Biology subjects

Connelly, J.

Publications and source records attributed to Connelly, J..

15 recordsLinked to original sources

bioRxiv2026

Explainable machine learning relates histological to genomic pathology

Background & AimsHaematoxylin and eosin (H&E) staining remains the diagnostic gold standard for solid cancers, including hepatocellular carcinoma, and is increasingly complemented by genomic profiling for precision medicine. Inferring genomic alterations directly from H&E images could streamline testing, but heterogeneity and biases in human training data limit interpretation of genotype-phenotype associations. Here, we aimed to relate histologic to genomic pathology to provide biological explainability for mutation prediction models and assess the impact of germline variation on model performance. MethodsWe analysed 597 murine liver tumours with matched whole-genome sequencing and histopathology (163,835 image tiles; 22.9 million nuclei). Our controlled in vivo design accounted for germline variation, biological sex, and causal mutagen (N-diethylnitrosamine), removing confounding factors present in human cohorts. We trained and evaluated deep learning and supervised machine learning models to predict germline variation and cancer driver alterations from H&E. ResultsModelling accurately predicted germline and somatic alterations from histology, at both locus-specific and genome-wide scales. Quantitative image analysis revealed an unexpected association between Egfr driver mutations and hepatic steatosis, linking genotype to an interpretable morphological phenotype. While model performance declined when applied to tumours from unrepresented genetic backgrounds, this limitation was biologically informative, revealing strain-dependent differences in tumour evolution, notably the prevalence of whole-genome duplication. ConclusionsMachine learning integration of histological and genomic pathology enables accurate, interpretable inference of genetic alterations from H&E, potentially reducing reliance on costly ancillary molecular assays. Our predictions are supported by human-interpretable biological features, addressing concerns around "black-box" technologies. However, caution is required when applying such methods to samples with a genetic background that, even if closely related, is beyond the genetic horizon of training data.

pathology↗

bioRxiv2026

Computational modeling and preclinical validation support targeting somatic instability for Huntington's disease treatment

Huntingtons disease (HD) is caused by an expanded CAG trinucleotide repeat within the huntingtin (HTT) gene. Genetic modifiers of disease onset and progression in HD implicate somatic instability (SI) of the expanded CAG repeat as a key pathogenic driver, with MSH3 emerging as a leading therapeutic target. Reducing SI, particularly in the most affected neuronal cell type, medium spiny neurons (MSNs) of the striatum, is thus a rational therapeutic strategy for HD. To inform the development of an SI-targeted therapy, we generated a computational model simulating SI in MSNs to infer therapeutic effects on MSN survival resulting from an intervention that reduces SI. The model takes advantage of HD patient data to predict therapeutic benefit across a range of inherited CAG lengths and ages of intervention, considering the degree of target engagement regionally and per cell. To target SI experimentally, we designed an artificial microRNA to lower MSH3 mRNA (miMSH3) after delivery with AAV-DB-3, a previously described MSN-targeting AAV capsid variant. AAV-DB-3.miMSH3 achieved from 48 to 94% MSH3 mRNA reduction in MSNs of nonhuman primates (NHPs), which, when modeled, would reduce the composite Unified Huntington Disease Rating Scale change over baseline from 50 to over 120% as well as delay motor symptom onset by many years. AAV-DB-3.miMSH3 also showed robust target engagement in vivo with up to 46% reduction in SI in HdhQ111 mice. The integration of preclinical experimental data and the computational model support the translational potential of AAV-DB-3.miMSH3 as a disease-modifying therapy applicable for HD patients with a broad range of inherited repeat lengths. One Sentence SummaryPredictive modeling to guide therapeutic targeting of disease modifiers in Huntingtons disease.

neuroscience↗

bioRxiv2025

Germline and somatic variation influence nuclear-mitochondrial crosstalk in tumourigenesis

Hepatocellular carcinoma is the most prevalent primary liver cancer and arises from hepatocytes, which are very metabolically active and contain abundant mitochondria. Despite extensive characterisation of nuclear driver genes and pathways, the role of crosstalk between the mitochondrial and nuclear genomes in mitochondrial mutagenesis remains poorly understood. To address this, we leveraged a chemical carcinogenesis model of liver cancer in four mouse strains. We analysed hundreds of tumours with paired whole-genome sequencing and total RNA sequencing to comprehensively characterise mitochondrial mutations and expression. Following de novo assembly of strain-specific mitochondrial genomes, we devised an accurate and efficient heteroplasmy detection approach and analysed mitochondrial DNA (mtDNA) content and transcription. Across strains, there was high concordance of the number of heteroplasmies, mutation signatures, and variant allele frequencies. In contrast, the heteroplasmy rate and genic locations differed between strains, suggesting strain-specificity in DNA repair mechanisms in early tumour development. Independent of age or environmental factors, tumours had lower mtDNA content than adjacent normal liver, suggesting mitochondrial loss during tumourigenesis. Additionally, within individual strains, mtDNA content differences were associated with nuclear driver gene choice. Our comprehensive characterisation of mitochondrial genomes and expression patterns demonstrates that both germline and somatic genetic variation influence tumour mtDNA content, mutation patterns, and gene expression.

cancer biology↗

bioRxiv2025

Genetic background sets the trajectory of cancer evolution

Human cancers are heterogeneous. Their genomes evolve from genetically diverse germlines in complex and dynamic environments, including exposure to potential carcinogens. This heterogeneity of humans, our environmental exposures, and subsequent tumours makes it challenging to understand the extent to which cancer evolution is predictable. Addressing this limitation, we re-ran early tumour evolution hundreds of times in diverse, inbred mouse strains, capturing genetic variation comparable to and beyond that found in human populations. The sex, environment, and carcinogenic exposures were all controlled and tumours comprehensively profiled with whole genome and transcriptome sequencing. Within a strain, there was a high degree of consistency in the mutational landscape, a limited range of driver mutations, and all strains converged on the acquisition of a MAPK activating mutation with similar transcriptional disruption of that pathway. Despite these similarities in the phenotypic state of tumours, different strains took markedly divergent paths to reach that state. This included pronounced biases in the precise driver mutations, the strain specific occurrence of whole genome duplication, and differences in subclonal selection that reflected both cancer susceptibility and tumour growth rate. These results show that interactions between the germline genome and the environment are highly deterministic for the trajectory of tumour genome evolution, and even modest genetic divergence can substantially alter selection pressures during cancer development, influencing both cancer risk and the biology of the tumour that develops.

genomics↗

bioRxiv2024

RBM39 degrader invigorates natural killer cells to eradicate neuroblastoma despite cancer cell plasticity

The cellular plasticity of neuroblastoma is defined by a mixture of two major cell states, adrenergic (ADRN) and mesenchymal (MES), which may contribute to therapy resistance. However, how neuroblastoma cells switch cellular states during therapy remains largely unknown and how to eradicate neuroblastoma regardless of their cell states is a clinical challenge. To better understand the lineage switch of neuroblastoma in chemoresistance, we comprehensively defined the transcriptomic and epigenetic map of ADRN and MES types of neuroblastomas using human and murine models treated with indisulam, a selective RBM39 degrader. We showed that cancer cells not only undergo a bidirectional switch between ADRN and MES states, but also acquire additional cellular states, reminiscent of the developmental pliancy of neural crest cells. The lineage alterations are coupled with epigenetic reprogramming and dependency switch of lineage-specific transcription factors, epigenetic modifiers and targetable kinases. Through targeting RNA splicing, indisulam induces an inflammatory tumor microenvironment and enhances anticancer activity of natural killer cells. The combination of indisulam with anti-GD2 immunotherapy results in a durable, complete response in high-risk transgenic neuroblastoma models, providing an innovative, rational therapeutic approach to eradicate tumor cells regardless of their potential to switch cell states.

cancer biology↗

bioRxiv2023

Engineered human myogenic cells in hydrogels generate functional myofibers within dystrophic mouse muscle

Transplantation of human myogenic progenitor cells (MPCs) is a promising therapeutic strategy for treating muscle-wasting diseases, e.g., Duchenne muscular dystrophy (DMD). To increase engraftment efficiency of donor stem cells, modulation of host muscles is required, significantly limiting their clinical translation. Here we develop a clinically relevant transplantation strategy synergising hydrogel-mediated delivery and engineered human MPCs generated from CRISPR-corrected DMD patient-derived pluripotent stem cells (PSCs). We demonstrate that donor-derived human myofibers produce full-length dystrophin at 4 weeks and 5 - 6 months (long-term) post transplantation in the unmodulated muscles of the dystrophin-deficient mouse model of DMD. Remarkably, human myofibers are innervated by mouse motor neurons forming neuromuscular junctions and supported by vascularisation after long-term engraftment in dystrophic mice. PAX7+ cells of human origin replenish the satellite cell niche. There was no evidence of tumorigenesis in mice engrafted with hydrogel-encapsulated human MPCs. Our results provide a proof-of-concept in developing hydrogel-based cell therapy for muscle-wasting diseases.

cell biology↗

bioRxiv2023

Insight into the emerging insect to human pathogen Photorhabdus revealing geographic differences in immune cell tropism.

Photorhabdus asymbiotica is a species of the insect pathogenic Photorhabdus genus that has been isolated as an etiological agent in human infections. Since then, multiple isolates have been identified worldwide, however actual clinical infections have so far only been identified in North America, Australia and Nepal. Previous research on the clinical isolates had shown that the strains differed in their behaviour when infecting cultured human cells, based on the geographical site of isolation. In this study we investigate the differences between the pathogenic activities of P. asymbiotica isolates from different geographic locations. We present findings from the clinical isolates from Australia (Kingscliff) and North America (ATCC43949), and soil borne nematode isolates from Thailand (PB68) and Northern Europe non-clinical P. asymbiotica genospecies HIT and JUN. We also show the first findings from a new clinical isolate of P. luminescens (Texas), the first non-asymbiotica species to cause a human infection, confirming its ability to infect and survive inside human immune cells. Infection assays were done using both cultured cell lines (THP-1, CHO and HEK cells) and also primary immune cells, Peripheral Blood Mononuclear cells (PBMCs) isolated from human blood. Here for the first time, we show how P. asymbiotica, selectively infects certain immune cells while avoiding others, and that infectivity varies depending on growth temperature. We also show that the infected immune cells vary depending on the geographical location a strain is isolated from, and that the European HIT and JUN strains lack the ability to survive within mammalian cells in tissue culture.

microbiology↗

bioRxiv2022

Strand-resolved mutagenicity of DNA damage and repair

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here, we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small DNA adducts, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky adducts5. We find that DNA damage tolerance is also common during transcription, where RNA-polymerases frequently bypass lesions without triggering repair. At multiple genomic scales, we show the pattern of DNA damage induced mutations is largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can corrupt the fidelity of nucleotide excision repair and actively drive oncogenic mutagenesis. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance, and repair of DNA damage, thereby shaping cancer genome evolution.

genomics↗

bioRxiv2022

Evaluation of CA repeat arrays as targets for MeCP2 function in the brain

The DNA binding protein MeCP2 is reported to bind methylated cytosine in CG and CA motifs in genomic DNA, but it was recently proposed that arrays of tandemly repeated CA containing either methylated or hydroxymethylated cytosine are the primary targets for MeCP2 binding and function. Here we investigated the predictions of this hypothesis using a range of published datasets. We failed to detect enrichment of cytosine modification at genomic CA repeat arrays in mouse brain regions and found no evidence for preferential MeCP2 binding at CA repeats. Moreover, we did not observe a correlation between the CA repeat density near genes and their degree of transcriptional deregulation when MeCP2 was absent. Our results do not provide support for the hypothesis that CA repeats are key mediators of MeCP2 function. Instead, we found that CA repeats are subject to CAC methylation to a degree that is typical of the surrounding genome and contribute modestly to MeCP2-mediated modulation of gene expression in accordance with their content of this canonical target motif.

genomics↗

bioRxiv2022

Design of an Integrated Microvascularised Human Skin-on-a-Chip Tissue Equivalent Model

Tissue engineered skin constructs have been under development since the 1980s as a replacement for human skin tissues and animal models for therapeutics and cosmetic testing. These have evolved from simple single cell-assays to increasingly complex models with integrated dermal equivalents and multiple cell types including a dermis, epidermis and vasculature. The development of micro-engineered platforms and biomaterials has enabled scientists to better recreate and capture the tissue microenvironment in vitro, including the vascularization of tissue models and their integration into microfluidic chips. However, to date, microvascularised human skin equivalents in a microfluidic context have not been reported. Here we present the design of a novel skin-on-a-chip model integrating human derived primary and immortalized cells in a full thickness skin equivalent. The model is housed in a microfluidic device, in which a microvasculature was previously established. We characterize the impact of our chip design on the quality of the microvascular networks formed and evidence that this enables the formation of more homogenous networks. We developed a methodology to harvest tissues from embedded chips, after 14 days of culture, and characterize the impact of culture conditions and vascularization (including with pericyte co-cultures) on the stratification of the epidermis in the resulting skin equivalents. Our results indicate that vascularization enhances stratification and differentiation (thickness, architecture and expression of terminal differentiation markers such as involucrin and transglutaminase 1), allowing formation of more mature skin equivalents in microfluidic chips. The skin-on-a-chip tissue equivalents developed, thanks to their realistic microvasculature, may find application for the testing efficacy and safety of therapeutics delivered systemically, in a human context.

bioengineering↗

bioRxiv2021

Toll-like receptor 2 orchestrates a potent anti-tumor response in non-small cell lung cancer

Targeting early-stage lung cancer is vital to improve overall survival. We previously identified Toll-like receptor 2 (TLR2) as a regulator of oncogene-induced senescence (OIS) and the senescence-associated secretory phenotype (SASP), both key for tumor suppression. Here, we demonstrate that TLR2 is widely expressed in human lung tumor epithelium where it correlates with improved survival and clinical regression. Using genetically engineered mouse models of lung cancer we have shown that Tlr2 is a tumor suppressor in lung cancer initiation via regulation of proliferation and the SASP. The SASP is integral in the regulation of immune surveillance of premalignant cells, and we observe impaired myeloid derived immune surveillance following Tlr2 loss. Lastly, we show that administration of a synthetic Tlr2 agonist significantly reduces preinvasive lung tumor growth. Our data highlight an unexpected tumor surveillance pathway in early-stage lung cancer with therapeutic potential. Statement of significanceLung cancer is a major cancer of unmet need. This study identifies a novel tumor suppressor mechanism in lung cancer. Not only does this highlight a potential therapeutic target for early-stage disease but also multiple secreted candidate biomarkers that could be exploited to augment lung cancer screening approaches.

cancer biology↗

bioRxiv2021

Radio-pathomic maps of cell density identify glioma invasion beyond traditional MR imaging defined margins

Current MRI signatures of brain cancer often fail to identify regions of hypercellularity beyond the contrast enhancing region. Therefore, this study used autopsy tissue samples aligned to clinical MRIs in order to quantify the relationship between intensity values and cellularity, as well as to develop a radio-pathomic model to predict cellularity using MRI data. This study used 93 samples collected at autopsy from 44 brain cancer patients. Tissue samples were processed, stained for hematoxylin and eosin (HE) and digitized for nuclei segmentation and cell density calculation. Pre- and post-gadolinium contrast T1-weighted images (T1, T1C), T2 fluid-attenuated inversion recovery (FLAIR) images, and apparent diffusion coefficient (ADC) images calculated from diffusion imaging were collected from each patients final acquisition prior to death. In-house software was used to align tissue samples to the FLAIR image via manually defined control points. Mixed effect models were used to assess the relationship between single image intensity and cellularity for each image. An ensemble learner was trained to predict cellularity using 5 by 5 voxel tiles from each image, employing a 2/3-1/3 train-test split for validation. Single image analyses found subtle associations between image intensity and cellularity, with a less pronounced relationship within GBM patients. The radio-pathomic model was able to accurately predict cellularity in the test set (RMSE = 1015 cells/mm2) and identified regions of hypercellularity beyond the contrast enhancing region. We concluded that a radio-pathomic model for cellularity is able to identify regions of hypercellular tumor beyond traditional imaging signatures.

neuroscience↗

bioRxiv2020

Accurate Genomic Variant Detection in Single Cells with Primary Template-Directed Amplification

Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here we present primary template-directed amplification (PTA), a new isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the new types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a new tool for mapping genome-wide interactions of mutagens with single living human cells at base pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate whole genome amplification, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

genomics↗

bioRxiv2020

Neuronal non-CG methylation is an essential target for MeCP2 function

SUMMARYDNA methylation is implicated in neuronal biology via the protein MeCP2, mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 corepressor complexes to methylated cytosine in the CG dinucleotide, but also to non-CG methylation, which is abundant specifically in neuronal genomes. To test the biological significance of its dual binding specificity, we replaced the MeCP2 DNA binding domain with an orthologous domain whose specificity is restricted to mCG motifs. Knock-in mice expressing the domain-swap protein displayed severe Rett syndrome-like phenotypes, demonstrating that interaction with sites of non-CG methylation, specifically the mCAC trinucleotide, is critical for normal brain function. The results support the notion that the delayed onset of Rett syndrome is due to the late accumulation of both mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2-null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of particular relevance to the Rett syndrome phenotype.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗

bioRxiv2020

Distinct genetic pathways define pre-leukemic and compensatory clonal hematopoiesis in Shwachman-Diamond syndrome

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome with predisposition to developing leukemia. We found that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations in EIF6 or TP53. EIF6 mutations cause functional compensation for the germline deficiency by alleviating the SDS ribosome joining defect, improving translation, and reducing p53 activation. TP53 mutations decrease checkpoint activation without affecting ribosome assembly. We link development of leukemia with acquisition of biallelic TP53 alterations. Our results define distinct pathways of clonal selection driven by germline fitness constraint and provide a mechanistic framework for clinical surveillance.

cancer biology↗