNA-targeted deletion of FANCA exon 2 and a portion of exon 3, or by Cas9 single guide RNA-targeted cleavage of FANCA exon 4 to promote mutagenic end-joining. Resulting FANCA mutations were verified by a combination of PCR deletion or mismatch cleavage screening, DNA sequencing and Western blot analysis, to confirm the presence of allelic mutations and the loss of FANCA protein expression (Figure 3A and 3B)(21). We were able to generate only single allele FANCA deletions in the HPV+ sporadic HNSCC cell line UM-SCC-47.…”

“…FANC transgene complementation of FA patient-derived cell lines conferred MMC resistance, with 3-to-10-fold higher MMC IC 50 values. In contrast, FANCA deletion in sporadic HNSCC cell lines sensitized cells to MMC, with 14-to-44-fold lower MMC IC 50 values (Table S2 and Table 1 of (21)).…”

“…FANCA -mutant sublines were developed in sporadic HNSCC by Cas9-mediated gene editing using dual guide RNA-targeted Cas9 deletion of a 5’ portion of FANCA, or single guide-targeted FANCA exon 4 cleavage to promote mutagenic end joining (21). Clonally-derived FANCA -mutant sublines were identified and characterized by colony deletion-specific PCR screening and/or Sanger sequencing prior to transgene complementation (21). FANC transgene complementations were performed by lenti- or retroviral transduction and by Cas9/Cas12a/CpfI-gRNA-targeted FANC transgene insertion into chromosome 4q Safe Harbor Site 231 (SHS231; (22)).…”

“…Retroviral transduction of the FANCA transgene vector S11FAIN was performed as previously described (21). Chromosomal SHS231 safe harbor site transgene cassettes were electroporated into FANC -deficient cells with expression plasmids encoding SHS231-specific gRNAs and Cas9 or Cas12a/Cpf1 nuclease.…”