Biology subjects

Downes, M.

Publications and source records attributed to Downes, M..

15 recordsLinked to original sources

bioRxiv2026

Scalable 3D cell-interaction analysis via supercell graphs for prostate cancer risk stratification

Cellular interactions underlie fundamental biological processes but are not fully represented in conventional 2D histology images. While 3D pathology allows for more-accurate construction of cell-level graphs, machine-learning models are computationally unwieldy and prone to overfitting, especially when dealing with small cohorts. Here, we introduce SCALE3D, a SuperCell graph Analysis framework for LargE 3D pathology datasets. In SCALE3D, spatially adjacent and morphologically similar cells are grouped into functional "supercells." Supercell subtypes are defined via morphology-based clustering and 3D graphs connecting these supercells are used to model their interactions. Validation was performed with 76 radical prostatectomy specimens from patients with known 5-year biochemical recurrence (BCR) outcomes. SCALE3D-derived features achieve higher performance for BCR prediction than established 3D nuclear and glandular morphological features. Combining these complementary features further improves prediction performance. Compared to individual cell-level 3D graphs, SCALE3D maintains comparable prognostic performance with improved noise tolerance while reducing computational times by up to 1,000-fold.

pathology↗

bioRxiv2026

CA19-9 induces microenvironment remodeling in pancreatic ductal adenocarcinoma

Durable therapeutic efficacy remains a major barrier to improving outcomes for patients with pancreatic ductal adenocarcinoma (PDAC). An immunosuppressive tumor microenvironment (TME) is a hallmark of PDAC and has been demonstrated to be a dominant driver of therapeutic resistance. The aberrant glycan CA19-9 is prevalent in PDAC and drives tumor progression, but the paracrine mechanisms by which it contributes to TME remodeling are unknown. To address this, we mapped TME changes and performed functional analyses using a genetically engineered mouse model (GEMM) harboring KrasG12D mutation and inducible CA19-9 expression. Elevation of CA19-9 led to expansion of antigen-presenting cancer associated fibroblasts (apCAFs) and regulatory T cells (Tregs), which can drive immunosuppression. Antibody blockade of CA19 -9 resulted in significant restoration of normal histology and decreased apCAF and Treg populations. We dissected the paracrine signaling mechanisms that drive this TME remodeling in vitro using mouse and human organoid mono- and co-culture models as well as in vivo using GEMMs and syngeneic orthotopic transplantation models. CA19-9 induced IL1a and TGFb expression, reprogramming pancreatic mesothelial cells into apCAFs in vitro, which in turn directly ligated naive Cd4+ T cells resulting in Treg differentiation in co-cultures. Antibody blockade of IL1a and TGFb in mice led to reduced apCAF and Treg differentiation. We previously reported that CA19-9 modification of the secreted Fbln3 protein increased Egfr engagement and now find that the induction of IL1a and TGFb expression by CA19-9 is dependent on Fbln3 hyperactivation of EGFR signaling. Genetic depletion of Fbln3 led to reduced tumor progression and increased Cd8+ T cell infiltration in mice. Together these findings identify a previously unknown signaling axis driving immunosuppressive phenotypes in PDAC, uncovering multiple potential nodes to relieve the immunosuppressive pressures within the PDAC TME.

cancer biology↗

bioRxiv2026

HDAC Inhibition Sensitizes Pancreatic Tumors to DNA Damage by Global Redistribution of the Transcriptional Machinery

The DNA damage response (DDR) is critical for pancreatic ductal adenocarcinoma (PDAC) development and therapeutic responses, including to genotoxic agents. While epigenetic modulators have been shown to contribute to the DDR, how chromatin regulation dictates responses to DNA damage in PDAC remains incompletely understood. Here, we identify Class I histone deacetylases (HDACs) as critical regulators of the DDR. HDAC1/2 directs the genomic distribution of H3K27ac, ensuring sufficient BRD4 and RNA polymerase II (Pol II) occupancy at DDR gene promoters. HDAC inhibition by entinostat shifts the balance of H3K27 acetylation preferentially towards intergenic regions, diverting BRD4 and Pol II from promoters, thereby suppressing DDR gene expression. In line with this, HDAC inhibition heightens DNA damage and sensitizes PDAC to diverse DNA-damaging and DDR-targeting agents. Since the clinical development of HDAC inhibitors has been limited by systemic toxicity, we developed bottlebrush prodrug (BPD) nanoparticles for tumor-selective entinostat delivery. Entinostat-BPD achieved tumor-specific HDAC inhibition while displaying potent efficacy and reduced systemic toxicity. These findings reveal an HDAC-dependent DDR vulnerability and offer combinational and precision targeting strategies to facilitate clinical translation and improve PDAC patient outcomes. SIGNIFICANCE STATEMENTThe ability of tumor cells to tolerate DNA damage limits the efficacy of many anticancer therapies. Our study reveals that pancreatic cancer cells enforce this resistance by sustaining expression of DNA damage response (DDR) genes through Class I histone deacetylases (HDACs). HDACs maintain genome-wide acetylation patterns required for efficient recruitment of the transcriptional machinery to DDR genes. Pharmacological HDAC inhibition disrupts this process and sensitizes pancreatic cancer cells to diverse DNA-damaging agents. To overcome systemic toxicity that limits translational potential, we further establish a bottlebrush prodrug nanoparticle platform that enables tumor-selective HDAC inhibition. Given the central role of the DDR in cancer, targeting HDAC-mediated DDR regulation through drug combinations and precision delivery may have broad therapeutic relevance across cancer types.

cancer biology↗

bioRxiv2026

CA19-9 promotes liver metastasis of pancreatic cancer through E-selectin mediated extravasation

Pancreatic ductal adenocarcinoma (PDAC) frequently metastasizes to the liver, which drives patient mortality. CA19-9 is elevated in most PDAC tumors and is widely used as a clinical biomarker. Elevated serum levels are associated with poor outcomes. However, whether CA19-9 functionally contributes to metastatic progression has not been fully defined, in part because mice lack endogenous CA19-9 expression. Here, using syngeneic murine PDAC cells engineered to express CA19-9, we investigated its functional role in liver metastasis. In splenic injection models, CA19-9 expression markedly increased liver metastatic burden by promoting both metastatic seeding and subsequent metastatic outgrowth. In vitro, CA19-9 enhanced tumor cell adhesion to endothelial cells through interaction with E-selectin. Metastatic seeding of CA19-9-expressing cells was reduced by genetic deletion of E-selectin or antibody neutralization of either CA19-9 or E-selectin in vivo. Therapeutic targeting of CA19-9 with a neutralizing antibody markedly reduced liver metastatic burden after metastatic seeding. CA19-9 expression increased AKT signaling in PDAC cells and liver metastases, and CA19-9 levels correlated with AKT activation in human PDAC tissues. These findings show that CA19-9 promotes PDAC liver metastasis through E-selectin-dependent metastatic seeding and AKT-associated metastatic outgrowth, highlighting CA19-9 as a functional mediator of PDAC metastasis and a potential therapeutic target.

cancer biology↗

bioRxiv2026

A rare human TNFAIP3 variant reveals how A20 abundance is regulated by TAX1BP1

Variants in TNFAIP3 (encoding A20) are among the most consistent non-MHC genetic associations with human autoimmune disease. Moreover, TNFAIP3 haploinsufficiency confers an autosomal dominant autoinflammatory and autoimmune disease. Whilst these findings strongly suggest that quantitative changes in A20 predispose to pathology, there is currently no evidence that post-transcriptional modifications regulate A20 abundance. We used human reverse genetics approach to investigate this question. From a cohort of patients with complex immune diseases, we identified an ultrarare hypomorphic TNFAIP3 variant (A20S254R) that is both hypomorphic and prone to phosphorylation. One of five carriers of this variant was distinguished by autoimmunity and an NF-kB gain-of-function transcriptional signature. A search for modifiers identified a variant in TAX1BP1 (p.Leu207Ile). We discovered that TAX1BP1 normally retrains A20 phosphorylation. Furthermore, TAX1BP1 preferentially binds phospho-A20, which explains the enhanced interaction between TAX1BP1L207I and A20S254R. A20 phosphorylation regulates its abundance, not by MALT1-mediated degradation, but probably via autophagy. Thus, while A20 phosphorylation has been implicated in A20 protease activity, an epistatic interaction between rare human genetic variants reveals how phosphorylation also regulates A20 abundance.

immunology↗

bioRxiv2025

Detection of prostate cancer in 3D pathology datasets via generative immunolabeling

Recent advancements in nondestructive 3D pathology offer a complement to standard histology by enabling comprehensive volumetric analyses of intact clinical specimens (e.g. biopsies). Prior studies have demonstrated the added prognostic value of 3D pathology for prostate cancer risk stratification by correlating 3D microarchitectural features with long-term patient outcomes. However, these analyses relied on coarse manual annotations of cancer-enriched regions for downstream analysis without fine-grained delineation between often-intermixed cancerous and benign glands. To address these limitations, we have developed a 3D computational pipeline: Synthetic Immunolabeling for Generative Heatmaps of Tumor (SIGHT). SIGHT relies on deep learning-based 3D image translation models, trained in a fully supervised fashion, to convert H&E-analog 3D pathology datasets into multiplexed 3D immunofluorescence datasets that facilitate tumor detection. Our implementation of SIGHT synthetically labels two cytokeratin markers that are differentially expressed in cancerous and benign prostate glands, which are used to generate explainable 3D heatmaps of cancer-enriched regions in prostate tissues. Validation of SIGHT against ground-truth annotations from a panel of genitourinary pathologists yields an average F1 score of 0.88 which is comparable to the average inter-pathologist agreement F1 score of 0.90. To demonstrate the value of SIGHT, we developed machine classifiers of recurrence risk based on 3D glandular histomorphometric features from 75 patients. Volumetric glandular analysis in SIGHT-identified cancer-enriched regions vs. all tissue regions yields an average Kaplan-Meier hazard ratio of 3.57 (1.6 - 7.9 CI) vs. 0.92 (0.45 - 1.89 CI).

pathology↗

bioRxiv2024

Identification of molecular and functional subtypes using chronic pancreatitis patient-derived organoid models

Chronic pancreatitis (CP) affects [~]3 million people worldwide, yet altering the course of disease is challenging. We developed a patient-derived organoid (PDO) platform to investigate the molecular pathogenesis of this disease and identify therapeutic strategies. We generated 36 PDOs from patients with idiopathic, hereditary, and alcohol-related CP with high genetic concordance. PDOs retained inflammation-associated transcriptional and proteomic features. Transcriptomic profiling revealed three molecular subtypes of CP independent of etiology. We discovered widespread dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in half of the CP PDOs, including those with wildtype CFTR. Clinically available CFTR modulators stabilized mutant or wildtype CFTR, restored CFTR function, and decreased mitogenic and inflammatory signaling. This work provides the first comprehensive PDO platform for modeling CP. We demonstrate the utility of this platform for precision therapeutic investigations. Our findings reveal CFTR modulators as a broadly applicable and effective therapeutic strategy.

cell biology↗

bioRxiv2023

Inhibiting Stromal Class I HDACs Curbs Pancreatic Cancer Progression

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

cancer biology↗

bioRxiv2022

A Synthetic ERRα Agonist Induces an Acute AerobicExercise Response and Enhances Exercise Capacity

Repetitive physical exercise induces physiological adaptations in skeletal muscle that improves exercise performance and is effective for the prevention and treatment of several diseases. Here we report the identification of a synthetic agonist for the orphan nuclear receptor ERR (estrogen receptor-related receptor ), SLU-PP-332, that activates an acute aerobic exercise genetic program in skeletal muscle in an ERR-dependent manner. SLU-PP-332 increases mitochondrial function and cellular respiration consistent with induction of this genetic program. When administered to mice, SLU-PP-332 increased the type IIa oxidative skeletal muscle fibers and enhanced exercise endurance. These data indicate the feasibility of targeting ERR for development of compounds that act as exercise mimetics that may be effective in treatment of numerous metabolic disorders and to improve muscle function in the aging.

pharmacology and toxicology↗

bioRxiv2021

The autophagy initiating kinase ULK1 is required for pancreatic cancer cell growth and survival

Amongst cancer subtypes, pancreatic ductal adenocarcinoma (PDA) has been demonstrated to be most sensitive to autophagy inhibition, which may be due to unique metabolic rewiring in these cells. The serine/threonine kinase ULK1 forms the catalytic center of a complex mediating the first biochemical step of autophagy. ULK1 directly recieves signals from mTORC1 and AMPK to trigger autophagy under stress and nutrient poor conditions. Studies in genetic engineered mouse models of cancer have revealed that deletion of core downstream autophagy genes (ATG5, ATG7) at the time of tumor iniation leads to a profound block in tumor progression leading to the development of autophagy inhibitors as cancer therapeutics. However, most preclinical studies and all clinical studies have relied on non-specific lysomotropic agents such as chloroquine and its derivatives, whose toxicity and off-target issues preclude further clinical development and which do not represent the impact of solely biochemically disrupting the autophagy pathway. Furthermore, druggable targets in the core autophagy pathway are quite limited, with ULK1 and ULK2 representing the only protein kinases in the pathway. Here we explore the genetic requirement for ULK1 and ULK2 in human PDA cancer cell lines and xenografts, and take advantage of new small molecule ULK1 inhibitors to demonstrate that ULK inhibition can overcome autophagy induction triggered by PDA therapeutics including chemotherapy and MEK inhibition. Finally we show that ULK inhibitors increase MHC Class I in PDA cells, suggestion a potential therapeutic avenue for such agents in combination with checkpoint immunotherapy.

cancer biology↗

bioRxiv2021

Bisphenol A derivatives act as novel coactivator binding inhibitors for estrogen receptor β

Bisphenol A and its derivatives are recognized endocrine disruptors based on their complex effects on estrogen receptor (ER) signaling. While the effects of bisphenol derivatives on ER have been thoroughly evaluated, how these chemicals affect ER{beta} signaling is not well understood. Herein, we identified novel ER{beta} ligands by screening a chemical library of bisphenol derivatives. Many of the compounds identified showed intriguing dual activities as ER agonists and ER{beta} antagonists. Docking simulations suggested that these compounds act as coactivator binding inhibitors (CBIs). Direct binding experiments using wild-type and mutated ER{beta} demonstrated the presence of a second ligand interaction position at the coactivator binding site in ER{beta}. Our study is the first to propose that bisphenol derivatives act as CBIs, presenting a critical view point for future ER signaling-based drug development.

biochemistry↗

bioRxiv2021

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection

Summary ParagraphWe identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone1, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications2, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry3. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir4. Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginones translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

microbiology↗

bioRxiv2020

Circadian benefits of exercise training reflect metabolic and structural flexibility

Structured AbstractO_ST_ABSObjectiveC_ST_ABSExercise is a critical component of a healthy lifestyle and a key strategy for the prevention and management of metabolic disease. Identifying molecular mechanisms underlying adaptation in response to chronic physical activity is of critical interest in metabolic physiology. Circadian rhythms broadly modulate metabolism, including muscle substrate utilization and exercise capacity. Here, we define the molecular and physiological changes induced across the daily cycle by voluntary low intensity daily exercise. MethodsWildtype c57BL6/J male and female mice were housed with or without access to a running wheel for six weeks. Maximum running speed was measured at four different zeitgeber times (ZTs, hours after lights on) using either electrical or manual stimulation to motivate continued running on a motorized treadmill. RNA isolated from plantaris muscles at six ZTs was sequenced to establish the impact of daily activity on genome-wide transcription. Patterns of gene expression were analyzed using Gene Set Enrichment Analysis (GSEA) and Detection of Differential Rhythmicity (DODR). Blood glucose, lactate, and ketones, and muscle and liver glycogen were measured before and after exercise. ResultsWe demonstrate that the use of mild electrical shocks to motivate running negatively impacts maximum running speed and describe a manual method to motivate running in rodent exercise studies. Using this method, we show that time of day influences the increase in exercise capacity afforded by six weeks of voluntary wheel running: when maximum running speed is measured at the beginning of the nighttime active period in mice, there is no measurable benefit from a history of daily voluntary running, while maximum increase in performance occurs at the end of the night. We show that daily voluntary exercise dramatically remodels the muscle circadian transcriptome. Finally, we describe daily rhythms in carbohydrate metabolism associated with the timedependent response to moderate daily exercise. ConclusionsCollectively, these data indicate that chronic nighttime physical activity dramatically remodels daily rhythms of muscle gene expression, which in turn support daily fluctuations in exercise performance.

physiology↗

bioRxiv2020

Oxidized Lipids and CD36-Mediated Lipid Peroxidation in CD8 T Cells Suppress Anti-Tumor Immune Responses

T cell metabolic fitness plays a pivotal role in anti-tumor immunity and metabolic deregulation causes T cell dysfunction (i.e., exhaustion) in cancer. We identify that the scavenger receptor CD36 limits anti-tumor CD8+ T cell effector functions through lipid peroxidation. In murine tumors, oxidized phospholipids (OxPLs) were highly abundant and CD8+ TILs increased uptake and accumulation of lipids and lipid peroxidation. Functionally exhausted CD8+ TILs substantially increased CD36 expression and CD36-deficient CD8+ TILs had more robust anti-tumor activity and cytokine production than wild-type cells. We further show that CD36 promotes uptake of oxidized low-density lipoproteins (OxLDL) and induces lipid peroxidation in CD8+ TILs, and OxLDL inhibits CD8+ T cell functions in a CD36-dependent manner. Moreover, glutathione peroxidase 4 (GPX4) over-expression lowers lipid peroxidation and restores functionalities in CD8+ TILs. These results define a key role for an oxidized lipid-CD36 axis in promoting intratumoral CD8+ T cell dysfunction.

immunology↗

bioRxiv2019

Obesity Potentiates TH2 Immunopathology via Dysregulation of PPAR

How obesity affects immune function is not well understood. Clinically, obesity is strongly associated with severe TH2 immunopathology1-3, though the physiological, cellular, and molecular underpinnings of this association remain obscure. Here, we demonstrate that obese mice are susceptible to severe atopic dermatitis (AD), a major manifestation of TH2 immunopathology and disease burden in humans4,5. Mechanistically, we show that dysregulation of the nuclear hormone receptor (NHR) PPAR{gamma} (peroxisome proliferator-activated receptor gamma) in T cells is a causal link between obesity and the increased TH2 immunopathology. We find that PPAR{gamma} oversees a cellular metabolic transcriptional program that restrains nuclear gene expression of the chief TH2 priming and effector cytokine interleukin-4 (IL-4). Accordingly, thiazolidinediones (TZDs), potent PPAR{gamma} agonists, robustly protect obese mice from TH2 immunopathology. Collectively, these findings establish PPAR{gamma} as a molecular link between obesity and TH2 immune homeostasis and identify TZDs as novel therapeutic candidates for TH2 immunopathology. Fundamentally, these findings demonstrate that shifting physiologic metabolic states can shape the tone of adaptive immune responses to modulate differential disease susceptibility.

immunology↗