Multilayered Omics-Based Analysis of a Head and Neck Cancer Model of Cisplatin Resistance Reveals Intratumoral Heterogeneity and Treatment-Induced Clonal Selection.

Platinum-based drugs, in particular cisplatin (cis-diamminedichloridoplatinum(II), CDDP), are used for treatment of squamous cell carcinoma of the head and neck (SCCHN). Despite initial responses, CDDP treatment often results in chemoresistance, leading to therapeutic failure. The role of primary resistance at subclonal level and treatment-induced clonal selection in the development of CDDP resistance remains unknown. By applying targeted next-generation sequencing, fluorescence hybridization, microarray-based transcriptome, and mass spectrometry-based phosphoproteome analysis to the CDDP-sensitive SCCHN cell line FaDu, a CDDP-resistant subline, and single-cell derived subclones, the molecular basis of CDDP resistance was elucidated. The causal relationship between molecular features and resistant phenotypes was determined by siRNA-based gene silencing. The clinical relevance of molecular findings was validated in patients with SCCHN with recurrence after CDDP-based chemoradiation and the TCGA SCCHN dataset. Evidence of primary resistance at clonal level and clonal selection by long-term CDDP treatment was established in the FaDu model. Resistance was associated with aneuploidy of chromosome 17, increased copy-numbers and overexpression of the gain-of-function (GOF) mutant variant p53 siRNA-mediated knockdown established a causal relationship between mutant p53 and CDDP resistance. Resistant clones were also characterized by increased activity of the PI3K-AKT-mTOR pathway. The poor prognostic value of GOF variants and mTOR pathway upregulation was confirmed in the TCGA SCCHN cohort. Our study demonstrates a link of intratumoral heterogeneity and clonal evolution as important mechanisms of drug resistance in SCCHN and establishes mutant GOF variants and the PI3K/mTOR pathway as molecular targets for treatment optimization. .