Autophagy is the main driver of radioresistance of HNSCC cells in mild hypoxia.

Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1ß leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.

Myoferlin: a potential marker of response to radiotherapy and survival in locally advanced rectal cancer.

PURPOSE: Patients with locally advanced rectal cancer often require neo-adjuvant chemoradiotherapy to downstage the disease, but the response is variable with no predictive biomarkers. We have previously revealed through proteomic profiling that myoferlin is associated with response to radiotherapy. The aims of this study were to further validate this finding and explore the potential for myoferlin to act as a prognostic and/or therapeutic target. MATERIALS AND METHODS: Immunohistochemical analysis of a tissue microarray for 111 patients was used to validate the initial proteomic findings. Manipulation of myoferlin was achieved using siRNA, a small molecular inhibitor (wj460) and a CRISPR-Cas9 knockout cell line. Radiosensitisation following treatment was assessed using 2D clonogenic assays, 3D spheroid models and patient derived organoids. Underlying mechanisms were investigated using electrophoresis, immunofluorescence and immunoblotting. RESULTS: Analysis of both the diagnostic biopsy and tumour resection samples confirmed that low myoferlin expression correlated with a good response to neoadjuvant LCRT. High myoferlin expression was associated with spread to local lymph nodes and worse 5-year survival (p = 0.01, HR 3.5, 95%CI [1.27, 10.04]). This was externally validated using the S:CORT database. Quantification of myoferlin using immunoblotting in immortalised colorectal cancer cell lines and organoids demonstrated that high myoferlin expression was associated with increased radioresistance. Biological and pharmacological manipulation of myoferlin resulted in significantly increased radiosensitivity across all cell lines in 2D and 3D models. Following irradiation, myoferlin knockdown cells had a significantly impaired ability to repair DNA double strand breaks. This appeared to be mediated via non-homologous end-joining. CONCLUSIONS: We have confirmed that high expression of myoferlin in rectal cancer is associated with poor response to neoadjuvant therapy and worse long-term survival. Furthermore, the manipulation of myoferlin led to increased radiosensitivity in vitro. This suggests that myoferlin could be targeted to enhance the sensitivity of rectal cancer patients to radiotherapy and further work is required.

Inhibition of key DNA double strand break repair protein kinases enhances radiosensitivity of head and neck cancer cells to X-ray and proton irradiation.

Ionising radiation (IR) is widely used in cancer treatment, including for head and neck squamous cell carcinoma (HNSCC), where it induces significant DNA damage leading ultimately to tumour cell death. Among these lesions, DNA double strand breaks (DSBs) are the most threatening lesion to cell survival. The two main repair mechanisms that detect and repair DSBs are non-homologous end joining (NHEJ) and homologous recombination (HR). Among these pathways, the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA dependent protein kinase catalytic subunit (DNA-Pkcs) play key roles in the sensing of the DSB and subsequent coordination of the downstream repair events. Consequently, targeting these kinases with potent and specific inhibitors is considered an approach to enhance the radiosensitivity of tumour cells. Here, we have investigated the impact of inhibition of ATM, ATR and DNA-Pkcs on the survival and growth of six radioresistant HPV-negative HNSCC cell lines in combination with either X-ray irradiation or proton beam therapy, and confirmed the mechanistic pathway leading to cell radiosensitisation. Using inhibitors targeting ATM (AZD1390), ATR (AZD6738) and DNA-Pkcs (AZD7648), we observed that this led to significantly decreased clonogenic survival of HNSCC cell lines following both X-ray and proton irradiation. Radiosensitisation of HNSCC cells grown as 3D spheroids was also observed, particularly following ATM and DNA-Pkcs inhibition. We confirmed that the inhibitors in combination with X-rays and protons led to DSB persistence, and increased micronuclei formation. Cumulatively, our data suggest that targeting DSB repair, particularly via ATM and DNA-Pkcs inhibition, can exacerbate the impact of ionising radiation in sensitising HNSCC cell models.

Targeting OGG1 and PARG radiosensitises head and neck cancer cells to high-LET protons through complex DNA damage persistence.

Complex DNA damage (CDD), containing two or more DNA lesions within one or two DNA helical turns, is a signature of ionising radiation (IR) and contributes significantly to the therapeutic effect through cell killing. The levels and complexity of CDD increases with linear energy transfer (LET), however, the specific cellular response to this type of DNA damage and the critical proteins essential for repair of CDD is currently unclear. We performed an siRNA screen of ~240 DNA damage response proteins to identify those specifically involved in controlling cell survival in response to high-LET protons at the Bragg peak, compared to low-LET entrance dose protons which differ in the amount of CDD produced. From this, we subsequently validated that depletion of 8-oxoguanine DNA glycosylase (OGG1) and poly(ADP-ribose) glycohydrolase (PARG) in HeLa and head and neck cancer cells leads to significantly increased cellular radiosensitivity specifically following high-LET protons, whilst no effect was observed after low-LET protons and X-rays. We subsequently confirmed that OGG1 and PARG are both required for efficient CDD repair post-irradiation with high-LET protons. Importantly, these results were also recapitulated using specific inhibitors for OGG1 (TH5487) and PARG (PDD00017273). Our results suggest OGG1 and PARG play a fundamental role in the cellular response to CDD and indicate that targeting these enzymes could represent a promising therapeutic strategy for the treatment of head and neck cancers following high-LET radiation.

KH-like Domains in PARP9/DTX3L and PARP14 Coordinate Protein-Protein Interactions to Promote Cancer Cell Survival.

Certain members of the ADP-ribosyltransferase superfamily (ARTD or PARP enzymes) catalyse ADP-ribosylation in response to cellular stress, DNA damage and viral infection and are upregulated in various tumours. PARP9, its binding partner DTX3L and PARP14 protein levels are significantly correlated in head and neck squamous cell carcinoma (HNSCC) and other tumour types though a mechanism where PARP9/DTX3L regulates PARP14 post-transcriptionally. Depleting PARP9, DTX3L or PARP14 expression in HNSCC or HeLa cell lines decreases cell survival through a reduction of proliferation and an increase in apoptosis. A partial rescue of survival was achieved by expressing a PARP14 truncation containing a predicted eukaryotic type I KH domain. KH-like domains were also found in PARP9 and in DTX3L and contributed to protein-protein interactions between PARP9-DTX3L and PARP14-DTX3L. Homodimerization of DTX3L was also coordinated by a KH-like domain and was disrupted by site-specific mutation. Although, cell survival promoted by PARP14 did not require ADP-ribosyltransferase activity, interaction of DTX3L in vitro suppressed PARP14 auto-ADP-ribosylation and promoted trans-ADP-ribosylation of PARP9 and DTX3L. In summary, we characterised PARP9-DTX3L-PARP14 interactions important to pro-survival signalling in HNSCC cells, albeit in PARP14 catalytically independent fashion.

Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR-edited cells.

BACKGROUND: Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population-level trials. As a result, a proportion of patients are under- or over-dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy. Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear. PURPOSE: The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects. METHODS: Normal human cells (RPE1), genetically modified to introduce defects in DNA double-strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE-LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12 keV/µm) and alpha particles (129 keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild-type cells to repair-deficient cells, and RBE values were calculated as the ratio of the MID of X-ray and particle irradiated cells. 53BP1 foci were used to quantify radiation-induced DSBs and their repair following irradiation. RESULTS: Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM-/- SER = 2.0 and Lig4-/- SER = 1.8), with genes associated with HR having smaller effects (BRCA1-/- SER = 1.2). Wild-type cells showed RBEs of 1.1, 1.3, 5.0 for low- and high-LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR-defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ-defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR-defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects. CONCLUSIONS: DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality.

DNA damage and repair dependencies of ionising radiation modalities.

Radiotherapy is utilised in the treatment of ∼50% of all human cancers, which predominantly employs photon radiation. However, particle radiotherapy elicits significant benefits over conventional photons due to more precise dose deposition and increased linear energy transfer (LET) that generates an enhanced therapeutic response. Specifically, proton beam therapy (PBT) and carbon ion radiotherapy (CIRT) are characterised by a Bragg peak, which generates a low entrance radiation dose, with the majority of the energy deposition being defined within a small region which can be specifically targeted to the tumour, followed by a low exit dose. PBT is deemed relatively low-LET whereas CIRT is more densely ionising and therefore high LET. Despite the radiotherapy type, tumour cell killing relies heavily on the introduction of DNA damage that overwhelms the repair capacity of the tumour cells. It is known that DNA damage complexity increases with LET that leads to enhanced biological effectiveness, although the specific DNA repair pathways that are activated following the different radiation sources is unclear. This knowledge is required to determine whether specific proteins and enzymes within these pathways can be targeted to further increase the efficacy of the radiation. In this review, we provide an overview of the different radiation modalities and the DNA repair pathways that are responsive to these. We also provide up-to-date knowledge of studies examining the impact of LET and DNA damage complexity on DNA repair pathway choice, followed by evidence on how enzymes within these pathways could potentially be therapeutically exploited to further increase tumour radiosensitivity, and therefore radiotherapy efficacy.

The Cellular Response to Complex DNA Damage Induced by Ionising Radiation.

Radiotherapy (ionising radiation; IR) is utilised in the treatment of ~50% of all human cancers, and where the therapeutic effect is largely achieved through DNA damage induction. In particular, complex DNA damage (CDD) containing two or more lesions within one to two helical turns of the DNA is a signature of IR and contributes significantly to the cell killing effects due to the difficult nature of its repair by the cellular DNA repair machinery. The levels and complexity of CDD increase with increasing ionisation density (linear energy transfer, LET) of the IR, such that photon (X-ray) radiotherapy is deemed low-LET whereas some particle ions (such as carbon ions) are high-LET radiotherapy. Despite this knowledge, there are challenges in the detection and quantitative measurement of IR-induced CDD in cells and tissues. Furthermore, there are biological uncertainties with the specific DNA repair proteins and pathways, including components of DNA single and double strand break mechanisms, that are engaged in CDD repair, which very much depends on the radiation type and associated LET. However, there are promising signs that advancements are being made in these areas and which will enhance our understanding of the cellular response to CDD induced by IR. There is also evidence that targeting CDD repair, particularly through inhibitors against selected DNA repair enzymes, can exacerbate the impact of higher LET, which could be explored further in a translational context.

ATM Regulates Differentiation of Myofibroblastic Cancer-Associated Fibroblasts and Can Be Targeted to Overcome Immunotherapy Resistance.

Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. SIGNIFICANCE: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

Factors affecting the radiation response in glioblastoma.

Glioblastoma (GBM) is a highly invasive primary brain tumor in adults with a 5-year survival rate of less than 10%. Conventional radiotherapy with photons, along with concurrent and adjuvant temozolomide, is the mainstay for treatment of GBM although no significant improvement in survival rates has been observed over the last 20 years. Inherent factors such as tumor hypoxia, radioresistant GBM stem cells, and upregulated DNA damage response mechanisms are well established as contributing to treatment resistance and tumor recurrence. While it is understandable that efforts have focused on targeting these factors to overcome this phenotype, there have also been striking advances in precision radiotherapy techniques, including proton beam therapy and carbon ion radiotherapy (CIRT). These enable higher doses of radiation to be delivered precisely to the tumor, while minimizing doses to surrounding normal tissues and organs at risk. These alternative radiotherapy techniques also benefit from increased biological effectiveness, particularly in the case of CIRT. Although not researched extensively to date, combining these new radiation modalities with radio-enhancing agents may be particularly effective in improving outcomes for patients with GBM.

Effectiveness of PARP inhibition in enhancing the radiosensitivity of 3D spheroids of head and neck squamous cell carcinoma.

A critical risk factor for head and neck squamous cell carcinoma (HNSCC), particularly of the oropharynx, and the response to radiotherapy is human papillomavirus (HPV) type-16/18 infection. Specifically, HPV-positive HNSCC display increased radiosensitivity and improved outcomes, which has been linked with defective signalling and repair of DNA double-strand breaks (DSBs). This differential response to radiotherapy has been recapitulated in vitro using cell lines, although studies utilising appropriate 3D models that are more reflective of the original tumour are scarce. Furthermore, strategies to enhance the sensitivity of relatively radioresistant HPV-negative HNSCC to radiotherapy are still required. We have analysed the comparative response of in vitro 3D spheroid models of oropharyngeal squamous cell carcinoma to x-ray (photon) irradiation and provide further evidence that HPV-positive cells, in this case now grown as spheroids, show greater inherent radiosensitivity compared to HPV-negative spheroids due to defective DSB repair. We subsequently analysed these and an expanded number of spheroid models, with a particular focus on relatively radioresistant HPV-negative HNSCC, for impact of poly(ADP-ribose) polymerase (PARP) inhibitors (olaparib and talazoparib) in significantly inhibiting spheroid growth in response to photons but also proton beam therapy. We demonstrate that in general, PARP inhibition can further radiosensitise particularly HPV-negative HNSCC spheroids to photons and protons leading to significant growth suppression. The degree of enhanced radiosensitivity was observed to be dependent on the model and on the tumour site (oropharynx, larynx, salivary gland, or hypopharynx) from which the cells were derived. We also provide evidence suggesting that PARP inhibitor effectiveness relates to homologous recombination repair proficiency. Interestingly though, we observed significantly enhanced effectiveness of talazoparib versus olaparib specifically in response to proton irradiation. Nevertheless, our data generally support that PARP inhibition in combination with radiotherapy (photons and protons) should be considered further as an effective treatment for HNSCC, particularly for relatively radioresistant HPV-negative tumours.

Overcoming the Impact of Hypoxia in Driving Radiotherapy Resistance in Head and Neck Squamous Cell Carcinoma.

Hypoxia is very common in most solid tumours and is a driving force for malignant progression as well as radiotherapy and chemotherapy resistance. Incidences of head and neck squamous cell carcinoma (HNSCC) have increased in the last decade and radiotherapy is a major therapeutic technique utilised in the treatment of the tumours. However, effectiveness of radiotherapy is hindered by resistance mechanisms and most notably by hypoxia, leading to poor patient prognosis of HNSCC patients. The phenomenon of hypoxia-induced radioresistance was identified nearly half a century ago, yet despite this, little progress has been made in overcoming the physical lack of oxygen. Therefore, a more detailed understanding of the molecular mechanisms of hypoxia and the underpinning radiobiological response of tumours to this phenotype is much needed. In this review, we will provide an up-to-date overview of how hypoxia alters molecular and cellular processes contributing to radioresistance, particularly in the context of HNSCC, and what strategies have and could be explored to overcome hypoxia-induced radioresistance.

USP9X Is Required to Maintain Cell Survival in Response to High-LET Radiation.

Ionizing radiation (IR) principally acts through induction of DNA damage that promotes cell death, although the biological effects of IR are more broad ranging. In fact, the impact of IR of higher-linear energy transfer (LET) on cell biology is generally not well understood. Critically, therefore, the cellular enzymes and mechanisms responsible for enhancing cell survival following high-LET IR are unclear. To this effect, we have recently performed siRNA screening to identify deubiquitylating enzymes that control cell survival specifically in response to high-LET α-particles and protons, in comparison to low-LET X-rays and protons. From this screening, we have now thoroughly validated that depletion of the ubiquitin-specific protease 9X (USP9X) in HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells using small interfering RNA (siRNA), leads to significantly decreased survival of cells after high-LET radiation. We consequently investigated the mechanism through which this occurs, and demonstrate that an absence of USP9X has no impact on DNA damage repair post-irradiation nor on apoptosis, autophagy, or senescence. We discovered that USP9X is required to stabilize key proteins (CEP55 and CEP131) involved in centrosome and cilia formation and plays an important role in controlling pericentrin-rich foci, particularly in response to high-LET protons. This was also confirmed directly by demonstrating that depletion of CEP55/CEP131 led to both enhanced radiosensitivity of cells to high-LET protons and amplification of pericentrin-rich foci. Our evidence supports the importance of USP9X in maintaining centrosome function and biogenesis and which is crucial particularly in the cellular response to high-LET radiation.

Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer.

Previous work utilizing proteomic and immunohistochemical analyses has identified that high levels of acid ceramidase (AC) expression confers a poorer response to neoadjuvant treatment in locally advanced rectal cancer. We aimed to assess the radiosensitising effect of biological and pharmacological manipulation of AC and elucidate the underlying mechanism. AC manipulation in three colorectal cancer cell lines (HT29, HCT116 and LIM1215) was achieved using siRNA and plasmid overexpression. Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological AC inhibitors. Using clonogenic assays, we demonstrate that an siRNA knockdown of AC enhanced X-ray radiosensitivity across all colorectal cancer cell lines compared to a non-targeting control siRNA, and conversely, AC protein overexpression increased radioresistance. Using CRISPR gene editing, we also generated AC knockout HCT116 cells that were significantly more radiosensitive compared to AC-expressing cells. Similarly, two patient-derived organoid models containing relatively low AC expression were found to be comparatively more radiosensitive than three other models containing higher levels of AC. Additionally, AC inhibition using carmofur and LCL521 in three colorectal cancer cell lines increased cellular radiosensitivity. Decreased AC protein led to significant poly-ADP ribose polymerase-1 (PARP-1) cleavage and apoptosis post-irradiation, which was shown to be executed through a p53-dependent process. Our study demonstrates that expression of AC within colorectal cancer cell lines modulates the cellular response to radiation, and particularly that AC inhibition leads to significantly enhanced radiosensitivity through an elevation in apoptosis. This work further solidifies AC as a target for improving radiotherapy treatment of locally advanced rectal cancer.

FLASH Radiotherapy: Current Knowledge and Future Insights Using Proton-Beam Therapy.

FLASH radiotherapy is the delivery of ultra-high dose rate radiation several orders of magnitude higher than what is currently used in conventional clinical radiotherapy, and has the potential to revolutionize the future of cancer treatment. FLASH radiotherapy induces a phenomenon known as the FLASH effect, whereby the ultra-high dose rate radiation reduces the normal tissue toxicities commonly associated with conventional radiotherapy, while still maintaining local tumor control. The underlying mechanism(s) responsible for the FLASH effect are yet to be fully elucidated, but a prominent role for oxygen tension and reactive oxygen species production is the most current valid hypothesis. The FLASH effect has been confirmed in many studies in recent years, both in vitro and in vivo, with even the first patient with T-cell cutaneous lymphoma being treated using FLASH radiotherapy. However, most of the studies into FLASH radiotherapy have used electron beams that have low tissue penetration, which presents a limitation for translation into clinical practice. A promising alternate FLASH delivery method is via proton beam therapy, as the dose can be deposited deeper within the tissue. However, studies into FLASH protons are currently sparse. This review will summarize FLASH radiotherapy research conducted to date and the current theories explaining the FLASH effect, with an emphasis on the future potential for FLASH proton beam therapy.

Base excision repair and its implications to cancer therapy.

Base excision repair (BER) has evolved to preserve the integrity of DNA following cellular oxidative stress and in response to exogenous insults. The pathway is a coordinated, sequential process involving 30 proteins or more in which single strand breaks are generated as intermediates during the repair process. While deficiencies in BER activity can lead to high mutation rates and tumorigenesis, cancer cells often rely on increased BER activity to tolerate oxidative stress. Targeting BER has been an attractive strategy to overwhelm cancer cells with DNA damage, improve the efficacy of radiotherapy and/or chemotherapy, or form part of a lethal combination with a cancer specific mutation/loss of function. We provide an update on the progress of inhibitors to enzymes involved in BER, and some of the challenges faced with targeting the BER pathway.

The radiobiology of HPV-positive and HPV-negative head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with reported incidences of ~800 000 cases each year. One of the critical determinants in patient response to radiotherapy, particularly for oropharyngeal cancers, is human papillomavirus (HPV) status where HPV-positive patients display improved survival rates and outcomes particularly because of increased responsiveness to radiotherapy. The increased radiosensitivity of HPV-positive HNSCC has been largely linked with defects in the signalling and repair of DNA double-strand breaks. Therefore, strategies to further radiosensitise HPV-positive HNSCC, but also radioresistant HPV-negative HNSCC, have focussed on targeting key DNA repair proteins including PARP, DNA-Pk, ATM and ATR. However, inhibitors against CHK1 and WEE1 involved in cell-cycle checkpoint activation have also been investigated as targets for radiosensitisation in HNSCC. These studies, largely conducted using established HNSCC cell lines in vitro, have demonstrated variability in the response dependent on the specific inhibitors and cell models utilised. However, promising results are evident targeting specifically PARP, DNA-Pk, ATR and CHK1 in synergising with radiation in HNSCC cell killing. Nevertheless, these preclinical studies require further expansion and investigation for translational opportunities for the effective treatment of HNSCC in combination with radiotherapy.

Inhibition of ATM Increases the Radiosensitivity of Uveal Melanoma Cells to Photons and Protons.

Treatment of uveal melanoma (UM) is generally successful, with local primary tumour control being at 90%-95%. Localized radiotherapy in the form of plaque brachytherapy or proton beam radiotherapy is the most common treatment modality in the UK. However, the basic mechanisms of radiation response, DNA repair and tissue reactions in UM have not been well documented previously. We have investigated the comparative radiosensitivity of four UM cell lines in response to exogenous radiation sources (both X-rays and protons), and correlated this with DNA repair protein expression and repair efficiency. We observed a broad range of radiosensitivity of different UM cell lines to X-rays and protons, with increased radioresistance correlating with elevated protein expression of ataxia telangiectasia mutated (ATM), a protein kinase involved in the signaling and repair of DNA double strand breaks. The use of an ATM inhibitor in UM cell lines enhanced radiosensitivity following both X-ray and proton irradiation, particularly in cells that contained high levels of ATM protein which are otherwise comparatively radioresistant. In proton-irradiated compared with non-irradiated primary enucleated UM patient samples, there was no significant difference in ATM protein expression. Our study therefore suggests that ATM is a potential target for increasing the radiosensitivity of more resistant UM subgroups.

Targeting DNA Double-Strand Break Repair Enhances Radiosensitivity of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma to Photons and Protons.

The response of head and neck squamous cell carcinoma (HNSCC) to radiotherapy depends on human papillomavirus type 16 (HPV) status, and where improved outcome and survival is observed in HPV-positive disease. However, strategies to further radiosensitise the tumours, particularly relatively radioresistant HPV-negative HNSCC, are actively being sought. The impact of targeting the major protein kinases involved in the signaling of DNA double-strand break (DSB) repair, namely ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the catalytic subunit of DNA-dependent protein kinase (DNA-Pkcs), on the radiosensitisation of HNSCC cells was examined. The response to both conventional photon radiotherapy, but also proton beam therapy, was analysed by clonogenic assays and 3D spheroid growth. We observed that inhibition of ATM, ATR, and particularly DNA-Pkcs, caused a significant reduction in HNSCC cell survival post-irradiation with both photons and protons, with less of an impact on the most radiosensitive HPV-positive cell line. The inhibition of DNA-Pkcs and, to a lesser extent ATM, in combination with radiation was also more effective at inhibiting the growth of 3D spheroids derived from relatively radioresistant HPV-negative HNSCC. Similar effects of the inhibitors were observed comparing photon and proton irradiation, demonstrating the potential for targeting DSB repair as an effective combination treatment for HNSCC.

Base Excision Repair in Chromatin and the Requirement for Chromatin Remodelling.

Base excision repair (BER) is a co-ordinated DNA repair pathway that recognises and repairs chemically modified bases and DNA single strand breaks. It is essential for the maintenance of genome integrity and thus in the prevention of the development of human diseases, including premature ageing, neurodegenerative diseases and cancer. Within the cell, DNA is usually packaged with histone proteins to form chromatin which imposes major constraints on the capacity of cells to perform BER. Therefore chromatin remodelling, stimulated through histone post-translational modifications (PTMs) or ATP-dependent chromatin remodelling complexes (ACRs), are required to stimulate access to the DNA damage and therefore enhance the BER process. Despite this, the molecular mechanisms through which this is co-ordinated and the specific enzymes that promote chromatin remodelling required for BER remain elusive. In this review, we summarise the multitude of in vitro studies utilising mononucleosome substrates containing site-specific DNA base damage that demonstrate the requirement for chromatin remodelling to facilitate BER, particularly in occluded regions. We also highlight preliminary evidence to date for the identity of ACRs, their mechanisms and the role of histone PTMs in modulating the cellular capacity for BER.