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.

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.

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.

Radiotherapy and the cellular DNA damage response: current and future perspectives on head and neck cancer treatment.

Incidences of head and neck squamous cell carcinoma (HNSCC) have been on the rise in the last few decades, with a significant risk factor being human papillomavirus (HPV) type-16/18 infection, particularly in the development of oropharyngeal cancers. Radiotherapy (RT) is an important treatment modality for HNSCC, where it promotes extensive cellular DNA damage leading to the therapeutic effect. It has been well-established that HPV-positive HNSCC display better response rates and improved survival following RT compared to HPV-negative HNSCC. The differential radiosensitivity has been largely associated with altered cellular DNA damage response mechanisms in HPV-positive HNSCC, and particularly with the signaling and repair of DNA double strand breaks. However, other factors, particularly hypoxia present within the solid cancer, have a major impact on relative radioresistance. Consequently, recent approaches aimed at enhancing the radiosensitivity of HNSCC have largely centered on targeting key proteins involved in DNA repair, DNA damage checkpoint activation, and hypoxia signaling. These studies have utilised in vitro and in vivo models of HPV-positive and HPV-negative HNSCC and examined the impact of specific inhibitors against the targets in combination with radiation in suppressing HNSCC cell growth and survival. Here, accumulating evidence has shown that targeting enzymes including poly (ADP-ribose) polymerase, ataxia telangiectasia and Rad-3 related, DNA-dependent protein kinase catalytic subunit, and checkpoint kinase 1 can radiosensitise HNSCC cells which should be taken forward in further preclinical studies, with the goal of optimizing the future effective RT treatment of HNSCC.

Molecular and epigenetic regulatory mechanisms of normal stem cell radiosensitivity.

Ionizing radiation (IR) therapy is a major cancer treatment modality and an indispensable auxiliary treatment for primary and metastatic cancers, but invariably results in debilitating organ dysfunctions. IR-induced depletion of neural stem/progenitor cells in the subgranular zone of the dentate gyrus in the hippocampus where neurogenesis occurs is considered largely responsible for deficiencies such as learning, memory, and spatial information processing in patients subjected to cranial irradiation. Similarly, IR therapy-induced intestinal injuries such as diarrhea and malabsorption are common side effects in patients with gastrointestinal tumors and are believed to be caused by intestinal stem cell drop out. Hematopoietic stem cell transplantation is currently used to reinstate blood production in leukemia patients and pre-clinical treatments show promising results in other organs such as the skin and kidney, but ethical issues and logistic problems make this route difficult to follow. An alternative way to restore the injured tissue is to preserve the stem cell pool located in that specific tissue/organ niche, but stem cell response to ionizing radiation is inadequately understood at the molecular mechanistic level. Although embryonic and fetal hypersensity to IR has been very well known for many decades, research on embryonic stem cell models in culture concerning molecular mechanisms have been largely inconclusive and often in contradiction of the in vivo observations. This review will summarize the latest discoveries on stem cell radiosensitivity, highlighting the possible molecular and epigenetic mechanism(s) involved in DNA damage response and programmed cell death after ionizing radiation therapy specific to normal stem cells. Finally, we will analyze the possible contribution of stem cell-specific chromatin's epigenetic constitution in promoting normal stem cell radiosensitivity.

Transient PP2A inhibition alleviates normal tissue stem cell susceptibility to cell death during radiotherapy.

Unintended outcomes of cancer therapy include ionizing radiation (IR)-induced stem cell depletion, diminished regenerative capacity, and accelerated aging. Stem cells exhibit attenuated DNA damage response (DDR) and are hypersensitive to IR, as compared to differentiated non-stem cells. We performed genomic discovery research to compare stem cells to differentiated cells, which revealed Phosphoprotein phosphatase 2A (PP2A) as a potential contributor to susceptibility in stem cells. PP2A dephosphorylates pATM, γH2AX, pAkt etc. and is believed to play dual role in regulating DDR and apoptosis. Although studied widely in cancer cells, the role of PP2A in normal stem cell radiosensitivity is unknown. Here we demonstrate that constitutively high expression and radiation induction of PP2A in stem cells plays a role in promoting susceptibility to irradiation. Transient inhibition of PP2A markedly restores DNA repair, inhibits apoptosis, and enhances survival of stem cells, without affecting differentiated non-stem and cancer cells. PP2Ai-mediated stem cell radioprotection was demonstrated in murine embryonic, adult neural, intestinal, and hematopoietic stem cells.

Histone H3 Lysine 9 Acetylation Obstructs ATM Activation and Promotes Ionizing Radiation Sensitivity in Normal Stem Cells.

Dynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.

Aneuploidogenic effects and DNA oxidation induced in vitro by differently sized gold nanoparticles.

Gold nanoparticles (Au NPs) are used in many fields, including biomedical applications; however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms is available. For this reason, experiments in human primary lymphocytes and murine macrophages (Raw264.7) were performed exposing cells to spherical citrate-capped Au NPs with two different nominal diameters (5 nm and 15 nm). The proliferative activity, mitotic, apoptotic, and necrotic markers, as well as chromosomal damage were assessed by the cytokinesis-block micronucleus cytome assay. Fluorescence in situ hybridization with human and murine pancentromeric probes was applied to distinguish between clastogenic and aneuploidogenic effects. Our results indicate that 5 nm and 15 nm Au NPs are able to inhibit cell proliferation by apoptosis and to induce chromosomal damage, in particular chromosome mis-segregation. DNA strand breaks were detected by comet assay, and the modified protocol using endonuclease-III and formamidopyrimidine-DNA glycosylase restriction enzymes showed that pyrimidines and purines were oxidatively damaged by Au NPs. Moreover, we show a size-independent correlation between the cytotoxicity of Au NPs and their tested mass concentration or absolute number, and genotoxic effects which were more severe for Au NP 15 nm compared to Au NP 5 nm. Results indicate that apoptosis, aneuploidy, and DNA oxidation play a pivotal role in the cytotoxicity and genotoxicity exerted by Au NPs in our cell models.