Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 22 November 2020 / Approved: 30 November 2020 / Online: 30 November 2020 (11:55:45 CET)

A peer-reviewed article of this Preprint also exists.

@Article{genes12040548, AUTHOR = {Errazquin, Ricardo and Sieiro, Esther and Moreno, Pilar and Ramirez, María José and Lorz, Corina and Peral, Jorge and Ortiz, Jessica and Casado, José Antonio and Roman-Rodriguez, Francisco J. and Hanenberg, Helmut and Río, Paula and Surralles, Jordi and Segrelles, Carmen and Garcia-Escudero, Ramon}, TITLE = {Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia}, JOURNAL = {Genes}, VOLUME = {12}, YEAR = {2021}, NUMBER = {4}, ARTICLE-NUMBER = {548}, URL = {https://www.mdpi.com/2073-4425/12/4/548}, ISSN = {2073-4425}, 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.}, DOI = {10.3390/genes12040548} } @Article{genes12040548, AUTHOR = {Errazquin, Ricardo and Sieiro, Esther and Moreno, Pilar and Ramirez, María José and Lorz, Corina and Peral, Jorge and Ortiz, Jessica and Casado, José Antonio and Roman-Rodriguez, Francisco J. and Hanenberg, Helmut and Río, Paula and Surralles, Jordi and Segrelles, Carmen and Garcia-Escudero, Ramon}, TITLE = {Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia}, JOURNAL = {Genes}, VOLUME = {12}, YEAR = {2021}, NUMBER = {4}, ARTICLE-NUMBER = {548}, URL = {https://www.mdpi.com/2073-4425/12/4/548}, ISSN = {2073-4425}, 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.}, DOI = {10.3390/genes12040548} }

Journal reference: GENES 2021
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.

Subject Areas

Fanconi anemia; gene editing; FANCA; head and neck cancer; CRISPR/Cas9

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