Biology subjects

Graham, T.

Publications and source records attributed to Graham, T..

At least 19 recordsLinked to original sources

bioRxiv2026

A COJEC-chemotherapy resistant model of Th-ALK(F1174L)/MYCN neuroblastoma offers insights into tumour immune evasion and development of the bone marrow metastatic niche

Multi-agent COJEC chemotherapy is the main-stay of induction treatment for patients diagnosed with high-risk neuroblastoma. However, at least 10% of patients will be primary refractory to chemotherapy and only 50% achieve 5-year overall survival. The bone marrow is the most frequent site of metastasis in these patients. Novel approaches are required to improve response rates but the inter- and intra- patient tumour heterogeneity and dynamics of the neuroblastoma immune microenvironment makes anticipation of resistance phenotypes incredibly challenging. We present here a novel immunocompetent C57 Bl/6 model of Th-ALK(F1174L)/MYCN neuroblastoma, in which spontaneous abdominal tumours are driven by expression of mutant Anaplastic Lymphoma Kinase and over-expression of Mycn in the neural crest. We have used this model to generate a personalised dosing schedule inducing COJEC-chemotherapy resistance, in which individual mice receive chemotherapy cycles dependent upon the progression of their neuroblastoma tumours. Using both single cell RNA sequencing and spatial immunophenotyping gave us extraordinary precision in our comprehensive analysis of the tumour intrinsic and microenvironmental factors associated with COJEC resistance. We found that the resistance phenotype was driven by Cdk8 upregulation in adrenergic and mesenchymal tumour cells. Infiltration of immunosuppressive myeloid-derived immune cells and remodeling of the tumour-associated stroma further contributed to COJEC resistance. In the bone marrow we observed expansion of neutrophils and evidence of NETosis associated with micro-metastatic disease. Our results further endorse the development of CDK8-targeting therapeutics for neuroblastoma patients which might boost the anti-tumour immune response. Additional studies will be required to define the roles of neutrophils and neutrophil NETosis in neuroblastoma progression and metastasis. Our C57 Bl/6 model will be pivotal in future preclinical studies of immune-modulating therapeutics.

cancer biology↗

bioRxiv2026

Epigenetic evolution of colorectal cancer and its microenvironment reveals new vulnerabilities

Epigenetics is central to tumorigenesis, but the co-evolution of the cancer epigenome and its microenvironment is severely understudied. Here, we measure chromatin accessibility and transcriptome, at single cell resolution, of a set of normal colon, primary colorectal cancers and metastases, and identify recurrent epigenetic alterations in tumour cells. We also found that the normal epithelium adjacent to the cancer had recurrent epigenetic alterations associated with inflammatory programs that were partially shared with tumour cells. Distinct tumour-intrinsic transcription factor binding programs were associated with differential abundance of malignant stroma cell identities. We then leveraged matched patient-derived organoids, to assess the functional impact of the most recurrent epigenetic alterations on cancer cell viability, using CRISPR interference. We found a set of epigenetic-driven cancer dependencies, related to developmental reprogramming and cellular stress resilience, representing new potential therapeutic targets.

cancer biology↗

bioRxiv2026

Whole genome sequencing of endometrial cancer identifies novel subgroups, drivers, and actionable alterations

Endometrial cancer (EC) is the most common gynaecological malignancy in high income countries, and is increasing in incidence. While molecular stratification has improved its management, precision care is hampered by incomplete characterization of the EC genome. We address this by analysis of whole genome sequencing (WGS) of 665 ECs generated by the UK Genomics England 100,000 Genome Project (100kGP). 5% of cases were associated with germline pathogenic variants in cancer genes, including BRCA1 which we confirmed predisposes to EC. We identified 107 putative coding driver genes, 35% of which had no prior established role in EC. Novel structural variants included gains of MYCN and loss of its negative regulator NEDD4.1 which were significantly mutually exclusive in copy number (CN) high tumours. Immunogenomic analysis confirmed selection for driver alterations of low immunogenicity based on patient HLA haplotype, and pervasive immune escape through multiple mechanisms. Unsupervised clustering of mutational signatures and genomic alterations identified known and novel molecular subgroups, including a CN-high subset with mutational signatures of homologous recombination deficiency (HRD) and favourable outcome. Independent prognostic value of single nucleotide variant (SNV) burden, CN burden and multiple coding drivers, along with the identification of targetable molecular alterations in over one-third of cases, underscores the promise of WGS for precision medicine in EC.

genomics↗

bioRxiv2026

Tumour evolution as ground truth for cancer whole-genome sequencing

Cancer genomes are shaped by evolutionary processes that couple mutagenesis, clonal selection, chromosomal instability, spatial growth and treatment response into structured genomic patterns, yet current benchmarking strategies largely ignore this evolutionary dependency. Here, we present SCOUT, a large-scale synthetic whole-genome sequencing resource of over 200 samples, designed for systematic benchmarking of tumour genomic analysis and evolutionary inference under controlled evolutionary ground truth. Unlike conventional task-specific simulations, SCOUT models tumour evolution as a latent generative process that simultaneously shapes mutations, copy-number alterations, variant allele frequencies, mutational signatures and clonal architectures. SCOUT recapitulates key features of solid and haematological malignancies, including driver mutations, chromosomal instability, intratumour heterogeneity, spatial sampling and treatment-associated evolutionary dynamics in tumour and matched-normal longitudinal and multi-region sequencing designs. Using SCOUT, we benchmarked widely used methods for somatic variant detection, copy-number analysis, mutational signature inference and tumour evolutionary reconstruction. Across analytical tasks, performance deteriorated in low-purity, highly subclonal and structurally complex tumours, while spatial sampling bias and hypermutation generated spurious evolutionary signals that confounded tumour interpretation across multiple inference layers. Evolutionary simulations further distinguished lineage-restricted genetic bottlenecks from multi-lineage resistance dynamics associated with tumour plasticity. Tumour purity consistently exerted a stronger effect on inference accuracy than sequencing depth. Together, our results establish evolutionary ground truth as a prerequisite for reproducible benchmarking and biologically interpretable analysis of cancer whole-genome sequencing data.

bioinformatics↗

bioRxiv2025

Methylation dynamics in the decades preceding acute myeloid leukaemia

DNA methylation is emerging as a highly sensitive and specific marker of cancer initiation and progression. How these cancer-specific methylation changes are established in the decades before cancer, however, remains largely unknown. Here, we use a unique collection of longitudinal blood samples collected annually up to 15 years prior to a diagnosis of acute myeloid leukaemia to sensitively track the dynamics of DNA methylation changes at high temporal resolution. We identify thousands of differentially methylated regions (DMRs) that exhibit altered patterns of methylation up to 10 years before acute myeloid leukaemia (AML) diagnosis. Most of these DMRs are strongly associated with expanding clones carrying somatic driver mutations. We identify a subset of epigenetic driver DMRs characterised by recurrent CpG alterations that are highly shared across pre-AML cases. These are likely to reflect early epigenetic reprogramming associated with AML development. We also reveal large numbers of stochastic passenger CpGs whose differential methylation results from hitch-hiking with clonal expansions driven by somatically acquired genetic events. These passenger CpGs can be exploited for lineage tracing to discover clonal expansions driven by missing driver mutations. Our findings show widespread changes in methylation patterns during the early stages of cancer development which could be utilised for risk prediction and therapeutic intervention.

genomics↗

bioRxiv2025

Single cell multi-omic whole genome sequencing and chromatin accessibility profiling reveals genome-epigenome coevolution in colorectal cancer

Epigenetic alterations co-evolve with genetic mutations to drive carcinogenesis and treatment response. Resolving genome-epigenome coevolution requires accurate multi-omic single cell measurement. Here we develop a new technology called "double ATAC" (dATAC) for high-throughput, high-quality, concurrent whole genome sequencing and chromatin accessibility profiling of individual somatic cells. dATAC is a "one pot" method that uses two rounds of tagmentation to sequentially label open chromatin regions and then the whole genome, and produces data of the same quality as current leading single-omic methods. Using colorectal cancer as a model system, we apply dATAC to reveal convergent reorganisation of the epigenome across expanding drug-resistant clones during 5-FU chemotherapy exposure, remarkable stoichiometry of chromatin accessibility at somatic copy number alterations, and the clonal expansion of copy-number altered T cells in the stroma of metastatic disease. dATAC is a robust single cell technology to accurately profile genome-epigenome coevolution across tissues, diseases and species.

molecular biology↗

bioRxiv2024

Long deletion signatures in repetitive genomic regions track somatic evolution and enable sensitive detection of microsatellite instability

Deficiency in the mismatch repair system (MMRd) causes microsatellite instability (MSI) in cancers and determines eligibility for immunotherapy. Here, we show that MMRd tumours harbour long-deletion signatures ([≥]2-5+ base pairs deleted in repetitive regions), which provide new insights into MSI evolution and enable sensitive MSI detection particularly in challenging clinical samples. Long deletions, accumulated through stepwise DNA slippage errors, are significantly more prevalent in metastatic MMRd tumours compared to primary tumours. Importantly, we show that long-deletion signatures harbour features that are distinct from background noise, making them robustly detectable even in shallow whole genome sequencing (sWGS, [~]0.1X coverage) of formalin-fixed samples. We constructed a machine learning classifier that uses these distinct features to detect Microsatellite Instability in LOw-quality (MILO) samples. MILO achieved 100% accuracy in detecting MSI in sWGS data with only 2%-15% tumour purity and demonstrated promise in identifying MMRd clones in precancerous intestinal lesions. We propose that MILO could be clinically used for the sensitive monitoring of MMRd cancer evolution from early to late stages, using minimal sequencing data from both archival and fresh-frozen samples with low tumour content. SignificanceMutational signatures characterised by long deletions in repetitive genomic regions provide a sensitive route to detect and track MMRd clone evolution, even with low purity shallow whole genome sequencing data.

bioinformatics↗

bioRxiv2023

Epigenetic heritability of cell plasticity drives cancer drug resistance through one-to-many genotype to phenotype mapping

Cancer drug resistance is multi-factorial, driven by heritable (epi)genetic changes but also phenotypic plasticity. Here we dissect it by perturbing colorectal cancer patient-derived organoids longitudinally with drugs in sequence. Combining longitudinal tracking, single cell omics, evolutionary modelling, and machine leaning, we found that different targeted drugs select for distinct subclones, supporting rationally designed drug sequences. The cellular memory was encoded as a heritable epigenetic configuration, from which multiple transcriptional programmes could run, supporting a one-to-many (epi)genotype-to- phenotype map that explains how clonal expansions and plasticity manifest together. This may ensure drug resistance subclones can exhibit distinct phenotypes in changing environments while still preserving the cellular memory encoding for their selective advantage. Chemotherapies resistance was instead entirely driven by plasticity. Inducing further chromosomal instability before drug application changed clonal evolution but not convergent transcriptional programmes. Collectively, our data show how genetic and epigenetic alterations are selected to "permissive epigenome" enabling phenotypic plasticity.

cancer biology↗

bioRxiv2023

NMR structure of the Orf63 pro-lytic protein from lambda bacteriophage

The orf63 gene resides in a region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed during infection. In lambda phage and Shiga toxin (Stx) producing phages found in enterohemorrhagic E. coli (EHEC) associated with food poisoning, Orf63 expression reduces the host survival and hastens the period between infection and lysis thereby giving it pro-lysogenic qualities. The NMR structure of dimeric Orf63 reveals a fold consisting of two helices and one strand that all make extensive intermolecular contacts. Structure-based data mining failed to identify any Orf63 homolog beyond the family of temperate bacteriophages. A machine learning approach was used to design an amphipathic helical ligand that bound a hydrophobic cleft on Orf63. This approach may open a new path towards designing therapeutics that antagonize the contributions of Stx phages in EHEC outbreaks.

biochemistry↗

bioRxiv2023

Adaptive therapy achieves long-term control of chemotherapy resistance in high grade ovarian cancer

Drug resistance results in poor outcomes for most patients with metastatic cancer. Adaptive Therapy (AT) proposes to address this by exploiting presumed fitness costs incurred by drug-resistant cells when drug is absent, and prescribing dose reductions to allow fitter, sensitive cells to re-grow and re- sensitise the tumour. However, empirical evidence for treatment-induced fitness change is lacking. We show that fitness costs in chemotherapy-resistant ovarian cancer cause selective decline and apoptosis of resistant populations in low-resource conditions. Moreover, carboplatin AT caused fluctuations in sensitive/resistant tumour population size in vitro and significantly extended survival of tumour-bearing mice. In sequential blood-derived cell-free DNA and tumour samples obtained longitudinally from ovarian cancer patients during treatment, we inferred resistant cancer cell population size through therapy and observed it correlated strongly with disease burden. These data have enabled us to launch a multicentre, phase 2 randomised controlled trial (ACTOv) to evaluate AT in ovarian cancer.

cancer biology↗

bioRxiv2022

Whole genome sequencing of 2,023 colorectal cancers reveals mutational landscapes, new driver genes and immune interactions

To characterise the somatic alterations in colorectal cancer (CRC), we conducted whole-genome sequencing analysis of 2,023 tumours. We provide the most detailed high-resolution map to date of somatic mutations in CRC, and demonstrate associations with clinicopathological features, in particular location in the large bowel. We refined the mutational processes and signatures acting in colorectal tumorigenesis. In analyses across the sample set or restricted to molecular subtypes, we identified 185 CRC driver genes, of which 117 were previously unreported. New drivers acted in various molecular pathways, including Wnt (CTNND1, AXIN1, TCF3), TGF-{beta}/BMP (TGFBR1) and MAP kinase (RASGRF1, RASA1, RAF1, and several MAP2K and MAP3K loci). Non-coding drivers included intronic neo-splice site alterations in APC and SMAD4. Whilst there was evidence of an excess of mutations in functionally active regions of the non-coding genome, no specific drivers were called with high confidence. Novel recurrent copy number changes included deletions of PIK3R1 and PWRN1, as well as amplification of CCND3 and NEDD9. Putative driver structural variants included BRD4 and SOX9 regulatory elements, and ACVR2A and ANKRD11 hotspot deletions. The frequencies of many driver mutations, including somatic Wnt and Ras pathway variants, showed a gradient along the colorectum. The Pks-pathogenic E. coli signature and TP53 mutations were primarily associated with rectal cancer. A set of unreported immune escape driver genes was found, primarily in hypermutated CRCs, most of which showed evidence of genetic evasion of the anti-cancer immune response. About 25% of cancers had a potentially actionable mutation for a known therapy. Thirty-three of the new driver genes were predicted to be essential, 17 possessed a druggable structure, and nine had a bioactive compound available. Our findings provide further insight into the genetics and biology of CRC, especially tumour subtypes defined by genomic instability or clinicopathological features.

genomics↗

bioRxiv2021

A gene coevolution network provides insight into eukaryotic cellular and genomic structure and function

Orthologous gene coevolution--which refers to gene pairs whose evolutionary rates covary across speciation events--is often observed among functionally related genes. We present a comprehensive gene coevolution network inferred from the examination of nearly three million orthologous gene pairs from 332 budding yeast species spanning [~]400 million years of eukaryotic evolution. Modules within the network provide insight into cellular and genomic structure and function, such as genes functioning in distinct cellular compartments and DNA replication. Examination of the phenotypic impact of network perturbation across 14 environmental conditions using deletion mutant data from the bakers yeast Saccharomyces cerevisiae suggests that fitness in diverse environments is impacted by orthologous gene neighborhood and connectivity. By mapping the network onto the chromosomes of S. cerevisiae and the opportunistic human pathogen Candida albicans, which diverged [~]235 million years ago, we discovered that coevolving orthologous genes are not clustered in either species; rather, they are most often located on different chromosomes or far apart on the same chromosome. The budding yeast coevolution network captures the hierarchy of eukaryotic cellular structure and function, provides a roadmap for genotype-to-phenotype discovery, and portrays the genome as an extensively linked ensemble of genes.

evolutionary biology↗

bioRxiv2021

Germline loss-of-function variants in the base-excision repair gene MBD4 cause a Mendelian recessive syndrome of adenomatous colorectal polyposis and acute myeloid leukaemia

Inherited defects in base-excision repair (BER) predispose to adenomatous polyposis and colorectal cancer (CRC), yet our understanding of this important DNA repair pathway remains incomplete. By combining detailed clinical, histological and molecular profiling, we reveal biallelic germline loss-of-function (LOF) variants in the BER gene MBD4 to predispose to adenomatous polyposis and -uniquely amongst CRC predisposition syndromes- to myeloid neoplasms. Neoplasms from MBD4-deficient patients almost exclusively accumulate somatic CpG>TpG mutations, resembling mutational signature SBS1. MBD4-deficient adenomas harbour mutations in known CRC driver genes, although AMER1 mutations were more common and KRAS mutations less frequent. We did not find an increased risk for colorectal tumours in individuals with a monoallelic MBD4 LOF variant. We suggest that this condition should be termed MBD4-associated neoplasia syndrome (MANS) and that MBD4 is included in testing for the genetic diagnosis of polyposis and/or early-onset AML.

genetics↗

bioRxiv2020

The P4-ATPase Drs2 regulates the homeostasis of Atg9

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether transported vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are just partially understood. Here we systematically investigate the interactome that regulates the function of MTCs. We found that P4-ATPases, a family of lipid transporters involved in the biogenesis of vesicles, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We used the lipid flippase Drs2 as a model to investigate the mechanism and biological relevance of such interplay during the transport of Atg9 vesicles. Binding to the N-terminal tail of Drs2 stabilizes TRAPPIII on membrane compartments loaded with Atg9 and it is required for the delivery of Atg9 during selective autophagy, a role that is independent of previously reported functions of the P4-ATPase. This mechanism relies on the I(S/R)TTK motif nested in the N-terminal tail cavity of Drs2, a motif that is required for the interaction with MTCs.

cell biology↗

bioRxiv2020

Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells

The SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with currently 29 million confirmed cases and close to a million deaths. At this time, there are no FDA-approved vaccines or therapeutics for COVID-19, but Emergency Use Authorization has been granted for remdesivir, a broad-spectrum antiviral nucleoside analog. However, remdesivir is only moderately efficacious against SARS-CoV-2 in the clinic, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. We identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5 A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivirs apparent potency 25-fold. We therefore suggest that the FDA-approved Hepatitis C therapeutics Epclusa (velpatasvir/sofosbuvir) and Zepatier (elbasvir/grazoprevir) should be fast-tracked for clinical evaluation in combination with remdesivir to improve treatment of acute SARS-CoV-2 infections.

microbiology↗

bioRxiv2020

Single-Strand DNA Breaks Cause Replisome Disassembly

DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet CMG eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks", which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates beyond the nick on double-stranded DNA and is then actively removed from chromatin by the p97 ATPase. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they argue that CMG cannot be deposited on dsDNA while cells resolve replication stress. HighlightsO_LIThe structures of leading and lagging strand collapsed forks are different C_LIO_LICMG passively "runs off" the broken DNA end during leading strand fork collapse C_LIO_LICMG is unloaded from duplex DNA after lag collapse in a p97-dependent manner C_LIO_LINicks are uniquely toxic lesions that cause fork collapse and replisome disassembly C_LI

molecular biology↗

bioRxiv2018

Point of Care Diagnostic Deployment and Targeted Treatment of Antimicrobial Resistant Non-Typhoidal Salmonella: a New Mathematical Model and Cost-Benefit Analysis

Invasive non-typhoidal Salmonella (NTS) is among the leading causes of blood stream infections in sub-Saharan Africa and other developing regions, especially among pediatric populations. Invasive NTS can be difficult to treat and have high case-fatality rates, in part due to emergence of strains resistant to broad-spectrum antibiotics. Furthermore, improper treatment contributes to increased antibiotic resistance and death. Point of care (POC) diagnostic tests that rapidly identify invasive NTS infection, and differentiate between resistant and non-resistant strains, may greatly improve patient outcomes and decrease resistance at the community level. Here we present for the first time a model for NTS dynamics in high risk populations that can analyze the potential advantages and disadvantages of four strategies involving POC diagnostic deployment, and the resulting impact on antimicrobial treatment for patients. Our analysis strongly supports the use of POC diagnostics coupled with targeted antibiotic use for patients upon arrival in the clinic for optimal patient and public health outcomes. We show that even the use of imperfect POC diagnostics can significantly reduce total costs and number of deaths, provided that the diagnostic gives results quickly enough that patients are likely to return or stay to receive targeted treatment.

epidemiology↗

bioRxiv2018

Reply: Neutral tumor evolution?

Mutation, selection and neutral drift shape the cancer evolutionary process1. The role of selection has received particular interest, but inferring the presence and strength of selection during tumour growth remains challenging. Recently, we analysed the frequency distribution of subclonal mutations in many cancers and found that in approximately 30% of cases the observed distribution was entirely consistent with a simple model of neutral evolution2. Thus, we concluded that neutral evolution, perhaps surprisingly, provides an adequate explanation of the intra-tumour heterogeneity present in a significant proportion of cancers.\n\nTarabichi and colleagues [bioRxiv: 2017/06/30/158006] question the robustness of the method we presented in Williams et al. 20162 to identify neutral cancer evolution from variant allele frequency (VAF) distributions. Their critique has four main points that we addre ...

cancer biology↗