Impaired DNA double-strand break repair and effective radiosensitization of HPV-negative HNSCC cell lines through combined inhibition of PARP and Wee1

Agnes Oetting; Sabrina Christiansen; Fruzsina Gatzemeier; Sabrina Köcher; Lara Bußmann; Arne Böttcher; Katharina Stölzel; Anna Sophie Hoffmann; Nina Struve; Malte Kriegs; Cordula Petersen; Christian Betz; Kai Rothkamm; Henrike Barbara Zech; Thorsten Rieckmann

doi:10.1016/j.ctro.2023.100630

Impaired DNA double-strand break repair and effective radiosensitization of HPV-negative HNSCC cell lines through combined inhibition of PARP and Wee1

Clin Transl Radiat Oncol. 2023 Apr 20:41:100630. doi: 10.1016/j.ctro.2023.100630. eCollection 2023 Jul.

Authors

Agnes Oetting^{1

2}, Sabrina Christiansen^{1

2}, Fruzsina Gatzemeier^{1

2}, Sabrina Köcher^{1

2}, Lara Bußmann^{2

3}, Arne Böttcher², Katharina Stölzel², Anna Sophie Hoffmann², Nina Struve^{1

3}, Malte Kriegs¹, Cordula Petersen¹, Christian Betz², Kai Rothkamm¹, Henrike Barbara Zech^{1

2

3}, Thorsten Rieckmann^{1

2}

Affiliations

¹ Department of Radiotherapy, University Medical Center Hamburg Eppendorf, Germany.
² Department of Otorhinolaryngology, University Medical Center Hamburg Eppendorf, Germany.
³ Mildred-Scheel Cancer Career Center HaTriCS4, University Medical Center Hamburg-Eppendorf, Germany.

Abstract

Objectives: In head and neck squamous cell carcinoma (HNSCC), tumors negative for Human Papillomavirus (HPV) remain a difficult to treat entity and the morbidity of current multimodal treatment is high. Radiotherapy in combination with molecular targeting could represent suitable, less toxic treatment options especially for cisplatin ineligible patients. Therefore, we tested dual targeting of PARP and the intra-S/G2 checkpoint through Wee1 inhibition for its radiosensitizing capacity in radioresistant HPV-negative HNSCC cells.

Materials and methods: Three radioresistant HPV-negative cell lines (HSC4, SAS, UT-SCC-60a) were treated with olaparib, adavosertib and ionizing irradiation. The impact on cell cycle, G2 arrest and replication stress was assessed through flow cytometry after DAPI, phospho-histone H3 and γH2AX staining. Long term cell survival after treatment was determined through colony formation assay and DNA double-strand break (DSB) levels were assessed through quantification of nuclear 53BP1 foci in cell lines and patient-derived HPV± tumor slice cultures.

Results: Wee1 and dual targeting induced replication stress but failed to effectively inhibit radiation-induced G2 cell cycle arrest. Single as well as combined inhibition increased radiation sensitivity and residual DSB levels, with the largest effects induced through dual targeting. Dual targeting also enhanced residual DSB levels in patient-derived slice cultures from HPV-negative but not HPV+ HNSCC (5/7 vs. 1/6).

Conclusion: We conclude that the combined inhibition of PARP and Wee1 results in enhanced residual DNA damage levels after irradiation and effectively sensitizes radioresistant HPV-negative HNSCC cells. Ex vivo tumor slice cultures may predict the response of individual patients with HPV-negative HNSCC to this dual targeting approach.

Keywords: Cell-cycle checkpoint; DNA repair; Head and neck cancer; PARP; Radiosensitization; Radiotherapy; Wee1.