Whole Genome CRISPR-Cas9 Screen Identifies STING as a Determinant of Intrinsic Radiosensitivity in Head and Neck Squamous Cell Carcinoma

Abstract

Radiation therapy is a critical component of curative therapy for HNSCC. However, the mechanisms of radioresistance in HPV-Negative HNSCC, a molecularly distinct disease with poor prognosis relative to HPV-Positive HNSCC, are incompletely understood. Although RNA interference based screening methods have been used to understand resistance to radiotherapy, they are hampered by off-target effects and partial target suppression. The recent introduction of CRISPR technology has provided a more efficient method for gene targeting and suppression. As such we hypothesized that a whole genome CRISPR-Cas9 screen in HPV-Negative HNSCC would identify novel components of the cellular response to ionizing radiation. A whole genome CRISPR-Cas9 library containing 123,411 guide RNAs (gRNAs) targeting 19,050 genes was amplified, sequenced to ensure diversity, and packaged into lentivirus. HPV-Negative HNSCC cells (160 million) were transduced with lentivirus in a pooled format and irradiated. Following irradiation, 50 million cells per condition were collected (400x coverage) and gRNA enrichment or depletion determined via MAGeCK analysis of next-generation sequencing (NGS) results. Clonogenic assay (CA) was used to determine the effects of gene knockout (KO) on radiation and cisplatin sensitivity. Immunoblotting was performed to determine effects of knockout on protein expression. Tumor growth delay assays were performed using tumor cells implanted subcutaneously in athymic nude mice. Cas9 expression alone showed no effect on radiosensitivity by CA indicating feasibility to proceed with genetic screening. Analysis of NGS results show the top-ranked gRNAs enriched after radiation target STING (stimulator of interferon genes; p < 0.001; FDR <0.01), suggesting that STING KO imparts a radioresistant phenotype. Isogenic STING KO cell lines were established by CRISPR-Cas9 KO. CA shows that STING KO significantly enhanced radiation survival in multiple KO lines by a factor of 5-10. STING KO also conferred resistance to cisplatin treatment (3-7 fold) in CAs suggesting that STING regulates an intrinsic response to DNA damage. To determine the effects of STING KO on in vivo radiosensitivity, tumor growth delay assays were performed in immunodeficient mice. STING KO tumors were significantly more radioresistant when compared to isogenic STING proficient tumors (p< 0.05) with a dose modifying factor of 0.35. Whereas STING has previously been shown to regulate the extrinsic (immune-based) response of tumors to radiation, our data suggest that STING, as identified by a whole genome CRISPR-Cas9 screen, regulates the intrinsic response to radiation in HNSCC. This supports future investigation of strategies activating the STING pathway in combination with radiotherapy to enhance therapeutic response.