Biology subjects

Harrington, K. J.

Publications and source records attributed to Harrington, K. J..

11 recordsLinked to original sources

bioRxiv2026

Intratumoural oncolytic HSV-1 reshapes the local and systemic immune landscape through CD8+ T cell reprogramming

Most oncolytic viruses are delivered by intratumoural injection, and local administration can induce regression of both injected and distant tumours in mice and patients. However, the mechanisms by which local viral infection reprograms systemic immunity remain poorly understood. Here we show that intratumoural RP1, an oncolytic HSV-1 encoding GM-CSF and GALV-GP-R-, drives regression of injected and uninjected murine melanoma tumours and prolongs survival. RP1 elicits coordinated CD4 and CD8 T cell infiltration accompanied by local cytokine remodelling, reshaping the immune landscape at both tumour sites. Leveraging the Timer of Cell Kinetics and Activity (Tocky) system and Kaede photoconvertible protein technology, we resolve the temporal dynamics of CD8 T cell responses following local virotherapy and identify two systemically induced, virus-driven CD8 T cell populations distinguished by TCR engagement kinetics: antigen-engaged Timer-positive viral-induced precursors (VIPs) and Timer-negative KLRG1 viral-induced terminal effectors (VITEs). Single-cell transcriptomic and pseudotime analyses reveal divergent differentiation trajectories; VIPs exhibit sustained antigen engagement within the tumour microenvironment (TME), a transcriptional programme associated with self-renewal, and preferential homing to draining lymph nodes. A VIP-associated gene signature correlates with clinical response to RP1 plus PD-1 blockade in the IGNYTE trial of RP1 and nivolumab in PD-1-refractory melanoma and is independently associated with response to immune checkpoint inhibitor (ICI) therapy in melanoma. These findings establish a mechanistic link between local oncolytic virotherapy, systemic CD8 T cell reprogramming, and durable regression of distant lesions in patients.

cancer biology↗

bioRxiv2026

Preclinical efficacy of a systemically-administered, second-generation STING agonist that promotes antitumour immunity in combination with radiotherapy

As potent triggers of innate immunity, STING agonists hold promise as active immunotherapeutic agents for cancer treatment. Second-generation STING agonists, suitable for systemic delivery, are being investigated in preclinical research and have entered clinical trials. Here, the novel synthetic STING agonist, BI-1703880 (STINGa), which was designed for intravenous delivery, was investigated for anti-tumour and immunological effects. We show that STINGa activates the STING pathway and results in a transient and dose-dependent upregulation and secretion of interferons and proinflammatory cytokines in vitro and in vivo. We show that intravenous administration of repeated dosing with low-dose STINGa is well tolerated. We report that radiotherapy (RT) and STING agonism synergizes to generate innate immune cell and CD8+ T cell responses that control tumour growth. Anti-tumour activity induced by combined RT / STINGa was reduced in mice lacking a functional immune system. RT / STINGa combination treatment also initiated development of protective immune memory. RT / STINGa upregulated PD-L1, PD-1 and CTLA-4 in the tumour microenvironment. Our findings show that combining RT / STINGa with immune checkpoint inhibitors further increases therapeutic benefit. Our data confirm STING as a therapeutic target in cancer and support the clinical development of BI-1703880 STING agonist, thereby suggesting radiotherapy as a potential combination for enhancing anti-tumour efficacy.

cancer biology↗

bioRxiv2026

Addition of chemotherapy to radiotherapy promotes progenitor-exhausted CD8⁺ T-cell clonal dominance in head and neck cancer

Concomitant chemoradiotherapy (CRT) is a standard-of-care for unresectable locally-advanced head and neck squamous cell carcinoma (LA-SCCHN), but its immune effects, particularly compared to radiotherapy (RT) alone, remain unclear. Using syngeneic murine models, we integrated Nr4a3-Tocky reporter analysis with single-cell transcriptomics and T-cell receptor clonotyping comprehensively to profile intratumoural CD8 T-cells following RT/CRT. We show that CRT uniquely drives robust antigen-specific clonal expansion and biases differentiation toward progenitor (or precursor) exhausted (TPEX) phenotypes, while RT favours terminal exhaustion (TEX). Single-cell analyses reveal CRT-induced clonal dominance within TPEX subsets, suggesting the potential for enhanced immune reinvigoration. In peripheral blood mononuclear cells (PBMCs) from patients treated with CRT, high levels of TEX cells were found early and at 3 months post-treatment, with delayed peripheral TPEX expansion at 3 months, indicating phased progenitor recovery. These findings demonstrate distinct immunological remodelling by CRT versus RT and underscore the critical importance of treatment timing for optimising combination immunotherapy strategies in LA-SCCHN.

cancer biology↗

bioRxiv2025

Multicellular Calcium Waves in Cancer-Associated Fibroblasts Regulate Neuronal Mimicry and Anisotropy Leading to Immune Exclusion

Stromal barriers exclude CD8+ T cells from accessing cancer cells and hamper immune-mediated tumour control. Through multi-pronged analysis of tumours that transition from immune inflamed to immune excluded, we reveal that the formation of stromal barriers is associated with the acquisition of neuronal gene expression programmes in cancer-associated fibroblasts (CAFs), including TUBB3 expression. This leads to neuronal mimicry, with stromal barrier formation underpinned by coordinated transient bursts of intracellular calcium release, similar to those observed in neuronal tissue. Blockade of calcium release through either pharmacological or molecular interventions, such as STC2 depletion, prevents CAF alignment and the build-up of CD8+ T cells at stromal boundaries. Nintedanib treatment prevents neuronal mimicry and restores immune-mediated tumour control. Thus, we uncover unexpected mimicry of neuronal behaviour in CAFs, document the mechanism by which it leads to immune exclusion, and identify ways to prevent the induction of neuronal mimicry and restore immune-mediated tumour control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/684281v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@938ee5org.highwire.dtl.DTLVardef@18a9c48org.highwire.dtl.DTLVardef@11a5982org.highwire.dtl.DTLVardef@ea6a79_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

ARID1A stabilises non-homologous end joining factors at DNA breaks induced by the G4 ligand pyridostatin

ARID1A is a subunit of the BAF chromatin remodelling complex that is frequently mutated in cancer. It is challenging to predict how ARID1A loss impacts cancer therapy response because it participates in many different cellular pathways. G quadruplex (G4) binding ligands, such as pyridostatin, have shown anticancer effects, but the pathways and genetic determinants involved in the response to G4 ligands are still not fully understood. Here, we show that ARID1A deficient cells are selectively sensitive to pyridostatin when compared with isogenic controls. Sensitivity to pyridostatin was apparent in ovarian and colorectal cancer cell line models, and in vivo studies suggest that G4 ligands hold promise for treating ARID1A deficient cancers. While we find that ARID1A impacts on pyridostatin-induced transcriptional responses, we find that pyridostatin-mediated toxicity in ARID1A-deficient cells is driven by defective DNA repair of topoisomerase-induced breaks. We show that ARID1A-deficient cells are unable to efficiently accumulate non-homologous end joining proteins on chromatin following pyridostatin exposure. These data uncover a role for ARID1A in the cellular response to G4 ligands, and link remodelling to G4 ligand-induced transcriptional and DNA damage responses.

molecular biology↗

bioRxiv2024

Combination of oncolytic Maraba virus with immune checkpoint blockade overcomes therapy resistance in an immunologically cold model of advanced melanoma with dysfunctional T cell receptor signalling.

BackgroundOver the past decade, cancer immunotherapies have revolutionised the treatment of melanoma; however, responses vary across patient populations. Recently, baseline tumour size has been identified as an independent prognostic factor for overall survival in melanoma patients receiving immune checkpoint inhibitors (ICIs). MG1 is a novel oncolytic agent with broad tumour tropism that has recently entered early phase clinical trials. The aim of this study was to characterise T cell responses in human and mouse melanoma models following MG1 treatment and to establish if features of the tumour immune microenvironment (TIME) at two distinct tumour burdens would impact the efficacy of oncolytic virotherapy. MethodsHuman 3D in vitro priming assays were performed to measure anti-tumour and anti-viral T cell responses following MG1 infection. TCR sequencing, T2 killing assay, and peptide recall assays were used to assess the evolution of the TCR repertoire, and measure specific T cell responses, respectively. In vivo, subcutaneous 4434 melanomas were characterised using RNAseq, immunohistochemistry (IHC), and flow cytometry. The effectiveness of intra-tumoural MG1 was assessed in advancing 4434 tumours and the generation of anti-tumour and anti-viral T cells measured by splenocyte recall assays. Finally, combination MG1 and -PD-1 therapy was investigated in advanced 4434 tumours. ResultsMG1 effectively primed functional cytotoxic T cells (CTLs) against tumour associated antigens (TAA) as well as virus-derived peptides, as assessed using peptide recall and T2 killing assays, respectively. TCR sequencing revealed that MG1-primed CTL comprised larger clusters of similar CDR3 amino acid sequences compared to controls. In vivo testing of MG1 demonstrated that MG1 monotherapy was highly effective at treating early disease, resulting in 90% cures; however, the efficacy of MG1 reduced as the disease burden (local tumour size) increased, and the addition of -PD-1 was required to overcome resistance in more advanced disease. Differential gene expression profiles revealed that increased tumour burden was associated with an immunologically colder TIME. Furthermore, analysis of TCR signalling in advancing tumours demonstrated a different dynamic of TCR engagement compared to smaller tumours, in particular a shift in antigen recognition by CD4+ cells, from conventional to regulatory subset. ConclusionCombination of MG1 with PD-1 overcomes therapy resistance in an immunologically cold model of advanced melanoma.

cancer biology↗

bioRxiv2023

PARP inhibitors enhance reovirus-mediated cell killing through the death-inducing signaling complex (DISC) with an associated NF-kappa B-regulated immune response.

Oncolytic Reovirus type 3 Dearing (RT3D), is a naturally occurring double-stranded (ds) RNA virus that is under development as an oncolytic immunotherapy We used an unbiased high-throughput cytotoxicity screen of different targeted therapeutic agents with the aim of identifying potential drug-viral sensitizers to enhance RT3D tumour killing. Talazoparib, a clinical poly(ADP)-ribose polymerase 1 (PARP-1) inhibitor, was identified as a top hit and found to cause profound sensitisation to RT3D. This effect was not seen with other classes of oncolytic virus and was not mediated by enhanced viral replication or PARP inhibitor-related effects on the DNA damage response. RT3D interacts with retinoic acid-induced gene-1 (RIG-I) and activates PARP-1, with consequent PARylation of components of the extrinsic apoptosis pathway. Pharmacological and genetic inhibition of PARP-1 abrogates this PARylation and increases levels of extrinsic apoptosis, NF-kB signalling and pro-inflammatory cell death. Direct interaction between PARP-1 and RIG-I following RT3D/talazoparib treatment is a key factor in activating downstream signaling pathways that lead to IFN-{beta} and TNF-/TRAIL production which, in turn, amplify the therapeutic effect through positive feedback. Critically, it was possible to phenocopy the effect of RT3D through the use of non-viral ds-RNA therapy and RIG-I agonism. In in vivo studies, we demonstrated profound combinatorial efficacy of RT3D and talazoparib in human A375 melanoma in immunodeficient mice. More impressively, in immunocompetent mouse models of 4434 murine melanoma, we achieved 100% tumour control and protection from subsequent tumour rechallenge with the combination regimen. Correlative immunophenotyping confirmed significant innate and adaptive immune activation with the combination of RT3D and PARP inhibition. Taken together, these data provide a clear line of sight to clinical translation of combined regimens of PARP inhibition or ds-RNA agonism, with either viral or non-viral agents, in tumour types beyond the relatively narrow confines of current licensed indications for PARP inhibition.

cancer biology↗

bioRxiv2023

Sculpting the tumour microenvironment by combining radiotherapy and ATR inhibition for curative-intent anti-PD-L1- and anti-NKG2A-based adjuvant immunotherapy

Despite some success in other cancer types, the results of combining radiotherapy/chemoradiotherapy and immune checkpoint blockade have been disappointing in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). For such a potentially radiocurable disease, there remains an imperative to explore novel combination approaches. Here, we show that combining ATR inhibition with radiotherapy (ATRi/RT) increases the frequency of highly activated NKG2A/PD-1 double-positive T cells in patients and in animal models of HNSCC. Addition of dual anti-NKG2A and anti-PD-1/-PD-L1 blockade to ATRi/RT in the adjuvant, post-radiotherapy setting induces a robust antitumour immune response in HNSCC preclinical models. Efficacy of the combination regimen relies on CD40/CD40L costimulatory-mediated infiltration of activated/proliferative/memory CD8 and CD4 conventional T cells with persistent or new T cell receptor (TCR) signalling, respectively, as defined by tracking of T cell dynamics. In this favourable therapeutic context, TCR sequencing shows increased richness of the TCR repertoire and the emergence of numerous and large TCR clusters that share antigen specificity in response to full combination therapy. Collectively, our data point towards promising combination approaches for future clinical testing in HNSCC.

immunology↗

bioRxiv2023

Spatially-resolved profiling of the T-cell receptor repertoire in archival clinical specimens using FUME-TCRseq

Genomic analysis of the T-cell receptor (TCR) reveals the strength, breadth and clonal dynamics of the adaptive immune response to pathogens or cancer. The diversity of the TCR repertoire, however, means that sequencing is technically challenging, particularly for samples with low quality, degraded nucleic acids. Here, we have developed and validated FUME-TCRseq, a robust and sensitive RNA-based TCR sequencing methodology that is suitable for formalin-fixed paraffin-embedded samples and low amounts of input material. FUME-TCRseq incorporates unique molecular identifiers into each molecule of cDNA, allowing correction for sequencing errors and PCR bias. We used RNA extracted from colorectal and head and neck cancers to benchmark the accuracy and sensitivity of FUME-TCRseq against existing methods, and found excellent concordance between the datasets. Furthermore, FUME-TCRseq detected more clonotypes than a commercial RNA-based alternative, with shorter library preparation time and significantly lower cost. The high sensitivity and the ability to sequence RNA of poor quality and limited amount enables quantitative analysis of small numbers of cells from archival tissue sections, which is not possible with other methods. To demonstrate this we performed spatially-resolved FUME-TCRseq of colorectal cancers using macrodissected archival samples, revealing the shifting T-cell landscapes at the transition to an invasive phenotype, and between tumour subclones containing distinct driver alterations. In summary, FUME-TCRseq represents an accurate, sensitive and low-cost tool for the characterisation of T-cell repertoires, particularly in samples with low quality RNA that have not been accessible using existing methodology.

immunology↗

bioRxiv2022

DNA-PKcs is required for cGAS/STING-dependent viral DNA sensing in human cells

To mount an efficient interferon response to virus infection, intracellular pattern recognition receptors (PRRs) sense viral nucleic acids and activate anti-viral gene transcription. The mechanisms by which intracellular DNA and DNA viruses are sensed are relevant not only to antiviral innate immunity, but also to autoinflammation and anti-tumour immunity through initiation of sterile inflammation by self-DNA recognition. The PRRs that directly sense and respond to viral or damaged self-DNA function by signalling to activate interferon regulatory factor (IRF)-dependent type one interferon (IFN-I) transcription. We and others have previously defined DNA-dependent protein kinase (DNA-PK) as an essential component of the DNA-dependent antiviral innate immune system. Here, we show that DNA-PK is essential for STING-dependent IFN-I responses in human cells during stimulation with exogenous DNA and infection with DNA viruses.

immunology↗

bioRxiv2022

CDK4/6 inhibition and dsRNA sensor agonism co-operate to enhance anti-cancer effects through ER stress and immune modulation of tumour cells

Cytoplasmic pattern recognition receptors (PRRs) for double-stranded RNA (RIG-I/MDA5) are key mediators of anti-viral responses. PRR agonists, such as dsRNA oncolytic Reovirus type 3 Dearing (Rt3D), potently activate RNA sensors. We used an unbiased cytotoxicity screen to reveal synergistic drug-virotherapy combinations and found potent effects of Rt3D combined with the CDK4/6 inhibitor, palbociclib. The combination augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and the expression and activation/signaling of RNA sensors. Combined Rt3D-palbociclib treatment potently increased interferon production and signaling, and knockdown studies implicated key UPR proteins and the RNA sensor, RIG-I, as essential to the phenotype observed. Further experiments, using canonical RIG-I agonists and an ER stress inducer, thapsigargin, confirmed cross-talk between RNA sensing and ER stress pathways that augmented cancer cell death and interferon production. Combined Rt3D-palbociclib also increased innate immune activation within tumour cells and IFN-induced HLA expression. Analysis of the immunopeptidome revealed changes to HLA-captured peptides with Rt3D-palbociclib, including altered expression of peptides from cancer/testis antigens (CTA) and endogenous retroviral elements (ERVs). Our findings highlight cross-talk between UPR signaling and RNA-mediated PRR activation as a means of enhancing anti-cancer efficacy with potential pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, and broadens the therapeutic remit of CDK4/6 inhibitors to include roles as both ER stress and dsRNA PRR sensitizers.

cancer biology↗