{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:53:59Z","timestamp":1785405239544,"version":"3.56.0"},"reference-count":31,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T00:00:00Z","timestamp":1778544000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MR\/V028944\/1"],"award-info":[{"award-number":["MR\/V028944\/1"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"award":["MR\/V028944\/1"],"award-info":[{"award-number":["MR\/V028944\/1"]}],"id":[{"id":"https:\/\/ror.org\/03x94j517","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1R01CA256854"],"award-info":[{"award-number":["1R01CA256854"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"award":["1R01CA256854"],"award-info":[{"award-number":["1R01CA256854"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>The use of proton beam therapy (PBT), as a more precision-targeted radiotherapy technique, is increasing in the treatment of head and neck squamous cell carcinoma (HNSCC). PBT benefits from the precise delivery of the radiation dose to the tumour via the Bragg peak. However, challenges still remain in the treatment of HNSCC with radiotherapy, particularly with tumour radioresistance and recurrence, requiring strategies leading to radiosensitisation. There are added complexities with the use of PBT given the increase in linear energy transfer (LET) at and around the Bragg peak, which can cause an altered cellular response compared to low-LET radiation. Nevertheless, targeting the cellular DNA damage response is considered an important strategy to enhance tumour cell killing caused by radiotherapy. Therefore, using specific inhibitors against the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA-dependent protein kinase catalytic subunit (DNA-Pkcs), we investigated their impact in radiosensitising HPV-negative HNSCC cells to PBT of increasing LET. We demonstrate that inhibitors against ATR (AZD6738), and particularly ATM (AZD1390) and DNA-Pkcs (AZD7648), could significantly decrease clonogenic survival of HNSCC cell lines following PBT at both low and relatively high LET (~2 keV\/\u00b5m and ~8 keV\/\u00b5m, respectively). We confirmed that the inhibitors in combination with PBT led to DSB persistence through neutral comet assays and monitoring \u03b3H2AX\/53BP1 foci. We also show that this strategy can enhance the sensitivity of patient-derived organoids of HNSCC to PBT of both low and high LET, highlighting this as a strategy which should be exploited further.<\/jats:p>","DOI":"10.3390\/cells15100879","type":"journal-article","created":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T06:42:18Z","timestamp":1779691338000},"page":"879","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Radiosensitisation of Head and Neck Cancer Cells to Protons of Increasing LET Through Targeting DNA Double Strand Break Repair"],"prefix":"10.3390","volume":"15","author":[{"given":"Elizabeth R.","family":"Dufficy","sequence":"first","affiliation":[{"name":"Department of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0344-892X","authenticated-orcid":false,"given":"Amalia","family":"Goula","sequence":"additional","affiliation":[{"name":"Department of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-1438-0756","authenticated-orcid":false,"given":"Emma","family":"Melia","sequence":"additional","affiliation":[{"name":"Department of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2512-6710","authenticated-orcid":false,"given":"Abigail","family":"Bellamy","sequence":"additional","affiliation":[{"name":"Department of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5052-1125","authenticated-orcid":false,"given":"Jason L.","family":"Parsons","sequence":"additional","affiliation":[{"name":"Department of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK"},{"name":"School of Physics and Astronomy, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"778","DOI":"10.1002\/ijc.33588","article-title":"Cancer statistics for the year 2020: An overview","volume":"149","author":"Ferlay","year":"2021","journal-title":"Int. J. Cancer"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Gauss, C., Stone, L.D., Ghafouri, M., Quan, D., Johnson, J., Fribley, A.M., and Amm, H.M. (2024). Overcoming Resistance to Standard-of-Care Therapies for Head and Neck Squamous Cell Carcinomas. Cells, 13.","DOI":"10.3390\/cells13121018"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.phrs.2016.01.001","article-title":"Cisplatin resistance and opportunities for precision medicine","volume":"106","author":"Amable","year":"2016","journal-title":"Pharmacol. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1056\/NEJMoa0912217","article-title":"Human papillomavirus and survival of patients with oropharyngeal cancer","volume":"363","author":"Ang","year":"2010","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1093\/jnci\/djn011","article-title":"Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial","volume":"100","author":"Fakhry","year":"2008","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1016\/j.radonc.2013.03.013","article-title":"HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity","volume":"107","author":"Rieckmann","year":"2013","journal-title":"Radiother. Oncol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Vitti, E.T., and Parsons, J.L. (2019). The Radiobiological Effects of Proton Beam Therapy: Impact on DNA Damage and Repair. Cancers, 11.","DOI":"10.3390\/cancers11070946"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1071","DOI":"10.1038\/nature08467","article-title":"The DNA-damage response in human biology and disease","volume":"461","author":"Jackson","year":"2009","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"BSR20222586","DOI":"10.1042\/BSR20222586","article-title":"DNA damage and repair dependencies of ionising radiation modalities","volume":"43","author":"Melia","year":"2023","journal-title":"Biosci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1615\/CritRevOncog.2022044635","article-title":"Improving Radiotherapy Response in the Treatment of Head and Neck Cancer","volume":"27","author":"Wicker","year":"2022","journal-title":"Crit. Rev. Oncog."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Sarna, R., Kubina, R., Pazdzior-Heiske, M., Halama, A., Chudy, P., Wala, P., Krzykawski, K., and Nowak, I. (2025). Molecular Targets for Pharmacotherapy of Head and Neck Squamous Cell Carcinomas. Curr. Issues Mol. Biol., 47.","DOI":"10.3390\/cimb47080609"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e3","DOI":"10.1017\/erm.2020.4","article-title":"The radiobiology of HPV-positive and HPV-negative head and neck squamous cell carcinoma","volume":"22","author":"Zhou","year":"2020","journal-title":"Expert Rev. Mol. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"81662","DOI":"10.18632\/oncotarget.20953","article-title":"Novel DNA targeted therapies for head and neck cancers: Clinical potential and biomarkers","volume":"8","author":"Glorieux","year":"2017","journal-title":"Oncotarget"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Lei, H., He, A., Jiang, Y., Ruan, M., and Han, N. (2022). Targeting DNA damage response as a potential therapeutic strategy for head and neck squamous cell carcinoma. Front. Oncol., 12.","DOI":"10.3389\/fonc.2022.1031944"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Hafsi, H., Dillon, M.T., Barker, H.E., Kyula, J.N., Schick, U., Paget, J.T., Smith, H.G., Pedersen, M., McLaughlin, M., and Harrington, K.J. (2018). Combined ATR and DNA-PK Inhibition Radiosensitizes Tumor Cells Independently of Their p53 Status. Front. Oncol., 8.","DOI":"10.3389\/fonc.2018.00245"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1597","DOI":"10.1080\/09553002.2019.1664787","article-title":"Radiosensitization of head and neck squamous cell carcinoma lines by DNA-PK inhibitors is more effective than PARP-1 inhibition and is enhanced by SLFN11 and hypoxia","volume":"95","author":"Lee","year":"2019","journal-title":"Int. J. Radiat. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1038\/bjc.2012.265","article-title":"Targeting radiation-resistant hypoxic tumour cells through ATR inhibition","volume":"107","author":"Pires","year":"2012","journal-title":"Br. J. Cancer"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1158\/1535-7163.MCT-16-0239","article-title":"Radiosensitization by the ATR Inhibitor AZD6738 through Generation of Acentric Micronuclei","volume":"16","author":"Dillon","year":"2017","journal-title":"Mol. Cancer Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1038\/s41420-024-02059-3","article-title":"Inhibition of key DNA double strand break repair protein kinases enhances radiosensitivity of head and neck cancer cells to X-ray and proton irradiation","volume":"10","author":"Fabbrizi","year":"2024","journal-title":"Cell Death Discov."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1038\/s41419-025-07435-0","article-title":"Targeting Chk1 and Wee1 kinases enhances radiosensitivity of 2D and 3D head and neck cancer models to X-rays and low\/high-LET protons","volume":"16","author":"Melia","year":"2025","journal-title":"Cell Death Dis."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"115100","DOI":"10.1016\/j.ejca.2024.115100","article-title":"Feasibility analysis of using patient-derived tumour organoids for treatment decision guidance in locally advanced head and neck squamous cell carcinoma","volume":"213","author":"Fisch","year":"2024","journal-title":"Eur. J. Cancer"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1038\/s41420-025-02635-1","article-title":"Radiobiological characterisation of a 28 MeV proton beam delivered by the MC-40 cyclotron","volume":"11","author":"Fabbrizi","year":"2025","journal-title":"Cell Death Discov."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1186\/s13014-015-0529-y","article-title":"CFAssay: Statistical analysis of the colony formation assay","volume":"10","author":"Braselmann","year":"2015","journal-title":"Radiat. Oncol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1016\/S0140-6736(25)01962-2","article-title":"Proton versus photon radiotherapy for patients with oropharyngeal cancer in the USA: A multicentre, randomised, open-label, non-inferiority phase 3 trial","volume":"407","author":"Frank","year":"2026","journal-title":"Lancet"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Vitti, E.T., Kacperek, A., and Parsons, J.L. (2020). Targeting DNA Double-Strand Break Repair Enhances Radiosensitivity of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma to Photons and Protons. Cancers, 12.","DOI":"10.3390\/cancers12061490"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"73925","DOI":"10.18632\/oncotarget.18034","article-title":"Identification of a novel ATM inhibitor with cancer cell specific radiosensitization activity","volume":"8","author":"Dohmen","year":"2017","journal-title":"Oncotarget"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1002\/gcc.22125","article-title":"Targeted Inhibition of ATR or CHEK1 Reverses Radioresistance in Oral Squamous Cell Carcinoma Cells with Distal Chromosome Arm 11q Loss","volume":"53","author":"Sankunny","year":"2014","journal-title":"Genes Chromosomes Cancer"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1667\/RR13904.1","article-title":"The Major DNA Repair Pathway after Both Proton and Carbon-Ion Radiation is NHEJ, but the HR Pathway is More Relevant in Carbon Ions","volume":"183","author":"Gerelchuluun","year":"2015","journal-title":"Radiat. Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1016\/j.ijrobp.2013.07.014","article-title":"Induction and persistence of large gammaH2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells","volume":"87","author":"Bracalente","year":"2013","journal-title":"Int. J. Radiat. Oncol. Biol. Phys."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1016\/j.radonc.2015.08.014","article-title":"Differential DNA repair pathway choice in cancer cells after proton- and photon-irradiation","volume":"116","author":"Fontana","year":"2015","journal-title":"Radiother. Oncol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.ijrobp.2013.09.041","article-title":"Deficiency in homologous recombination renders Mammalian cells more sensitive to proton versus photon irradiation","volume":"88","author":"Grosse","year":"2014","journal-title":"Int. J. Radiat. Oncol. Biol. Phys."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/10\/879\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T06:50:28Z","timestamp":1779691828000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/10\/879"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,12]]},"references-count":31,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["cells15100879"],"URL":"https:\/\/doi.org\/10.3390\/cells15100879","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,12]]}}}