Biology subjects

Kearney, C. J.

Publications and source records attributed to Kearney, C. J..

10 recordsLinked to original sources

bioRxiv2026

ICOS blockade alleviates immune suppression in breast tumor microenvironment

Immune checkpoint blockade has become a standard of care for triple-negative breast cancer, yet only around half of patients benefit. Compensatory immunoregulatory pathways may limit the efficacy of PD-1/PD-L1 blockade. ICOS, an inducible co-stimulatory receptor enriched on activated regulatory T cells (Tregs), represents a potential route of such immune regulation, but its role in breast cancer immunotherapy remains unclear. Here, we identify ICOS as selectively upregulated on tumor-infiltrating Tregs and show that ICOS Tregs localize within a tumor niche enriched for exhausted CD8 T cells. We confirmed this cellular and spatial association across 1,359 tumor regions from 279 triple-negative breast cancer patients profiled by imaging mass cytometry, and single-cell transcriptomic data from 129 breast cancer donors. Genetic ablation or pharmacological blockade of ICOS disrupted this intratumoral Treg niche, reducing Treg activation and tissue-residency programs while expanding cytotoxic CD8 T cells with a tissue-resident memory phenotype. In syngeneic orthotopic breast cancer models, ICOS blockade restricted primary tumor growth, reduced spontaneous metastasis and prolonged survival without disrupting systemic immune homeostasis. Therapeutic activity was observed with both neoadjuvant and adjuvant treatment, while combined ICOS and PD-1 blockade further enhanced tumor control. These findings support ICOS blockade as a therapeutic strategy to improve checkpoint immunotherapy.

cancer biology↗

bioRxiv2026

An MLL-Independent Function of Menin Promotes Resistance to MAPK-Targeted Therapy

BRAF mutant colorectal cancer (CRC) remains difficult to treat despite the clinical use of combined BRAF and EGFR inhibition, highlighting a need to define tumour-intrinsic mechanisms that limit therapeutic response. Here, using genome-wide CRISPR-Cas9 screening in BRAF-mutant CRC cells, we identify MEN1, encoding the chromatin-associated protein Menin, as a selective determinant of sensitivity to combined encorafenib and cetuximab (EC). MEN1 loss markedly enhanced EC-mediated inhibition of cell proliferation and ERK activity while having comparatively little effect in untreated cells, and re-expression of Menin restored resistance. Transcriptomic and chromatin profiling revealed that Menin supports the transcriptional response associated with MAPK signalling. Menin occupied promoters of MAPK/BRAF-responsive genes and EC treatment caused widespread displacement of Menin from chromatin. Phosphoproteomic analysis demonstrated extensive remodelling of MAPK signalling following EC treatment, whereas proximity proteomics showed that the Menin-associated protein complexes remained largely intact despite loss of Menin chromatin occupancy. Importantly, MLL1 loss did not reproduce the sensitising effect of MEN1 deletion, and pharmacological Menin inhibition with revumenib failed to phenocopy either genetic MEN1 loss or acute Menin degradation, indicating that this phenotype is independent of Menin-MLL activity. Together, these findings identify a previously unrecognised, MLL-independent role for Menin in buffering the response of BRAF-mutant CRC cells to MAPK pathway inhibition and suggest targeting Menin, rather than disruption of its interaction with MLL, may provide a strategy for enhancing the response to BRAF-targeted therapy for CRC.

cancer biology↗

bioRxiv2026

Scaffold-mediated delivery of a miRNA-29b mimic mitigates excessive extracellular matrix deposition and matrix contraction in wound healing applications

Disruption of the wound healing cascade can result in pathological outcomes, including fibrosis due to myofibroblast-mediated contraction and collagen deposition. Despite the clinical significance, effective treatments for fibrosis remain limited as current therapies often show inconsistent efficacy, adverse effects, and patient discomfort. Combinatorial therapeutic strategies integrating biomaterial scaffolds with gene delivery have shown promise in regenerative healing. MicroRNAs (miRNAs) are key regulators of fibrotic signalling in cells, including fibroblasts and myofibroblasts. Specifically, miRNA-29b is notable for downregulating pro-fibrotic genes, including collagen type I, reducing ECM accumulation, and limiting fibroblast/myofibroblast overactivation. In this context, the present work develops a collagen-GAG (CG) scaffold platform for delivery of miRNA-29b complexed GET nanoparticles to inhibit fibrosis. Initially, bioinformatic analysis of miRNA-29b validated its involvement in ECM-associated pathways and processes, followed by successful nanoparticle internationalisation in primary dermal fibroblasts. The anti-fibrotic efficacy of the optimised miRNA-29b nanoparticles was subsequently demonstrated by significant reductions in collagen deposition and -SMA expression, both key indicators of myofibroblast differentiation and fibrosis. The optimised miRNA-29b formulation was then incorporated into 3D porous collagen-GAG (CG) scaffolds, which modulated fibrotic gene expression while preserving scaffold structure conducive to fibroblast/myofibroblast infiltration and proliferation. Finally, functional outcomes of seeded TGF-{beta}-stimulated fibroblasts, including reduced matrix contraction, -SMA expression, and ECM deposition, were comparable to those observed in non-fibrotic conditions, thereby confirming the therapeutic potential of scaffold-mediated miRNA-29b delivery. Together, these findings demonstrate that scaffold-mediated miRNA-29b delivery represents a promising anti-fibrotic strategy for wound healing by mitigating myofibroblast activation, limiting matrix contraction, and preventing pathological ECM accumulation.

bioengineering↗

bioRxiv2026

ThermoClock: A Novel Automated Temperature Regulation Device that Can Model Circadian Entrainment and Disruption in 2D and 3D in Vitro Models

Circadian rhythms are critical for maintaining homeostasis and regulating physiological functions, and consequentially impact disease progression; yet, they remain largely overlooked in in vitro models used in preclinical research. One major barrier to rigorously testing the role of circadian rhythms in these models is the lack of accessible tools that seamlessly integrate into standard culture setups and are capable of sustainably delivering time cues to cells and tissues in long term experiments. Here, we present the ThermoClock, a low-cost, Arduino-based automated temperature control system capable of delivering independent temperature programs to multiple cultures simultaneously. Using circadian reporter U2OS cell lines (Bmal1:Luc and Per2:Luc), we demonstrated that ThermoClock-driven temperature cycles (36{degrees}C/38.5{degrees}C, 12h:12h) produced significantly higher amplitude entrainment than a programmable incubator delivering identical temperature trajectories, suggesting that the ramp time to setpoint is a critical determinant of entrainment strength. We further applied ThermoClock to skin explants from keratinocyte-specific Dbp:Luc reporter mice, showing that circadian temperature cycles (T24: 12h:12h and T25: 12.5h:12.5h) extended synchronized circadian rhythms ex vivo, while a shortened T-cycle (T20: 10h:10h) induced rhythm disruptions. We also observed reduced cell migration in T20 temperature-entrained explants wounded ex vivo, closely recapitulating attenuated wound healing observed in T20 light-cycle-disrupted mice in vivo. Finally, we show that wounding can act as a phase-resetting cue but its efficacy depends on pre-injury entrainment state, with circadian entrained tissues (T25) resisting reset, while disrupted (T20) and unentrained tissues showed resetting sensitivity. These findings establish ThermoClock as a versatile platform for incorporating circadian regulation and, for the first time, disruption into 2D and 3D in vitro systems and demonstrate that peripheral clock disruption and its functional consequences can be modeled ex vivo.

bioengineering↗

bioRxiv2025

Scaffold-mediated miRNA-155 inhibition promotes regenerative macrophage polarisation leading to anti-inflammatory, angiogenic, and neurogenic responses for wound healing

Chronic wounds represent a significant clinical challenge due to persistent inflammation and impaired nerve regeneration, both of which delay healing. Conventional treatments often yield limited success and inconsistent outcomes, especially in complex and recurring wounds. Combinatorial strategies that integrate biomaterial scaffolds with gene delivery offer a promising approach to promote tissue repair. MicroRNAs (miRNAs), particularly miRNA-155, have emerged as crucial regulators of wound healing. MiRNA-155 is highly expressed in inflammatory conditions and plays a key role in macrophage activation, polarisation, and nerve regeneration. In this context, this study introduces a miRNA-155 inhibitor-activated scaffold designed to modulate the chronic wound environment by inhibiting miRNA-155. MiRNA-155 inhibitor complexed GET nanoparticles were characterised and incorporated into collagen-glycosaminoglycan (CG) scaffolds. These supported dermal fibroblast and endothelial cell growth while enabling controlled inhibitor release. Scaffold-mediated miRNA-155 inhibition in both non-polarised (M0) and pro-inflammatory (M1) macrophages promoted anti-inflammatory (M2) polarisation. Macrophage secretome analysis showed reduced inflammatory cytokines and increased angiogenic growth factor secretion in both conditions. The regenerative potential of the miRNA-i-activated scaffold via macrophage polarisation was validated through inflammatory and angiogenic functional assays with endothelial cells. In parallel, scaffold-mediated miRNA-155 delivery to dorsal root ganglia (DRG) promoted neurogenic outcomes through enhanced axonal regrowth, essential for the synergistic repair of chronic wounds across the skin-nerve axis. In vivo implantation of miRNA-155 inhibitor-activated scaffolds in chicks demonstrated successful integration without disrupting vascular network formation. Collectively, these findings establish the miRNA-155 inhibitor-activated scaffold as a regenerative platform with anti-inflammatory, pro-angiogenic, and neurogenic outcomes, offering a multifaceted solution for chronic wound healing applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/682055v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1c6a6b6org.highwire.dtl.DTLVardef@eb04f9org.highwire.dtl.DTLVardef@1937f15org.highwire.dtl.DTLVardef@7c70d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Preprint: Triggered sequential viral-transduction from collagen-based scaffolds for tissue regeneration

Chronic wounds are a major healthcare issue that are recalcitrant to many traditional treatments. Increasingly, tissue engineering scaffolds are being developed and translated to promote their healing. To control signaling in the wound environment, gene therapy approaches are being explored, with adeno-associated virus (AAV) becoming increasingly popular. One critical challenge in chronic wound healing is that the wounds do not progress through the typical wound healing cascade, with signaling getting stuck in the inflammatory/immature tissue formation phase. This motivated us to develop a system capable of triggered sequential release of viral vectors to drive coordinated signaling. By housing this system within a collagen-glycosaminoglycan (GAG) scaffold, we aim to provide a proven extracellular matrix template as well as the correct signaling profile for closure of chronic wounds. Our system consists of two alginate pockets within the collagen-GAG scaffold, which we use to control the release of AAV. The first pocket allows diffusion of one AAV therapeutic and the second pocket can be ultrasound-triggered using low-frequency stimulation to release the second therapeutic. Initially, we developed and characterized the system using a reporter AAV. At our high AAV loading, we got sustained release and GFP expression in HEK293T cells over 9 days from our system in vitro, but lower loading had minimal transduction. When this lower group was triggered with ultrasound, cells were successfully transduced. Finally, we demonstrated sequential release of AAV encoding clinically-relevant genes for angiogenesis. This system has the potential for broad applicability as it can be readily adapted to mimic a range of biological pathways.

bioengineering↗

bioRxiv2025

A TAK1Cytokine Toxicity Checkpoint Controls Anti-Cancer Immunity

The success of cancer immunotherapies is currently limited to a subset of patients, which underscores the urgent need to identify the processes by which tumours evade immunity. Through screening a kinome-wide CRISPR/Cas9 sgRNA library we identified MAP3K7 (TAK1) as a suppressor of CD8+ T cell mediated killing. We demonstrate that TAK1 acts as a cancer-intrinsic checkpoint by integrating signals from T cell-secreted TNF and IFNy effector cytokines to elicit a cytoprotective response. This cytoprotective response profoundly limits the anti-cancer activity these key effector molecules and completely abrogates bystander killing by perforin deficient T cells. Inhibition of the TAK1 checkpoint effectively redirects the combined TNF/IFNy pathway activation to promote inflammatory cell death via RIPK1 and Caspase-8 and simultaneously amplifies the output of the IFNy pathway, thereby priming cells for cytokine-induced cell death. Mechanistically, TAK1 deficiency led to proteasomal degradation of cFLIP, enhancing the formation of Complex II and subversion of other cytoprotective responses. Targeting the TAK1 checkpoint led to profound attenuation of tumour growth in immune competent mice, with minimal impact in immune deficient counterparts. Adoptive cell therapy led to preferential elimination of TAK1 deficient clones. Collectively, our study uncovers a cancer-intrinsic checkpoint controlled by TAK1 activity that switches TNF and IFNy responses from cytoprotective to apoptosis. Cancer cells exploit this to limit cell death in the presence of the cytotoxic lymphoctye cytokines TNF and IFN{gamma} and therapeutic intervention can fully unleash the impact of these effector molecules both on the direct target and bystander cells. These findings highlight the clinical development of TAK1 biologics as a potential strategy to improve cancer immunotherapies through harnessing and enhancing the cytotoxic potential of CTL-derived cytokines. In BriefDjajawi et al. identify TAK1 as a cancer-intrinsic cytokine toxicity checkpoint that limits the efficacy of anti-cancer immune responses. Cancer cells exploit TAK1 activity to confers resistance to CD8+ T cell-derived TNF and IFN{gamma}-induced apoptosis, limiting both direct and bystander killing. Mechanistically, TAK1 loss acts through 1. cFLIP and RIPK1 to promote cell death, 2. The inactivation of pro-survival signals and 3. Via amplification of the IFNy response. Targeting of this TAK1 checkpoint enhanced anti-tumour immunity in vivo and improved adoptive cellular therapy. These findings identify a strategy employed by transformed cells to avoid destruction by inflammatory cytokines and provide new therapeutic vulnerabilities for enhancing immunotherapies. HighlightsO_LICRISPR screens identify TAK1 as a tumour-intrinsic survival checkpoint limiting destruction by CD8+ T cells. C_LIO_LITAK1 protects tumour cells from combined TNF and IFN{gamma}-induced apoptosis. C_LIO_LITAK1 loss destabilizes cFLIP, priming cells for STAT1 and RIPK1-dependent cell death. C_LIO_LITAK1 loss promotes tumour control in immune competent animals and enhances the efficacy of adoptive cell therapy. C_LI

cancer biology↗

bioRxiv2021

BET Inhibition Enhances TNF Mediated Anti-Tumor Immunity

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance anti-tumor immunity and augment cancer immunotherapies. By screening a small molecule library of epigenetics-based therapeutics, BET bromodomain inhibitors (BETi) were identified as agents that promote the anti-tumor activity of CD8+ T-cells. BETi sensitized diverse tumor types to the cytotoxic effects of the pro-inflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-{kappa}B target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of pro-survival NF-{kappa}B signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific (TCB) antibodies increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune oncology agents. STATEMENT OF SIGNIFICANCEManipulating the epigenome is an evolving strategy to enhance anti-tumor immunity. We demonstrate that BET bromodomain inhibitors potently sensitize solid tumors to CD8+ T-cell killing in a TNF-dependent manner. This immunomodulatory mechanism can be therapeutically leveraged to augment immuno-oncology therapies, including TCB antibodies and immune checkpoint blockade.

cancer biology↗

bioRxiv2020

Epigenetic reprogramming of plasmacytoid dendritic cells drives type I interferon-dependent differentiation of acute myeloid leukemias for therapeutic benefit

Pharmacological inhibition of epigenetic enzymes can have therapeutic benefit, particularly against hematological malignancies. While these agents can affect tumor cell growth and proliferation, recent studies have demonstrated that pharmacological de-regulation of epigenetic modifiers may additionally mediate anti-tumor immune responses. Here we discovered a novel mechanism of immune regulation through the inhibition of histone deacetylases (HDACs). In a genetically engineered model of t(8;21) AML, leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor panobinostat required activation of the type I interferon (IFN) signaling pathway. Plasmacytoid dendritic cells (pDCs) were identified as the cells producing type I IFN in response to panobinostat, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated activation of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, while combined treatment of panobinostat and recombinant IFN improved therapeutic outcomes. These discoveries offer a new therapeutic approach for t(8;21) AML and demonstrate that epigenetic rewiring of pDCs enhances anti-tumor immunity, opening the possibility of exploiting this cell type as a new target for immunotherapy.

cancer biology↗

bioRxiv2019

Efficient CRISPR/Cas9 gene ablation in uncultured naive mouse T cells for in vivo studies

CRISPR/Cas9 technologies have revolutionised our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene deletion strategies in T cells require in vitro stimulation or culture that can both preclude studies of gene function within unmanipulated naive T cells and can alter subsequent differentiation. Here we demonstrate highly efficient gene deletion within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knock-out cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. This protocol thus expands CRISPR-based probing of gene function beyond models of robust T cell activation, to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumour models.

immunology↗