Oncotarget

Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical non-homologous end-joining pathway, but it may have roles in modulating gene expression and thus, the overall cellular response to DNA damage. Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Downregulation of pathways involved in biosynthesis were observed in both genotypes, but upregulated biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/{beta}-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trial as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment.

The smoke and cancer-associated lncRNA 1 (SCAL1) is an emergent biomarker in lung cancer. However, the precise role of SCAL1 as a mediator of tobacco smoke-induced lung carcinogenesis remains unclear. BEAS-2B cells were cultured and exposed to 20% cigarette smoke extract (CSE), followed by quantification of SCAL1. We evaluated the impact of SCAL1 on cell viability, ROS mitigation, cancer stemness, tumorigenic differentiation, cellular invasiveness, and apoptosis for different CSE incubation time points through SCAL1 expressional modulation using SCAL1-specific siRNAs and scrambled controls. We observed an upregulation of SCAL1 in cells exposed to CSE for 2, 4, and 6 hours, with the highest expression observed at 6 hours (p<0.001). Exposure of BEAS-2B cells to CSE showed the formation of focal adhesions and stress fibers resembling tunneling nanotubes. Intracellular ROS levels significantly increased upon CSE exposure compared to control cells (p<0.05). We found increased levels of anti-apoptotic and cancer stem (CSC) cell markers like BCL2, ALDH1A1, CD133, CD44, and TCTP and decreased levels of TP53 in CSE-exposed cells. Knockdown of SCAL1 using siRNA transfection reversed these effects at all time points. Additionally, we observed a significant decrease in the number of spheroid colonies in siSCAL1 (+) cells compared to siSCAL1 (-) cells (p<0.01) exposed to CSE. SCAL1 is pivotal in mediating cellular responses to cigarette smoke, leading to tumorigenic differentiation of BEAS-2B cells. Understanding the mechanisms could provide valuable insights into lung cancer pathogenesis and therapeutic approaches.

Multiple Myeloma is a plasma cell malignancy for which there is currently no cure, despite many novel therapies. TRAIL (Tumour necrosis factor-related apoptosis inducing ligand) is a promising anti-tumour agent although TRAIL-insensitive cells readily emerge when used as a single agent and its effects are limited due to many TRAIL-resistant cells which emerge soon after treatment. EZH2 is a H3K27 histone methyltransferase found to be overexpressed in many cancers including Multiple Myeloma. We tested the hypothesis that epigenetic reprogramming using the EZH2 inhibitor GSK343 would enhance TRAIL sensitivity, overcome TRAIL resistance, and target TRAIL-resistant quiescent cell populations. We show that GSK343 is a potent TRAIL sensitiser in TRAIL-sensitive RPMI 8226, NCI-H 929 and U266, and in TRAIL-resistant OPM-2 and JJN3, the latter showing very potent synergistic induction of apoptosis, primarily via caspase-8 activation but also via caspase-9. GSK343-enhancement of TRAIL responses was further enhanced in a 3D cell culture model of Multiple Myeloma in NCI-H 929 and U266. We show that in TRAIL-resistant sub-populations of Multiple Myeloma cells, GSK343 responses were completely attenuated in RPMI 8226 although synergistic enhancement of apoptosis was observed in NCI-H 929. Furthermore, following isolation of PKH26Hi quiescent cell populations, TRAIL responses and enhancement of TRAIL responses by GSK343 were completely attenuated. These studies show that EZH2 inhibition enhances TRAIL responses both in TRAIL-sensitive and TRAIL-resistant MM cell lines suspension culture and also in 3D cell culture to model the semi-solid Multiple Myeloma lesions in bone. Pre-existing TRAIL resistance was also enhanced by EZH2, and although synergistic enhancement of TRAIL responses by GSK343 was seen in NCI-H 929, responses were completely lost in TRAIL-resistant RPMI 8226 and also in quiescent cells. These studies highlight that although EZH2 inhibitors enhance TRAIL responses, acquired TRAIL resistance, and the presence of quiescent cells may mediate TRAIL-insensitivity in response to GSK343.

Genetic alterations that change the functions of p53 or other proteins in the p53 pathway contribute to a majority of cancers. Accordingly, many technological approaches and model systems have been employed to dissect the complex phenotypes of this critical tumor suppressor and its mutants. Studies of human p53 are commonly conducted in tumor-derived cell lines that retain wild type TP53 alleles and isogenic derivatives with engineered TP53 alterations. While this genetic approach has provided numerous insights, such studies are bound to paint an incomplete picture of p53 and its many effects on the cell. Given the preponderance of p53 pathway defects in cancer, it is reasonable to assume that cancers that arise without mutations in the TP53 coding sequence would very likely harbor other genetic or epigenetic alterations that effect the normal function of this pathway. One possible solution to this conundrum is to study p53 in cells that have been artificially immortalized. Unlike cells derived from tumors ex vivo, cells that have been immortalized in vitro are not shaped by evolutionary selection during tumorigenesis, and presumably retain many of the normal functions of p53 and other tumor suppressors. We report here a functional characterization of p53 in the immortalized human cell line hTERT-RPE1 and describe the dominant-negative effects of a heterozygous missense p53 A276P mutation that apparently arose during serial culture. Detailed studies of this contact mutant, also found in human tumors, demonstrate the practical utility of this model system for studying the complex phenotypes of human p53.

In the fusion-positive subset of rhabdomyosarcoma, the PAX3::FOXO1 oncoprotein is the most common fusion driver. We previously established a human myoblast system for inducible expression of PAX3::FOXO1. In the current study, we modulate PAX3::FOXO1 protein expression to understand the epigenetic and phenotypic functions at different PAX3::FOXO1 levels. Proliferative and oncogenic outcomes depend on PAX3::FOXO1 dosage in this system with transformation dominant at intermediate levels and growth suppression dominant at high levels. After prolonged PAX3::FOXO1 expression, there is dosage-dependent heterogeneity in single cell gene expression profiles. We observe a dosage-specific effect for PAX3::FOXO1 chromatin recognition and identify factors that modulate PAX3::FOXO1 chromatin binding. PAX3::FOXO1 dosage affects expression signatures related to cell cycle, epithelial-mesenchymal transition, and myogenesis. Whereas intermediate PAX3::FOXO1 expression maximizes chromatin binding to modulate gene expression, high PAX3::FOXO1 expression alters S phase progression and increases accessibility behind the replication fork. We conclude that PAX3::FOXO1 exerts dosage-dependent functions to influence epigenetic heterogeneity in fusion-positive rhabdomyosarcoma.

Although many cancer prognoses have improved in the past fifty years due to advancements in treatments, there has been little to no improvement in therapies for small cell lung cancer (SCLC) which currently has a five-year survival rate of less than 7%. One promising avenue to improve treatment for SCLC is to understand its underlying genetic alterations that drive its formation and growth. One such mutation in SCLC, which appears in many cancers, is of the Rb gene. When mutated, Rb causes hyperproliferation and loss of cellular identity. Normally Rb promotes differentiation by regulating lineage specific transcription factors including regulation of pluripotency factors such as Sox2. However, there is evidence that when certain tissues lose Rb, Sox2 becomes upregulated and promotes oncogenesis. To better understand the relationship between Rb and Sox2 and to uncover new treatments for SCLC we have studied the role of Sox2 in Rb loss initiated tumors by investigating both the tumor initiation in SCLC genetically engineered mouse models, as well as tumor maintenance in SCLC cell lines.

XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumor and hematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using hematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-{kappa}B(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision making in the personalized treatment of cancer patients undergoing treatment with selinexor.

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast S. cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides for basal reporter activity, the P53 RE enables LUC1 transactivation under the control of the human P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family proteins dependent expression was measured after shifting the yeast cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter can increase its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this improved accessibility, when present near the target binding site of P53 family transcription factors can be exploited in order to regulate the transcriptional activity of P53, P63 and P73 proteins.

Targeted inhibition of DNA damage response proteins has received significant clinical attention owing to the success of PARP inhibitors. Due to the loss of the G1/S checkpoint, cancer cells are reliant on the G2/M checkpoint to cope with elevated DNA replication stress. We previously demonstrated a single induction of 8-oxo-guanine at telomeres in cancer cells was sufficient to induce replication stress, but was well tolerated at the cellular level. Here, we found inhibition of ATR, Chk1, or Wee1 after induction of telomere oxidative stress significantly induced genome instability and reduced cell viability. This occurred at doses markedly less than those required to increase instability in non-cancer cells. We determined the mechanism of this instability is due to cells progressing through S-phase with telomere damage and exiting G2-phase prematurely, prolonging their mitosis. This study demonstrates targeted oxidative base damage at telomeres can enhance the therapeutic efficacy of ATR inhibition in cancer.

The fundamental transcription factor p53 regulates cellular processes and integrates signals of cellular stress, triggering a coordinated response to ensure survival of cells restored to healthy function and programmed death of those that couldnt be repaired. Unsurprisingly, this is one of the most mutated genes in human cancers, with most changes occurring in the DNA-binding domain of the protein. In this work, we take a genome-wide approach and use available resources to identify high confidence p53-target genes, that we examine in three breast cancer cell lines with different p53 status, wild type (MCF-7) and different mutations in the DNA-binding domain (MDA-MB231, T47D). Comparison of p53-targets expression in response to DNA damage by RNAseq and cellular assays reveals that MDA-MB231 have a severely impaired p53-dependent pathway functionality while T47D are much less affected. MDA-MB231 are more resistant to DNA damage yet unable to repair and able to override cell cycle arrest leading to survival while T47D are sensitive only to high dose and exposure to genotoxic agents. This data shows the variability of effects of different p53 mutations and highlight the importance of understanding the mechanisms of p53 in the context of genotoxicity-based treatment.

Bacterial polysaccharide vaccination is generally commenced within 6 months to 1 year following SCT. Antibody responses to these vaccines tend to be variable, raising concerns for inadequate protection from infection and the potential for repeated vaccination. This landmark analysis was performed to evaluate pathogen specific antibody titers, as well as helper T cell and B cell recovery from transplant and post-vaccination amongst patients surviving at least 6 months post allogeneic SCT. Antibody titers to various pneumococcal serotypes and Hemophilus influenza type B (HiB) followed a Power Law distribution in each individual at all time points evaluated. Distinct serotype vaccine responses were observed and antibody titer hierarchy developed early after vaccination and was maintained over time. When B cell recovery was modeled as a function of helper T cell reconstitution over time, the robustness of the antibody response was dependent on threshold values of cellular immune recovery. These suggest that antibody responses are a highly regulated dynamical system.

Inflammation is an early immune response against invading pathogens and damaged tissue. Although beneficial, uncontrolled inflammation leads to various diseases and may be fatal. Peroxynitrite (PN) is a major reactive nitrogen species (RNS) generated during inflammation. It produces various DNA lesions including labile 8-nitroguanine which spontaneously converts into abasic sites resulting in DNA strand breakage. Here, we report the discovery of a previously unrecognized function of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT or MGMT). We showed that hAGT through its active site nucleophilic Cys145 thiolate can spontaneously react with 8-nitroguanine in DNA to form a stable DNA-protein crosslink (DPC). Interestingly, the process of DPC formation provides protection from PN-mediated genome instability. The Cys145-mutant of hAGT failed to form DPC and provide protection against inflammation-associated, PN-mediated cytotoxicity. Gel shift, dot blot and UV-Vis assays showed formation of a covalent linkage between PN-damaged DNA and hAGT through its active site Cys145. Finally, expression of hAGT was found to be significantly increased by induced macrophages and PN. The data presented here clearly demonstrated hAGT as a dual function protein that along with DNA repair is capable of maintaining genomic integrity and providing protection from the toxicity caused by PN-mediated DNA damage. Although DPCs may seem detrimental, there are multiple systems in place in normal cells for their repair.

In the era of personalized medicine as well as precision medicine, targeted therapy has become an integral part of cancer treatment in conjunction with conventional chemo- and radiotherapy. We designed aptamer-siRNA chimeras that can specifically target cancers expressing EpCAM, a stem cell marker and deliver the specific siRNA required for therapy response. The siRNAs were chosen against PLK1, BCL2 and STAT3 as these oncogenes play prominent role in tumour progression of several cancers. Targeted delivery of EpCAM-siRNA chimeras resulted in cell death in several cancer cell lines such as cancers of the breast, lung, head and neck, liver and retinoblastoma. In vivo analysis of EpCAM-siRNA chimera mediated silencing on RB xenografts tumour model showed increased tumor reduction in all the three EpCAM-siRNA treated conditions. However, regulation of PLK1 exhibited higher efficacy in tumour reduction. Therefore. We studied signaling mechanism using global phosphoproteomics analysis. An increased P53 mediated downstream signalling pathway might have enabled increased apoptosis in the cancer cells. In conclusion, this study demonstrated the efficacy of EpCAM aptamer chimeras coupled to siRNA gene silencing for targeted anti-cancer therapy.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC=\"FIGDIR/small/656199v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (48K):\norg.highwire.dtl.DTLVardef@1ce89daorg.highwire.dtl.DTLVardef@bc5daeorg.highwire.dtl.DTLVardef@aa4ceforg.highwire.dtl.DTLVardef@a1159e_HPS_FORMAT_FIGEXP M_FIG C_FIG Illustration showing how EpCAM aptamer-mediated silencing of PLK1 could control the cell cycle progression at multiple number of check points and induce apoptosis involving hyper and hypophosphorylation of variety of signalling molecules

Rhabdomyosarcoma (RMS) is a tumor which resembles skeletal muscle. Current treatments are limited to surgery and non-targeted chemotherapy, highlighting the need for alternative therapies. Differentiation therapy uses molecules that act to shift the tumor cells phenotype from proliferating to differentiated, which in the case of skeletal muscle includes exit from the cell cycle and potentially fusion into myofibers. We previously identified EphA7 expressed on terminally differentiated myocytes as a potent driver of skeletal muscle differentiation: stimulation of ephrin-A5-expressing myoblasts with EphA7 causes them to undergo rapid, collective differentiation. We therefore tested EphA7 as a candidate molecule for differentiation therapy on human RMS (hRMS) cell lines. Surprisingly, EphA7 had a lesser effect than ephrin-A5, a difference explained by the divergent suite of Ephs and ephrins expressed by hRMS. We show that in hRMS ephrin-A5 binds and signals to EphA8 and EphA7 binds and signals to ephrin-A2, and that Fc chimeras of both molecules are potent inhibitors of hRMS proliferation. These results identify key differences between hRMS and normal muscle cells and support further research into Eph:ephrin signaling as potential differentiation therapies. Summary statementThis study identifies EphA7 and ephrin-A5 as external regulators of rhabdomyosarcoma proliferation, highlighting ephrin-A5 as a potential candidate for differentiation therapy in future cancer treatments.

The phenotypic plasticity of cancer cells has recently emerged as an important factor of treatment failure. The mechanisms of phenotypic plasticity are not fully understood. One of the hypotheses is that the degree of chromatin accessibility defines the easiness of cell transitions between different phenotypes. To test this, a method to compare overall chromatin accessibility between cells in a population or between cell populations is needed. We propose to measure the chromatin accessibility of a cell by total fluorescence signal from nuclei stained with DNA-binding fluorescent molecules. This method is based on the existing data that some small molecules bind nucleosome-free DNA more easily than nucleosomal DNA. Thus, nuclear fluorescence of these molecules is proportional to the amount of nucleosome-free DNA, serving as a measure of chromatin accessibility. We optimized the method using several DNA binding molecules and known chromatin modulating agents. Using a set of tumor and non-tumor cells of different origins we observed the tendency to the higher chromatin accessibility of tumor versus non-tumor cells. Chromatin accessibility was also increased upon oncogene-induced transformation of mouse and human cells.

Non-Small Cell Lung Cancer (NSCLC) patients are diagnosed late when the disease has metastasized. Kras is a prevalent mutation in NSCLC besides EGFR and TP53 and targeted therapies against this have been challenging. We have explored deregulation of an endocytic adapter protein, Huntingtin Interacting Protein-1(HIP1) and its relevance in a Kras mutant lung adenocarcinoma cell line as a model system. HIP1 RNA expression is observed to be significantly reduced in high-grade and metastatic lung cancer patients as compared to low-grade tumours and this correlates with poor survival. HIP1 depletion followed by global proteome profiling in A549 cells identified metabolic pathways to be majorly upregulated, followed by RNA transport and surveillance, amongst others. HIP1 depletion also significantly increased anchorage independent growth and invasion of these cells. However, the EMT markers did not follow the canonical regulation. We observed E-Cadherin and Vimentin induction, which is suggestive of collective migration. Additionally, we observed a hypoxic microenvironment to induce HIP1 expression, mediated by Hypoxia Inducible Factor 2 (HIF2), suggesting that a HIF2-HIP1 axis can cause tumour suppression and needs further exploration.

Missense mutations in the DNA binding domain of p53 are observed frequently in Esophageal Squamous Cell Carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common hotspot p53 mutations while rarer mutations are poorly characterized. We had previously identified SMARCD1 as an oncogenic transcriptional target of rare non-hotspot p53 mutants detected from squamous cell carcinoma of the oral tongue (SCCOT). We now report the characterization of non-hotspot p53 mutations from ESCC. In-vitro tumorigenic assays performed following ectopic-expression of non-hotspot mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harbouring mutant p53. Of these, ARF6, C1QBP and TRIM23 were studied further due to their previously reported pro-oncogenic roles. Reverse transcription quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumor samples revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild type and several mutant p53 forms followed by RT-qPCR, Chromatin affinity-purification and Promoter-luciferase assays indicated the exclusive recruitment of p53 mutants - P190T and P278L, to the target genes leading to activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effect of the genes was confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of rare non-hotspot mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor specific p53 mutations.

Biochemical investigations of the hepatoprocarcinogen N-acetyl-2-aminofluorene (AAF) have shown that normal adult rat hepatocytes in primary culture express two sets of pharmacokinetic constants - designated Systems I and II, and Sites I and II - associated respectively with the metabolism (System I [high-affinity Km[APPARENT] and low-velocity VMAX[APPARENT]] and System II [low-affinity Km APPARENT] and high-velocity VMAX[APPARENT]]), and the macromolecular binding (Site I [high-affinity KD[APPARENT] and low capacity BMAX[APPARENT]] and Site II ([low-affinity KD[APPARENT] and high-capacity BMAX[APPARENT]]) of AAF. Additional findings - that genomically saturating levels of AAF-DNA adducts form far below reported extracellular AAF concentrations required to block replicative and repair DNA synthesis; and, that biphasic Site I and Site II BMAX[APPARENT] and KD[APPARENT] expression curves varied inversely with respect to time and magnitude during hepatocyte growth - led us to wonder how macromolecular binding constants are expressed during chemical hepatocarcinogenesis. These questions were addressed by Scatchard analysis measurements through five consecutive AAF feeding cycles. Notably, cultured premalignant hepatocytes displayed reduced and elevated BMAX[APPARENT] and KD[APPARENT] levels, respectively, akin to the Site I and Site II expression curves observed during hepatocyte growth transitions in vitro. In contrast, prominent hepatocellular functions such as N-OH-AAF production, DNA replication, cell aggregation and resistance to AAF toxicity displayed different temporal trajectories. Impact StatementStriking similarities are observed between both Site I and Site II BMAX and KD expression curves during in vitro and in vivo premalignant growth transitions. These new findings fit earlier ones that hepatocytes growing during carcinogen exposure manifest fewer intranuclear AAF-DNA adducts. How this phenomenon leads to malignancy remains unclear.

Osteosarcoma (OSA) is a relatively rare but aggressive bone malignancy that primarily affects children and adolescents and has not seen survival improvements in at least three decades. No clinical or pathological prognostic factor has been identified that can lead to improved outcomes. Further, the complex genomic architecture and genetic heterogeneity of OSA and the rare nature of the tumor have significantly hampered identification and validation of molecular predictors. Recent assessment of DNA methylation in OSA has associated patterns with clinically informative outcomes, yet models building on these findings that can be applied in the clinic have not been developed. To address this unmet need, we developed methylation-based models for individualized patient risk prognostication and treatment response prediction at the time of diagnosis. By applying a region-based method to assess methylation, we were able to greatly reduce dimensionality and soften CpG-level noise, yielding stable features for model construction. These models, based on variably methylated regions (VMRs), underwent comprehensive internal cross-validation, and when possible, external validation in a completely independent dataset that was generated using a different technology from the discovery dataset. Further, they were contextualized with an unsupervised exploration of the features, showing widespread global hypermethylation being associated with unfavorable outcomes. Additionally, survival-associated signal appeared to be concentrated within a smaller, more focal subset of features, while in contrast, the chemoresponse signal seemed to come from a larger, more diffuse feature set, potentially suggesting a shift in cell state. An examination of epigenetic age using a methylation-derived epigenetic clock was also shown to provide an orthogonal approach of investigating the methylation landscape with significant association to outcome. Finally, clustering phenotypes were compared between methylation and several other omics platforms (mRNA, miRNA, and copy number variation) and showed very little overlap with one another. Together, these findings establish a foundation for methylation-based outcome stratification at the time of diagnosis and underscore the need for continued investigation including validation in larger and prospective cohorts.

Fanconi anaemia (FA) is a rare, recessive, genetic disorder characterised by a predisposition to cancer. Patients with FA are 700 times more likely to develop head and neck squamouscell carcinomas including oral epithelial dysplasia (OED), a potentially malignant disorder of the oral mucosa. This increased likelihood suggests that the molecular mechanism responsible for dysplastic transformation may involve defects in the FA pathway. In this study, the significance of ataxia telangiectasia and Rad3-related protein (ATR), which is responsible for homologous recombination repair (HRR), was investigated. ATR protects against mutations in both normal squamous cells and cancer cells by inducing HRR, and thus is associated with radiotherapy resistance. This investigation was designed to study the effects of heat on DNA repair specific to the FA pathway. Western blotting was carried out to determine whether heat affects the ATR pathway, followed by survival assays to determine the viability of cells after heat treatment and then compared to cells treated with a specific ATR inhibitor. This project aims to discover whether heat could be used as a non-invasive treatment to increase the sensitivity of tumour cells towards radiotherapy leading to an improved treatment plan for patients suffering from head and neck cancers.