IntroductionIncidences of head and neck squamous cell carcinoma (HNSCC) are on the rise, currently standing at 890,000 cases and 450,000 deaths per year worldwide [1]. Conventional X-ray radiotherapy is frequently used for the treatment of HNSCC, however patients can suffer from acute and long term adverse side effects, but also many tumours are inherently resistant to the treatment. Proton beam therapy (PBT) is increasingly being used as an alternative form of radiotherapy for HNSCC patients [2], as this is more precision targeted and the radiation dose can be largely confined to the tumour via the Bragg peak. However, there are still major biological and clinical uncertainties with PBT and how this may differ to conventional radiotherapy, largely due to the increases in linear energy transfer (LET) at and around the Bragg peak. Increases in LET lead to enhanced relative biological effectiveness (RBE), somewhat reflected in the RBE of 1.1 for PBT that is used clinically, although this is highly debated [3, 4]. Consequently, further studies are needed to understand the comparative radiobiology of X-rays versus PBT in well characterised HNSCC cell models, but then to identify and utilise targeted drugs/inhibitors against specific cellular pathways that enhance the effectiveness of the radiotherapy treatments and enhance patient survival.One of the most promising strategies to enhance the effectiveness of radiotherapy is to target the cellular DNA damage response. The major pathways involved in the repair of DNA double strand breaks (DSBs) through non-homologous end-joining (NHEJ) and homologous recombination (HR), and specific enzymes co-ordinating these pathways such as poly(ADP-ribose) polymerase-1 (PARP-1), ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and DNA protein kinase (DNA Pk), continue to be investigated as targets for radiosensitisation [5, 6]. Indeed, we have previously demonstrated the effect of targeting these proteins on both 2D monolayers and 3D spheroids models of HNSCC to enhance both X-ray and PBT efficacy [7, 8]. Although this provides a promising avenue, an alternative strategy is to target the cell cycle and to prevent radiation-induced cell cycle arrest which tumour cells use for promoting efficient DNA damage repair, thus driving radiotherapy resistance. Therefore, inhibitors against cell cycle checkpoint kinases, such as Chk1 and Wee1, have been developed and which is given more importance due to the fact that the majority of HNSCC tumours harbour p53 mutations. Consequently, these tumours are reliant on the G2/M cell cycle checkpoint driven by Chk1 and Wee1.It has been previously shown that HNSCC cells have increased X-ray radiosensitivity when combined with the Chk1 inhibitors PF-00477736 [9] or MK-8776 [10]. More recently, it was observed that pre-treatment with AZD7762 significantly increased radiosensitivity of p53-mutant containing UMSCC-1 cells, whereas a mild radioprotective phenotype on p53-wild type cell lines (UMSCC-6 and UMSCC-47) was seen [11]. Unlike Chk1 inhibition, there is currently limited evidence for the use of Wee1 inhibitors in radiosensitising HNSCC tumour models. However, one study performed in oral cavity HNSCC cell lines showed an increased radiosensitivity following Wee1 inhibition (MK-1775) associated with significantly increased apoptotic cell death [12]. Despite this, there are multiple completed or ongoing phase I clinical trials with Wee1 inhibition to investigate the maximum tolerated dose for future use in combination with the current standard chemoradiation regime (either cisplatin or cetuximab; NCT03028766, NCT02585973, NCT02555644) [13].Interestingly, the potential for Chk1 and Wee1 inhibition to function in combination with PBT in radiosensitising HNSCC cells are currently lacking, and particularly to assess whether there is any impact of LET on the cellular response. One study has shown that the Chk1 inhibitor PF-00477736 effectively radiosensitises triple negative breast cancer (TNBC) cells to both X-rays and PBT, created through decreasing the proficiency of HR [14]. However, no studies examining Chk1 inhibition following PBT in HNSCC cell models are available, and also to our knowledge, there is no current evidence for the radiosensitising potential of Wee1 kinase inhibition in combination with PBT in any tumour type. In this study, we have characterised the radiosensitisation potential of both Chk1 and Wee1 checkpoint kinase inhibition (using MK-8776 and MK-1775, respectively) in response to X-rays and PBT (including an examination of both low and relatively high-LET protons) in 2D and 3D HNSCC models. We demonstrate that the radiosensitivity of HNSCC can be significantly enhanced following the inhibition of either Chk1 or Wee1, and that mechanistically this is associated with delayed DSB repair through reduced HR efficiency. Our findings suggest that this could represent a potential therapeutic avenue for optimising the treatment of HNSCC tumours using radiotherapy.Materials and methodsAntibodies and inhibitorsInhibitors for Chk1 (MK-8776) and Wee1 (MK-1775) were purchased from Selleck Chemicals (Munich, Germany). Antibodies against γH2AX (05-636; Merck-Millipore, Watford, UK), RAD51 (ab133534, Abcam, Cambridge, UK) were used for immunofluorescent staining, along with goat anti-mouse Alexa Fluor 555 and goat anti-rabbit Alexa Fluor 488 (Life Technologies, Paisley UK) secondary antibodies. The following primary antibodies were used for immunoblotting:- phosphorylated Chk1 (S296), Cdc2 (Y15), ATR (T1989), ATM (S1981) and Chk1 (S345), in addition to unmodified Chk1 and Wee1 (2349, 4539, 58014, 13050, 2348, 2360 and 4936, respectively; Cell Signaling Technology, Massachusetts, USA); ATM and ATR (ab78 and ab2905, respectively; Abcam, Cambridge, UK); PARP-1 (sc-53643; Santa-Cruz Biotechnology, Heidelberg, Germany) and β-actin (A5441; Merck-Sigma, Gillingham, UK). The following primary antibodies were used in the DNA fibre spreading assay:- rat anti-BrdU (347580, BD Bioscience) and mouse anti-BrdU (ab6326, Abcam, Cambridge, UK).Cell culture, siRNA transfections and irradiationsFaDu and A253 were purchased from ATCC (Teddington, UK) and cultured in Minimal Essential Medium (MEM) and McCoy’s 5A modified medium, respectively. UMSCC12 were kindly provided by Prof. T Carey (University of Michigan, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM). All media was supplemented with 10% foetal bovine serum, 1x non-essential amino acids, 1x penicillin-streptomycin and 2 mM L-glutamine. For siRNA knockdowns, cells were seeded and after 24 h were transfected with a pool of four siRNA sequences targeting either CHEK1 or WEE1 (Horizon Discovery, Cambridge, UK), utilising RNAiMax (Life Technologies, Paisley, UK). Cells were treated with siRNA for 48 h before being utilised in immunoblotting and immunofluorescent staining, clonogenic survival assays, cell cycle analysis or spheroid growth assays.X-ray irradiations were performed at a dose rate of ~3 Gy/min using a 150 kV CellRad+ X-ray irradiator (Faxitron Bioptics, Tucson, AZ, USA). PBT irradiations were performed using the MC-40 cyclotron utilising a 28 MeV beam at a dose rate of ~5 Gy/min. A Monte Carlo simulation has been developed of the MC-40 beamline used in these studies using Geant4 [15]. This model consists of an accurate geometry of the system and validated beam line transport. A large uniform beam was achieved by passing the 28 MeV protons through a 100 μm thick Tantalum foil located 3.2 m from the cells. Accounting for the vacuum window and ionisation chambers, the primary beam has a kinetic energy of 25.7 MeV incident upon the cell dish. The cell dish base is modelled as 1.2 mm of polystyrene and the LET is calculated from energy deposits in water directly after the cell dish base. For low-LET data, the mean kinetic energy incident upon the cell layer is 22.9 MeV and the LET calculated to be 2.7 keV/um. For the relatively high-LET data, 4.5 mm of Perspex was placed before the cell dish, and a beam of 3.8 MeV protons is incident upon the cells with a mean LET of 10.8 keV/um.Cell viability and clonogenic survival assaysFor cell viability assays, 10,000 cells were treated with a serial dilution of either MK-8776 and MK-1775 (0.1–100 µM) in a 96-well plate 24 h after seeding, using either DMSO as a vehicle only control or hydrogen peroxide (10 mM) as a positive control. Cells were treated with these drugs for 72 h before viability was assessed. CellTiter Blue reagent (Promega, Wisconsin, USA) was added, cells were incubated at 5% CO2 at 37 °C for 2 h before measuring absorbance at a wavelength of 570 nm (A570), with the reference wavelength of 600 nm (R600). A570–R600 was calculated, and the survival of drug-treated cells relative to the DMSO control (after subtraction of the positive control) was determined.For clonogenic survival assays, single cells were seeded in media containing either MK-8776 or MK-1775 for 16 h prior to irradiation, using DMSO as a vehicle only control. Plating efficiencies for the cells were as follows: FaDu and UMSCC12 ( ~30%) and A253 ( ~10%). The numbers of cells seeded were doubled for each increase in radiation dose to allow for plating efficiencies. Following irradiation, the media was replaced and colonies left to form for 7–14 days before staining with 20% methanol and 0.5% crystal violet for 1 h. Colonies were counted using the GelCount colony counter (Oxford Optronix, Oxford, UK), and the surviving fraction was then calculated as colonies per treatment versus colonies in respective unirradiated control.Spheroid growth assaysCells were seeded at 500 cells/well in triplicate in ultra-low attachment plates (Greiner, Bio-One, Gloucestershire, UK) in 100 µl advanced DMEM F12 media containing 1% B-27 supplement, 0.5% N-2 supplement, 2 mM L-glutamine, 1x penicillin-streptomycin, 5 µg/ml heparin, 20 ng/µl epidermal growth factor (EGF) and 10 ng/µl fibroblast growth factor (FGF). Spheroids were left to form for 48 h before being treated with either MK-8776 or MK-1775, with DMSO as a vehicle only control, for 1 h. Immediately following irradiation, 60 µl of culture media was removed and replaced with 100 µl fresh media. Spheroids were then imaged at intervals up to day 13 post-seeding using the EVOS M5000 Imaging system (Life technologies, Paisley, UK). Spheroid diameter was determined using ImageJ, and converted to volume using the formula 4/3πr3.Patient-derived organoid generation and viability assaysTissue samples were immediately transferred into a tube containing RPMI1640 medium complemented with 2.5% penicillin/streptomycin and 1% gentamicin, and stored at 4 °C until required. All the following processing was performed on ice. The tissue was cut into small pieces (∼1 mm3), and enzymatically digested in tissue dissociation mix, consisting of Advanced DMEM/F12 medium complemented with 100 µg/ml DNAse I, 100 µg/ml Dispase (Corning, New York, USA) and 10 µM Y-27632 dihydrochloride (Abmole, Texas, USA). After incubation at 37 °C for 30 min, enzymatic dissociation was stopped on ice by adding basal media, consisting of Advanced DMEM/F12, 1 M HEPES, 1% GlutaMax, 1 mM N-Acetyl-L-cysteine, 1% penicillin/streptomycin, 10 nM gastrin and 5 µM Y-27632 dihydrochloride. The suspension was strained over 500 µm and 40 µm filters (pluriSelect), with fragments within this range being employed for organoid cultures. Fragments were then plated and cultured in organoid medium, consisting of basal media further supplemented with 1% N-2 supplement, 2% B-27 supplement, 20 ng/ml FGF-2, 50 ng/ml EGF, 0.5 μM TGF-β RI kinase VI inhibitor (SB431542) and conditioned medium from the cell line CRL-2376™ (1:100, ATCC, Manassas, VA, USA), containing Wnt-3A, R-spondin and Noggin; with 250 µg/ml Amphotericin B added for the first three days of fragment culture only. At 80% confluency, cells were initially seeded at 2000 cells in a 15 μl dome of Matrigel before being left to grow. RNAseq was performed which revealed very low (