RNA sequencing of Cal33 and SAS head and neck squamous cell carcinoma cell lines treated with GSK-J1 and radiation

Background: The sensitivity of head and neck squamous cell carcinoma (HNSCC) to ionizing radiation, among others, is determined by the number of cells with high clonogenic potential and stem-like features. These cellular characteristics are dynamically regulated in response to treat-ment and may lead to an enrichment of radioresistant cells with a cancer stem cell (CSC) pheno-type. Epigenetic mechanisms, particularly DNA and histone methylation, are key regulators of gene-specific transcription and cellular plasticity. Therefore, we hypothesized that specific epi-genetic targeting may prevent irradiation-induced plasticity and may sensitize HNSCC cells to radiotherapy. Methods: We compared the DNA methylome and intracellular concentrations of tricarboxylic acid cycle metabolites in radioresistant FaDu and Cal33 cell lines with their parental controls, as well as aldehyde dehydrogenase (ALDH)-positive CSCs with negative controls. Moreover, we conducted a screen of a chemical library targeting enzymes in-volved in epigenetic regulation in combination with irradiation and analyzed the clonogenic potential, sphere formation, and DNA repair capacity to identify compounds with both radiosensi-tizing and CSC-targeting potential. Results: We identified the histone demethylase inhibitor GSK-J1, which targets UTX (KDM6A) and JMJD3 (KDM6B), leading to increased H3K27 tri-methylation, heterochromatin formation, and gene silencing. The clonogenic survival assay after siRNA-mediated knock-down of both genes radiosensitized Cal33 and SAS cell lines. Moreover, high KDM6A expression in tissue sections of patients with HNSCC was associated with improved locoregional control after primary (n = 137) and post-operative (n = 187) radio/chemotherapy. Conversely, high KDM6B expression was a prognostic factor for reduced overall survival. Conclusions: Within this study, we investigated cellular and molecular mechanisms underlying irradiation-induced cellular plasticity, a key inducer of radioresistance, with a focus on epigenetic alterations. We identified UTX (KDM6A) as a putative prognostic and therapeutic target for HNSCC patients treated with radiotherapy. Overall design: The HNSCC cell lines SAS and Cal33 were treated with GSK-J1 (IC5, 4.5 µM) with or without a single 4 Gy x-ray irradiation. After 24 hours treatment, RNA was isolated using the RNeasy mini kit (Qiagen; #74104) according to the manufacturer's recommendation, in triplicates. RNA concentrations were measured with the NanoDrop ND-1000 (Peqlab). RNA sequencing was performed at the Genomics and Proteomics Core Facility (GPCF, DKFZ, Heidelberg, Germany). Libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Prep Kit following the manufacturer's instructions. Libraries concentration and quality control were performed using Qubit (Invitrogen) and Tapestation (Agilent). The samples were sequenced in a 2 × 100 bp paired-end setting on an Illumina HiSeq 4000 system according to the manufacturer's protocol.