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

Ricardo Errazquin,Ramon Garcia-Escudero,Jordi Surralles,Pilar Moreno,Francisco J Roman-Rodriguez,Jorge Peral,Esther Sieiro,Carmen Segrelles,Francisco J Roman-Rodriguez,José Antonio Casado,José Antonio Casado,José Antonio Casado,José Antonio Casado,Helmut Hanenberg,Helmut Hanenberg,Francisco J Roman-Rodriguez,María José Ramirez,Corina Lorz,Corina Lorz,Jessica Ortiz,Paula Río,Paula Río,Paula Río,Paula Río,Francisco J Roman-Rodriguez,Carmen Segrelles

doi:10.3390/genes12040548

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.

Highlights

Fanconi anemia (FA) is a rare DNA repair deficiency disorder characterized by hypersensitivity to DNA interstrand crosslink (ICL) agents and chromosome instability [1,2]
As only a handful of head and neck squamous cell carcinoma (HNSCC) cell lines derived from FA patients exists worldwide, and the characteristics of the cell lines including the genetic information and the sensitivity profiles to anticancer compounds are largely unknown [9,10,11], we propose in this report to use genetic engineering on wellestablished HNSCC cell lines in order to generate new FA-deficient cell line models for studying FA-HNSCC
Biallelic Mutation in the FANCA Gene by CRISPR/Cas9 Editing in Non-FA HNSCC Cell

Summary

Introduction

Fanconi anemia (FA) is a rare DNA repair deficiency disorder characterized by hypersensitivity to DNA interstrand crosslink (ICL) agents and chromosome instability [1,2].In most cases the disease is autosomal recessive, with the exception of FANCB, which isX-linked, and mutations in the oligomerization domain of RAD51/FANCR, which are dominant negative. In most cases the disease is autosomal recessive, with the exception of FANCB, which is. FA patients display varying degrees of developmental abnormalities, bone marrow failure (BMF), and increased cancer incidence. FA is due to functional inactivation of any one of 23 FA genes involved in DNA repair. FA-associated proteins interact in a pathway to repair ICL known as the FA pathway or the FA–BRCA pathway [2]. The pathway involves detection of the DNA crosslink at the stalled replication fork, unhooking of the crosslink, local generation of a double-strand break, and the use of homologous recombination (HR) proteins downstream to repair the break. The management of the BMF disease has remarkably improved over the last

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Conclusion