Abstract B024: CRISPR-Cas9 screening reveals a novel JAK1 dependent mechanism of radioresistance in head and neck squamous cell carcinoma
Authors: Kelley Vanessa , Baro Marta , Gasperi William , Phoomak Chatchai , Lee Hojin , Contessa Joseph 
Published: January 2025

Abstract
Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide, and local failure of disease following treatment with radiation therapy remains a major challenge for improving patient outcomes. We therefore designed a CRISPR-Cas9 pooled genetic screen to identify signaling mechanisms that regulate HNSCC radiosensitivity. Using a kinome gRNA library (763 genes with 8 gRNAs each), Cal27 and Detroit562 cells were screened using fractionated exposure to ionizing radiation as a selection pressure. Deep sequencing results identified significant enrichment or depletion of gRNAs for DNA damage signaling (ATM and DNAPK) and NFKB signaling (IRAK4, IRAK1, IKKA, IKKB), pathways known to regulate cell survival after radiation. Surprisingly, the screen also identified significant depletion of Janus kinase signaling (JAK1 and TYK2) to be a cause of radiation resistance. Independent knockout (KO) of Janus Kinase 1 (JAK1) in both Cal27 and Detroit562 cells caused radioresistance in vitro and in vivo. JAK1 KO prolongs G2 cell cycle arrest in HNSCC after treatment with radiation or after thymidine synchronization. JAK1 KO cells also have enhanced Rad51 foci formation indicative of homologous recombination usage for DNA repair. Consistent with an enhanced mitotic arrest, JAK1 KO causes the appearance of cells with 4N and 8N DNA content after exposure to radiation as measured by flow cytometry with propidium iodide or following 5-ethynyl-2’deoxyuridine (EdU) labeling. HNSCC cells with JAK1 KO, or treated with the JAK1 specific inhibitor Abrocitinib, formed significantly fewer micronuclei after radiation treatment consistent with a reduction in mitotic errors. Consistent with these findings, live cell imaging demonstrated cells with JAK1 KO undergo fewer mitotic catastrophe events. Loss of JAK1 function did not alter CDK1, but delayed activation of Aurora Kinase A (AURKA) and Polo-like Kinase 1 (PLK1), identifying a cell cycle signaling pathway regulated by JAK1. Live cell imaging also showed a more than doubling of the time required from chromatin condensation to sister chromatid separation, suggesting an associated defect in kinetochore assembly. The effects of inhibiting Kif18a, a kinesin that regulates kinetochore tension, in combination with radiation was therefore tested. Kif18 inhibition significant radiosensitized JAK1 KO HNSCC in clonogenic survival and xenograft models. In summary we have elucidated a JAK1-dependent mechanism for the regulation of AURKA/PLK1 signaling, a protective G2 arrest, and radioresistance. Furthermore, we provide a mechanistic and targeted pharmacologic approach for overcoming therapeutic resistance and enhancing radiation therapy in HNSCC.
Citation Format: Vanessa Kelley, Marta Baro, William Gasperi, Chatchai Phoomak, Hojin Lee, Joseph Contessa. CRISPR-Cas9 screening reveals a novel JAK1 dependent mechanism of radioresistance in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B024.