Treatment of head and neck squamous cell carcinoma (HNSCC) is characterized by high local recurrences mainly due to the DNA repair capacity of cancer cells. Selectively inhibiting these DNA repair mechanisms in combination with radiotherapy (RT) could increase locoregional tumor control. Moreover, recently we found that human papillomavirus positive (HPV+ )HNSCC cells have differences in their DNA repair efficiency compared to HPV- cells, suggesting differences in mechanisms of DNA repair and showing the need for different treatment approaches for HPV+ and HPV- HNSCC.
We performed a CRISPR/Cas9-based loss of function screen targeting 36 drugable genes in DNA damage response (DDR) to discover the most efficient therapeutic targets that are synthetically lethal in combination with RT for either HPV+, or HPV- tumors. We validated these results with commercially available drugs (NU7441 (DNA-PK inhibition); ABT-888 (PARP1/2 inhibition); AZD7762 (CHK1/2 inhibition)) by survival and clonogenic assays. We investigated the repair kinetics by gH2AX and RAD51 foci formation and the effect on cell cycle by flow cytometry. The therapeutic efficacy in cell line based and PDX mice models was assessed by tumor growth delay curves.
Our results revealed 14 hits for HPV+ and 18 hits for HPV- HNSCC, showing the presence of differences in DDR between HPV+ and HPV- HNSCC. Inhibition of PARP radiosensitized HPV+ HNSCC cells. This effect was due to p16-mediated inhibition of homologous recombination repair. This p16-dependent effect also translated in a slower tumor regrowth in vivo models. CHEK genes were important for survival of HPV- HNSCC. The majority of the overlapping genes were involved in the non-homologous end-joining pathway. Validation of these synthetic lethal hits with DNA-PK inhibitor (NU7441) radiosensitized HPV+ and HPV- HNSCC in vitro and in vivo.
Our results lead to robust assessment of novel targeted radiosensitizers for HPV+ and HPV- HNSCC.