Characterization of a head and neck cancer-derived cell line panel confirms the distinct TP53-proficient copy number-silent subclass

Abstract

Introduction: Head and neck squamous cell carcinomas (HNSCC) arise in the mucosal lining of the upper aerodigestive tract. Risk factors are exogenous carcinogen exposure, human papillomavirus (HPV) infection, and genetic predisposition such as Fanconi anemia (FA). Clinically, tumors are stratified based on stage, site and HPV-status. The majority of HPV-positive and -negative HNSCC is characterized by frequent copy number (CN) changes and an abrogated p53-pathway. A third genetically-defined HPV-negative subclass of HNSCC is emerging: tumors that lack gross chromosomal changes (CN-silent), are mostly TP53-proficient, and have a relatively favorable prognosis.

Methods: A representative panel of HPV-positive, HPV-negative and FA-HNSCC-derived cell lines was genetically characterized.

Results: Despite apparent differences in etiology, FA-HNSCC cell lines show comparable genetic alterations as sporadic non-FA-HNSCC-derived cell lines. Furthermore, we identified a near diploid CN-silent HPV-negative HNSCC line: VU-SCC-040. Molecular characterization uncovers the absence of TP53 mutations, a functional p53-pathway and a CASP8 mutation. TP53 gene knockout using CRISPR-Cas9 resulted in resistance to MDM2 inhibition. Whereas p53-status is often proposed as a predictive biomarker for treatment response, TP53-knockout did not change sensitivity to cisplatin, Chk1 and Wee1 inhibition. Additionally, 84 CN-silent tumors were identified in the HNSCC PanCancer cohort and shown to be enriched for female gender, HRAS and CASP8 mutations.

Conclusion: FA-derived HNSCC cell lines share comparable CN-profiles and mutation patterns as sporadic HPV-negative HNSCC. In contrast, a subclass of CN-silent, HPV-negative and TP53 wild-type HNSCC separates from the majority of HNSCC tumors. We show that VU-SCC-040 is a HNSCC cell model representative of this subclass.

Keywords: Copy number silent; Fanconi anemia; Head and neck squamous cell carcinoma; Low-coverage whole genome sequencing; TP53 wild-type; Target-enrichment sequencing; Targeted treatment; p53 pathway.

Figure 1. Low-coverage sequencing results of a representative panel of HNSCC cell lines.

Copy number profiles of a panel of HNSCC cell lines were obtained using low coverage whole-genome sequencing. Using the ACE package [19], the absolute copy numbers of all chromosomes per cell line was determined. Red represents a gain of the chromosomal region, blue a loss, white is neutral (no genomic aberration). All HNSCC lines, except for VU-SCC-040, showed large copy number aberrations with a high frequency of gains and losses. a ACE analysis revealed that 5 cell lines were near diploid, of which one HPV-positive cell line (UM-SCC-47) and one FA-HNSCC cell line (CCH-FAHNSCC-2). Interestingly, VU-SCC-040, a HPV-negative HNSCC line showed a copy number silent profile with just 3 single gains. b The majority of HNSCC lines (n=19) exhibited many copy number gains and losses, and became aneuploid to near 3N.

Figure 2. Haloplex targeted sequencing of frequently mutated genes in HNSCC.

Schematic overview of the gene mutations per cell line, obtained with targeted Haloplex sequencing. A red box represents a missense mutation, green a nonsense mutation, yellow a frameshift deletion, blue a frameshift insertion, purple a splice site mutation, grey an exomic deletion, orange both a missense mutation and a frameshift deletion, blue both a missense mutations and a splice site mutation. For CDKN2A encoding the p16 protein, the number between brackets indicates a single (1) or homozygous (2) loss of the locus. The mutation annotations per cell line and gene can be found in table S3.

Figure 3. Characterization of HPV-negative, CN-silent and TP53 wild-type cell line VU-SCC-040.

a Genomic CN-profile of copy number silent cell line VU-SCC-040, which only contains a single gain of 3q, 8q and 9p/q. b Western blot analysis of p53 and downstream target p21 in TP53-mutant cell lines VU-SCC-096, VU-SCC-120 and UM-SCC-22A, compared with CN-silent TP53 wild-type lineVU-SCC-040. VU-SCC-040 showed lower levels of p53 compared to the TP53 mutated cell lines, as expected. Furthermore, p21 expression was higher compared to the TP53 mutated cell lines, although UM-SCC-22A showed an intermediated p21 expression level. c-d Protein expression of p53 and p21 in TP53 wild-type cell line VU-SCC-040 (c) and TP53-null line UM-SCC-6 (d) was analyzed after treatment of cisplatin with the EC₉₀ concentration for the indicated timespans. Before treatment, levels of p53 and p21 are stable in VU-SCC-040. Upon treatment of cisplatin, p53 expression is increased within 4 hours, and p21 levels increase after 24 hours, strongly suggesting that this part of the pathway is intact. In UM-SCC-6, p53 expression is absent due to a frameshift insertion in exon 1 and consequently, p21 levels do not change upon treatment of cisplatin. e. 24 hours post-irradiation with 2, 4 and 6 Gy, an increase of p53 was observed for 4 and 6 Gy in VU-SCC-040. f. p53 protein expression was investigated in the parental VU-SCC-040^wt and CRISPR TP53-knockout line VU-SCC-040^TP53. Only a very low p53 expression was observed in the knockout line compared to the wild-type line, in line with the TIDE sequence analysis, and indicating that the majority of cells obtained a TP53 knockout. g. mRNA expression of TP53 was significantly reduced in the knockout cell line compared to the parental wild-type VU-SCC-040. Although this is not necessarily observed with a CRISPR-Cas9 approach, it confirms nonsense mediated decay of the RNA levels and the successful knockout of the gene. h. VU-SCC-040^wt cells are very vulnerable to MDM2 inhibition with Nutlin-3a, as expected for a cell with a functional p53-pathway. For the VU-SCC-040^TP53 knockout line, resistance to Nutlin-3a was obtained, in line with the EC₅₀-values of the TP53-mutant HNSCC lines (figure S2f–h). i.-k. Dose-response curves of VU-SCC-040^wt and VU-SCC-040^TP53 showing the relative cell viability after 72h treatment with a dilution range of Cisplatin (i), Chk1 inhibitor Rabusertib (LY2603618) (j) and Wee1 inhibitor Adavosertib (AZD1775 / MK-1775) (k). Surprisingly, no difference in response was found between VU-SCC-040^wt and VU-SCC-040^TP53, and the EC₅₀ values were within the range of TP53-mutant HNSCC cell lines, and sensitivity much higher than normal cells ([15,37] and van Harten et al., submitted). This indicates that the cell cycle is perturbed in VU-SCC-040, but apparently with an intact p53 pathway.