Abstract 3315: CRISPR-Cas9 screening reveals novel targets that govern HNSCC response to radiation therapy

Abstract

Abstract Purpose: Although Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide, the high failure rates for non-HPV associated tumors and the absence of new effective therapies has caused the survival rate for HNSCC to remain stagnant for the past decade. As radiation is a frontline treatment for HNSCC, we sought to uncover novel actionable targets that affect cellular responses to radiation therapy. Experimental Procedures: We performed a kinome-wide CRISPR-Cas9 knockout screen in UM-SCC47, FaDu, Cal27, and Detroit562 HNSCC cell lines using a multi-fraction radiation regimen as a selective pressure. These four cell lines were chosen to enhance molecular diversity as they represent the classical, atypical, basal, and mesenchymal transcriptional subtypes of HNSCC. sgRNAs from irradiated and unirradiated controls underwent Next Generation Sequencing, and the results were analyzed via MAGeCK analysis. This approach provided a systematic method to identify sgRNAs that were enriched (loss of gene causes radioresistance) or depleted (loss of gene causes radiosensitivity) following HNSCC cell radiation treatment. Results: Our results indicate that Cal27 and Detroit562 cell lines share multiple targets that govern cell responses to radiation despite the different mutational backgrounds and transcriptional subtypes. In particular, sgRNAs targeting ATM and PRKDC were significantly depleted in Cal27 and Detroit562 cells (FDR<0.01, p<0.01). ATM and PRKDC are regulators of the DNA damage response, and loss of function of these genes is known to radiosensitize cells. Therefore, depletion of sgRNAs targeting these genes serves as internal validation of our screening methodology. sgRNAs targeting JAK1 were enriched in the Cal27 and Detroit562 cell lines (FDR<0.01, p<0.01), suggesting that loss of JAK1 caused decreased radiation-induced cell death. As a role for JAK1 in the cell response to DNA damage has not been described, we sought to validate and characterize the role of JAK1 in HNSCC cell radiation treatment using JAK1 knockout (KO) cell lines. Clonogenic assays revealed that JAK1 KO cells were radioresistant when treated with both single and multi-fraction radiation regimens, validating the results of the CRISPR screen. We observed cell signaling changes consistent with loss of JAK1 including decreased phospho-STAT3 and IRF9 signaling. To uncover pathways that may contribute to JAK1 KO cell radioresistance, we performed bulk RNA sequencing of JAK1 KO and control cell lines. Our analysis revealed that JAK1 KO cells may undergo an epithelial-mesenchymal transition among other signaling changes. Conclusion: The use of a CRISPR-Cas9 screen with radiation as a selective pressure revealed both known and novel targets that govern HNSCC cell line responses to radiation therapy. Loss of JAK1 causes radioresistance, and its role in HNSCC DNA damage response warrants further investigation. Citation Format: Vanessa M. Kelley, Marta Baro, Aanchal Katoch, Chatchai Phoomak, Hojin Lee, Joseph Contessa. CRISPR-Cas9 screening reveals novel targets that govern HNSCC response to radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3315.