Senataxin modulates resistance to cisplatin through an R-loop mediated mechanism in HPV-associated Head and Neck Squamous Cell Carcinoma

Introduction: Oropharyngeal Squamous Cell Carcinoma (OPSCC) is a site defined subtype of head and neck cancer with two distinct clinical subtypes: HPV-associated (HPV+) and HPV-independent (HPV-); both of which are commonly treated with chemoradiotherapy involving cisplatin. Cisplatin creates DNA crosslinks, which lead to eventual cell death via apoptosis. Clinical outcomes in HPV- OPSCC are poor and although HPV+ has an improved response to therapy, a subset of patients suffer from distant metastases, with a poor prognosis. Therefore, there is a need to understand the molecular basis underlying treatment resistance. A common mechanism of chemotherapy resistance is upregulation of DNA repair, and a major source of endogenous DNA damage are DNA/RNA hybrids, known as R-loops. R-loops are three stranded DNA/RNA hybrids formed in the genome as a by-product of transcription and are normally transient; however, they can persist and become a source of genomic instability. The contribution of R-loops to the development of cisplatin resistance in OPSCC is unknown. Methods: HPV+ and HPV- cisplatin resistant cell lines were developed, and RNA-sequencing was used to investigate changes in gene expression. Changes in R-loop dynamics were explored using slot blots and DRIP-qPCR. The effect of depleting known R-loop regulators on cisplatin sensitivity was assessed using siRNA. R-loop burden in a cohort of HPV+ and HPV- OPSCC tumours was explored using S9.6 immunohistochemistry. Results: Development of cisplatin resistant clones led to changes in gene expression consistent with resistance, alongside alterations in the expression of known R-loop regulators. Both HPV+ and HPV- resistant cells had elevated global R-loop levels and in HPV+ resistant cells there was a corresponding upregulation of the R-loop resolving protein, senataxin, which was not observed in HPV- resistant cells. Depletion of senataxin led to increased sensitivity to cisplatin in both HPV+ and HPV- resistant cells, however, the effect was greater in HPV+ cells. Quantification of R-loop levels by S9.6 immunohistochemistry revealed that HPV+ tumours and tumours with bone metastases had a higher R-loop burden. Conclusion: R-loops are involved in modulating sensitivity to cisplatin and may represent a potential therapeutic target. ### Competing Interest Statement The authors have declared no competing interest.