Abstract 6947: CRISPR/Cas9 kinome screen identifies PNKP as a novel fitness gene in HPV-negative head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common malignancy worldwide. Most cases are linked to alcohol, tobacco, and areca nut, while a smaller proportion is driven by high-risk human papillomavirus (HPV). HPV-negative HNSCC is associated with poor prognosis with a 5-year survival rate of 40-50%. This dismal outcome has remained relatively static over the past several decades, highlighting the critical need for the discovery of novel targets and therapeutics to better manage HPV-negative HNSCC patients.Methodology: We performed parallel in vitro and in vivo CRISPR-Cas9 knockout kinome screens to identify kinases essential for the fitness of HPV-negative HNSCC cells. CAL27 Cas9 EGFP cells were transduced with a pooled sgRNA kinome library containing 6, 104 sgRNAs at a MOI of ∼0.4. After 7 days of puromycin selection, these transduced cells were cultured in vitro for either 7 or 14 days or implanted into the flanks of athymic nude mice, with tumor harvesting at 6 weeks. DNA was isolated from both cells and tumors, followed by next-generation sequencing to identify and quantify individual sgRNAs. Fitness genes identified in both the in vitro and in vivo screens were further validated using shRNA technology in a panel of HPV-negative HNSCC cell lines. Results: We identified 24 fitness genes that were common to both our in vitro and in vivo screening approaches. These kinases are primarily involved in pathways critical for DNA damage and repair, metabolism, and cellular proliferation. Among them, we prioritize the validation of polynucleotide kinase 3’phosphatase (PNKP) as an essential gene in a panel of HPV-negative HNSCC cell lines. shRNA-mediated knockdown of PNKP significantly reduced the cellular proliferation and clonogenic survival of CAL27, Detroit-562 and FaDu cells. Furthermore, PNKP knockdown severely impaired tumorigenicity in vivo, with CAL27 and FaDu xenografts showing markedly reduced tumor growth compared to controls. Phosphoproteomic analysis revealed that PNKP knockdown resulted in dephosphorylation of 762 unique sites (log2FC>1.5; FDR p<0.05), with a particular impact on proteins involved in nuclear and mitochondrial DNA repair pathways.Conclusion: Our study identified and confirmed PNKP as an essential gene for the survival of HPV-negative HNSCC cells. This discovery highlights the potential of PNKP as a novel therapeutic target, which may lead to the development of innovative therapeutics that could improve the clinical outcomes of HPV-negative HNSCC patients. Citation Format: Gurleen Kaur Tung, Kate Chatfield-Reed, Masaru Miyagi, Wendi Quinn O'Neill, Quintin Pan. CRISPR/Cas9 kinome screen identifies PNKP as a novel fitness gene in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6947.