Biology subjects

Coarfa, C.

Publications and source records attributed to Coarfa, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanosignaling Promotes Macrophage Apoptosis Resistance in Pulmonary Fibrosis via Metabolic Reprogramming

The mechanisms underlying the progression of pulmonary fibrosis in idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases remain unclear. Increased extracellular matrix stiffness is a hallmark of fibrotic lung diseases. Monocyte-derived macrophages can promote fibrosis progression. However, there is limited understanding of how the mechanical properties of the fibrotic microenvironment influence macrophage phenotypes. Profibrotic macrophages are apoptosis-resistant, and this phenotype is modulated by enhanced mitochondrial bioenergetics. The objective of the study was to determine how lung tissue stiffness impacts macrophage phenotypes and fibrotic progression. We demonstrate that mechanoactivated macrophages exhibit apoptosis-resistance, increased expression of the antiapoptotic protein Bcl-xL and increased mitochondrial oxidative phosphorylation. Critically, the metabolic reprogramming observed in mechanoactivated macrophages is dependent on increased glutaminolysis. Inhibition of glutaminolysis attenuated apoptosis resistance in mechanoactivated macrophages. Moreover, inhibition of Bcl-xL in vivo protected mice against experimental pulmonary fibrosis. Lastly, mechanoactivated primary IPF macrophages produce more profibrotic cytokines and promote extracellular matrix production in precision-cut lung slices. We describe a mechanism for acquired macrophage apoptosis resistance dependent on metabolic reprogramming regulated by extracellular matrix stiffness. Our results identify mechanoactivated apoptosis-resistant macrophages as pro-fibrotic mediators, suggesting a novel therapeutic target in IPF and related fibrotic disorders.

molecular biology↗

bioRxiv2026

Systemic interindividual epigenetic variants link periconceptional environment to human fetal development

At rare human genomic regions, DNA methylation states are established in the early embryo and maintained during cellular differentiation, yielding systemic (i.e. not tissue-specific) interindividual epigenetic variation. Previous screens for such correlated regions of systemic interindividual variation (CoRSIVs) were limited to White Americans. Here, we describe the first human CoRSIV screen including self-identified Black and White Americans. We integrate deep whole-genome bisulfite sequencing data for three tissues from each of ten Black and ten White donors in the NIH Genotype-Tissue Expression program. This approach identifies twice as many CoRSIVs among Black than White Americans. Establishment of CoRSIV methylation is sensitive to periconceptional environmental exposures including assisted reproduction, seasonal variation, and famine. CoRSIV-associated genes are enriched for GWAS variants linked to cancer and neurodevelopment. Although only 15% of Black CoRSIVs overlap with those among White individuals, both sets are associated with the same subfamilies of transposable elements. Within multiple cell lines, ranked enrichments of transcription factor binding to Black and White CoRSIVs are exquisitely coordinated and related to genome organization, indicating that CoRSIV methylation states established in the early embryo play an important role in guiding subsequent cellular differentiation.

Molecular Biology↗

bioRxiv2026

Alveolar niche disruption and aberrant epithelial reprogramming are early hallmarks of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease in which the earliest cellular events driving fibrosis remain poorly defined. Here, we analyzed lung samples from three independent and unique cohorts of patients with early disease and preserved lung function (Florence, NIH, Forli), applying an integrated multi-modal approach combining single-nucleus RNA sequencing, bulk transcriptomics, immunostaining, and spatial transcriptomics. Single nuclear RNA sequencing of samples obtained by diagnostic bronchoscopic cryobiopsy (Florence, n= 22) revealed that early IPF is characterized by a marked shift in alveolar epithelial composition, with loss of AT1 and AT2 cells and the emergence of aberrant basaloid cells and alveolar epithelial intermediate cells. These populations exhibited transcriptional programs associated with epithelial plasticity and profibrotic signaling and closely resembled those observed in end-stage IPF. Higher proportions of aberrant basaloid and alveolar epithelial intermediate cells were associated with subsequent disease progression, whereas AT2 cell abundance correlated with preserved lung function. Fibrotic CTHRC1+ fibroblasts are largely restricted to advanced disease, while endothelial remodeling and inflammatory fibroblast states are already evident in early IPF. Spatial transcriptomic analyses confirmed early disruption of the alveolar niche, with replacement of normal epithelial-capillary interactions by aberrant epithelial and venous endothelial cells (Forli, n= 24); the findings were replicated through single cell RNA sequencing of samples obtained by video assisted thoracoscopy two decades earlier (NIH n=9). Together, these findings identify that alveolar niche remodeling with loss of its normal components, and emergence of aberrant basaloid cells are features of early IPF, highlighting epithelial dysfunction as a key potential target for therapeutic interventions in early disease.

genomics↗

bioRxiv2026

Early Pulmonary Fibrosis is Defined by Niche- and Cell-Specific Molecular Programs

RationalePreclinical familial pulmonary fibrosis (FPF) represents an early stage of fibrotic lung disease, yet the compartment- and cell-specific molecular programs preceding fibrosis remain poorly understood. ObjectiveTo define spatially organized molecular signatures associated with preclinical FPF and identify tissue-informed circulating biomarkers linked to early fibrotic remodeling. MethodsWe performed integrated multi-omic profiling of histologically preserved and remodeled lung regions from subjects with preclinical FPF, Idiopathic Pulmonary Fibrosis (IPF), and controls using spatial transcriptomics, single-nucleus RNA sequencing (snRNAseq), and blood proteomics. Differential expression and pathway enrichment analyses were performed across spatial compartments and epithelial cell states. ResultsHistologically preserved lung regions in preclinical FPF demonstrated transcriptional abnormalities including stress-response, ciliary, and extracellular matrix-associated programs despite minimal architectural distortion. Spatial analyses identified alterations in alveolar niche molecular programs accompanied by increasing profibrotic signaling across preserved and tissue remodeled lung compartments. Compared with advanced IPF, preclinical FPF retained epithelial repair and surfactant-associated signatures. Integration with snRNAseq demonstrated enrichment of alveolar and airway epithelial cell dysregulated states associated with transitional phenotypes previously implicated in IPF. Compartment- and epithelial-associated transcriptional signatures identified in lung tissue were partially represented in the peripheral blood. ConclusionPreclinical FPF is characterized by compartment- and cell-specific molecular programs that precede established fibrosis. We identified distinct alveolar, airway, and vascular molecular signatures and epithelial remodeling states represented in the peripheral blood. These findings provide an initial framework for molecular classification of early stages of pulmonary fibrosis and support future studies evaluating minimally invasive approaches for disease stratification and precision therapeutics. At a Glance Commentary Scientific Knowledge on the SubjectThe molecular events preceding a diagnosis of pulmonary fibrosis remain poorly understood. Most mechanistic studies in Idiopathic Pulmonary Fibrosis (IPF) have relied on end-stage explanted lungs, limiting insight into the compartment- and cell-specific molecular programs associated with early stages of pulmonary fibrosis. What this study adds to the fieldUsing integrated spatial transcriptomics, single-cell sequencing, and peripheral blood proteomic profiling, we demonstrate that preclinical familial pulmonary fibrosis (FPF) is characterized by compartment- and cell-specific molecular programs that precede clinically detectable fibrosis. Spatial analyses identified distinct alveolar, airway, and vascular molecular signatures, while single cell analysis confirmed the presence of epithelial dysregulated states. These signatures are partially represented in the peripheral blood. Our findings provide an initial framework for biologically informed classification of early stages of pulmonary fibrosis and future minimally invasive approaches for disease stratification.

genomics↗

bioRxiv2025

Cell type-independent timekeeping gene modules enable embryonic stage prediction in zebrafish

Gene expression changes across embryonic development reflect both differentiation and genes whose expression varies strictly with developmental time, independent of cell type. Multiple embryonic timing systems set the onset and pace of developmental events, and blocking transcription arrests many of these events. However, the genes comprising the vertebrate embryonic timing system(s) remain largely unknown. To identify genes whose expression changes with time alone, we examine naive zebrafish embryonic explants that form only two tissue types yet maintain developmental timing, thus uncoupling developmental age from most differentiation programs. By comparing longitudinal gene expression in naive explants with Nodal-induced explants that differentiate into all three germ layers, we identify "timekeeping" genes whose temporal expression patterns vary strictly with developmental age. Consensus clustering of temporally dynamic genes identified 20 gene clusters, termed "chrono-constitutive modules" (CCMs), that maintain distinct schedules of expression regardless of tissue type. These CCM trajectories are similar in intact zebrafish embryos and single embryonic cells of multiple distinct lineages. Enrichment analysis of microRNA targets and transcription factor regulons within the CCMs further reveal distinct putative regulators of several modules. Strikingly, CCM expression patterns are also largely conserved during early development of another fish species, Japanese medaka. Machine learning models trained on only zebrafish CCM transcript levels accurately predict the developmental age of embryonic explants, intact embryos, and even individual embryonic cells, demonstrating their utility in developmental timekeeping. These results support the existence of transcriptional timekeeping during early development and demonstrate its utility in embryonic stage prediction.

developmental biology↗

bioRxiv2025

Individual Host Variation in Single-cell Responses to Enteroaggregative Escherichia coli Infection Modeled with Human Colon Organoids

Enteroaggregative Escherichia coli (EAEC) is a leading cause of diarrhea disease worldwide. Interestingly, EAEC disease outcomes vary dramatically among infected individuals, ranging from asymptomatic colonization to acute and chronic diarrhea. While previous research has focused on EAEC virulence factors, emerging evidence suggests that host genetic and cellular background may be equally important in determining disease susceptibility. To understand host-driven disease heterogeneity, we performed single-cell RNA sequencing on human colon organoids (HCOs) derived from three healthy adult donors following EAEC 042 infection. Surprisingly, despite comparable EAEC adherence across donors, we observed striking donor-specific responses to infection at multiple levels. A total of seven distinct colonic epithelial cell clusters were identified, with striking donor-specific baseline compositions. Following infection, the HCO donors exhibited three distinct response phenotypes evident in both cell type remodeling and gene regulation: hyperresponsive, intermediate, and non-responsive. We further observed extensive donor-specific cell-cell communication networks at baseline and after EAEC infection. This study suggests that the heterogeneity in host epithelial response to EAEC infection extends to the single-cell level. These findings establish a paradigm for understanding infectious disease susceptibility through the human organoid model system. IMPORTANCEUnderstanding why similar pathogen exposures produce vastly different clinical outcomes is critical for combating infectious diseases. By conducting single-cell RNA sequencing on human colon organoids derived from three different donors, we show that each donor possesses unique intrinsic cellular compositions and distinct transcriptomic responses to EAEC infection. These findings challenge the traditional pathogen-focused paradigm and highlight the critical role of host factors in infectious disease outcomes. This study also validates human organoids as a powerful platform for studying host heterogeneity, which may help predict disease susceptibility and optimize treatment for diverse patient populations in the future.

microbiology↗

bioRxiv2025

Sulfatase modifying factors control the timing of zebrafish gastrulation morphogenesis

To shape the emerging body plan, morphogenetic cell movements must be coordinated not only in space, but also in time. Convergence and Extension (C&E) movements that elongate the anteroposterior axis initiate with precise timing during vertebrate gastrulation, but the mechanisms controlling their onset remain unknown. We examined this question using zebrafish embryonic explants that faithfully recapitulate C&E cell movements and their precise timing in culture, in isolation from other gastrulation movements. We determined that new transcription is required at gastrulation onset for C&E in explants and identified sulfatase modifying factor 2 (sumf2) as a candidate trigger gene expressed at this time. sumf2 and its paralog sumf1 encode negative and positive regulators, respectively, of all sulfatase enzymes, which remove sulfate groups from their substrates, altering their biological activity. In zebrafish embryos and explants, sumf1 and sumf2 expression levels invert at gastrulation onset, predicting a reduction in sulfatase activity and consequent increase of substrate sulfation. We found that overexpressing sumf1 and sumf2 causes delayed or precocious C&E onset, respectively, whereas loss of sumf1 and sumf2 function shifts C&E timing in the opposite direction. We further identified Sulf1, an extracellular sulfatase that modifies heparan sulfate proteoglycans (HSPGs), as the key effector by which sumf1 and sumf2 control C&E timing. Accordingly, reduced or increased levels of sulfated heparan sulfate similarly shift C&E onset and suppress sumf1 and sumf2 mutant phenotypes. Together, our work supports a model in which sumf2 expression at zebrafish gastrulation onset reduces sulfatase activity, rewriting HSPG sulfation patterns to promote and/or permit C&E morphogenesis.

developmental biology↗

bioRxiv2025

Comprehensive Transcriptomic and Epigenomic Insights into Environmental Toxicant Exposures: The TaRGET II Resource

Environmental exposures to toxic chemicals can profoundly alter the transcriptome and epigenome in both humans and animals, contributing to disease development across the lifespan. To elucidate how early-life exposure to toxicants exerts such persistent effects, the Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription II (TaRGET II) Consortium generated a landmark resource comprising 2,564 epigenomes and 1,043 transcriptomes from longitudinal studies in mice. All data are publicly available through the TaRGET II data portal and the WashU Epigenome Browser. This resource from target (liver, brain, lung, heart) and surrogate (blood) tissues at weaning (3 weeks) and two adult time-points (5 and 10 months) characterized the molecular response to arsenic (As), lead (Pb), bisphenol-A (BPA), di-2-ethylhexyl phthalate(DEHP), tributyltin (TBT), tetrachlorodibenzo-p-dioxin (TCDD), and particulate matter with a diameter of <2.5m (PM2.5). The findings revealed persistent, toxicant-specific, sex-dependent epigenomic and transcriptomic perturbations, resulting in disrupted expression of 14,908 genes, altered chromatin accessibility at 87,409 regulatory elements, DNA methylation changes at 113,186 genomic regions, and chromatin state switching of histone modifications. The resulting high-resolution map of how environmental exposures reprogram the epigenome and transcriptome is broadly accessible via ToxiTaRGET database, offering unparalleled opportunities for the scientific community to investigate the molecular underpinnings of environmental toxicant exposures and their contributions to disease pathogenesis.

pharmacology and toxicology↗

bioRxiv2025

The impact of environmental exposures on the epigenomic and transcriptomic landscape of transposable elements

Transposable elements (TEs) are mobile DNA sequences that constitute a significant portion of mammalian genomes. While typically silenced by epigenetic mechanisms, mounting evidence indicates TEs can regulate gene expression and chromatin architecture. However, their regulatory roles under various environmental exposures remain largely unexplored. In this study, we investigate the regulatory functions of TEs in mouse liver tissue following early-life exposure to environmental toxicants, including arsenic (As), lead (Pb), bisphenol A (BPA), tributyltin (TBT), di-2-ethylhexyl phthalate (DEHP), tetrachlorodibenzo-p-dioxin (TCDD), and particulate matter less than 2.5 micrometers (PM2.5). These toxicants are linked to various health issues, including neurodevelopmental deficits, metabolic and immune dysfunction, and increased cancer risks. Integrative analysis of 351 multi-omics datasets from liver tissues of 5-month-old mice indicated that early-life environmental exposures significantly altered chromatin accessibility and expression of TEs in later life stage, revealing distinct exposure-specific signatures and sex-dependent responses. 6,699 TEs were identified with altered chromatin accessibility, mostly in non-coding regions, suggesting potential impact on gene regulation. Within these TEs, LINE elements were enriched in genes involved in metabolic pathways, while LTR elements, particularly the ORR1E subfamily, were predominantly associated with immune-related genes. Additionally, we identified 140 TE-gene chimeric transcripts with TE-derived novel transcription start sites, highlighting TE-contributed transcriptional plasticity. Our findings depict a comprehensive landscape of TE regulation under early-life toxicant exposures, offering insights into TEs biology and their impact on health and disease.

genomics↗

bioRxiv2025

Human bladder organoids model urinary tract infection and bacteriophage therapy

Urinary tract infections (UTIs), primarily caused by uropathogenic Escherichia coli (UPEC), are among the most common antibiotic-resistant infections. Despite this, currently available preclinical UTI models lack the breadth of morphotypic and heterogenous cell populations of the human bladder, impairing the development of novel therapies. To address these limitations, we developed human bladder organoids derived from the bladder stem cells of multiple healthy donors which recapitulate cellular diversity of the urothelium. Using bulk and single cell RNA-sequencing, we characterized organoid responses to UPEC and phage exposure individually and in combination to model phage therapy. Although phage minimally affected the uroepithelium in the absence of infection, during UTI, phage treatment reduced bacterial burdens and dampened inflammatory responses and barrier disruption. Collectively, our findings highlight human bladder organoids as a tool for capturing conserved and individual-specific uroepithelial responses to infection while also providing preclinical efficacy and safety testing for therapeutic development.

microbiology↗

bioRxiv2025

Macropinocytosis inhibition attenuates pro-fibrotic responses in lung fibroblasts and pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disorder with limited treatment options. Macropinocytosis is one of the key cellular processes involved in nutrient consumption from the extracellular environment under stress conditions. Here, we studied the role of macropinocytosis in lung fibroblast activation and experimental pulmonary fibrosis. We found that macropinocytosis is increased in human lung fibroblasts (HLFs) derived from IPF patients. The inhibition of macropinocytosis with 5-(n-ethyl-n-isopropyl)-amiloride (EIPA) significantly inhibited profibrotic responses in IPF-derived and TGF-{beta}1-stimulated HLFs. EIPA exerted antifibrotic effects by regulating amino acid (AA) uptake, mammalian target of rapamycin complex 1 (mTORC1) activation and mesenchyme homeobox1 (MEOX1) expression in activated HLFs. Both genetic and pharmacological inhibition of macropinocytosis significantly ameliorated pulmonary fibrosis in bleomycin (Bleo)-injured mice. Using IPF-derived precision cut lung slices (PCLS), we observed robust repression of profibrotic gene expression programs in EIPA-treated PCLS across different fibroblast subpopulations. Finally, we found that imipramine (Imi), a tricyclic antidepressant approved by the Food and Drug Administration (FDA), effectively inhibited macropinocytosis and ameliorated profibrotic responses in lung fibroblasts, Bleo-injured mice and IPF-derived PCLS. Taken together, our results suggest macropinocytosis inhibition as a potential therapeutic strategy to treat pulmonary fibrosis.

molecular biology↗

bioRxiv2025

Single cell transcriptomics in a treatment-segregated cohort exposes a STAT3-regulated therapeutic gap in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic pulmonary disease of unknown etiology. Since approved IPF drugs only slow disease progression, novel therapeutics are required that improve clinical outcomes. Here we report a single cell lung RNA-Seq and gene regulatory network analysis of the largest IPF cohort assembled to date. Segregating this cohort based on status of treatment with approved first-generation IPF antifibrotics (untreated, nintedanib- and pirfenidone-treated), we describe for the first time the transcriptional landscape of untreated IPF across 40 lung cell types, and the elements of this program that are impacted by these antifibrotics. On average, nearly 60% of the untreated IPF-dysregulated transcriptome is refractory to treatment with these drugs, a transcriptional deficit we refer to as the IPF therapeutic gap. Gene regulatory network analysis indicated a dominant functional footprint for the transcription factor STAT3 in both untreated IPF and the IPF therapeutic gap. Validating our analysis in a translational precision cut lung slice platform that recapitulates IPF explants, pharmacological inhibition of STAT3 reduced the IPF therapeutic gap in numerous lung cell types. Finally, we resolved a STAT3-anchored master regulatory network comprising numerous profibrotic transcription factors in IPF alveolar fibroblasts, a critical fibrotic lineage. Our study represents a comprehensive resource for translational lung fibrosis research and introduces a strategy for drug discovery that is adaptable to human disease more broadly.

cell biology↗

bioRxiv2025

Overcoming host restrictions to enable continuous passaging of human noroviruses in human intestinal enteroids

Human noroviruses (HuNoVs), the leading cause of viral gastroenteritis, can now be cultivated in human intestinal enteroids (HIEs). However, indefinite passaging of HuNoVs in HIEs remained a challenge, necessitating the use of patient stool samples as viral inocula. Using RNA-seq, we identified host restriction factors that might limit viral passaging. CXCL10, CXCL11, and CCL5 were among the most upregulated chemokines, suggesting their potential as host restriction factors. TAK-779, a CXCR3/CCR5/CCR2 antagonist, enhanced GII.3 HuNoV replication and viral spread in a dose- and time-dependent manner, enabling successful passaging of GII.3 HuNoV in two different HIE lines and generation of viral stocks. TAK-779 also enhanced replication of GI.1 and GII.17 strains, but not GII.4, suggesting strain-specific host interactions or immune evasion. This breakthrough in passaging provides critical insight into HuNoV-host interactions, establishes a scalable in vitro system for virus propagation, and opens avenues for structural, biochemical and therapeutic studies.

microbiology↗

bioRxiv2025

Early-Life Environmental Exposures Reprogram Epigenomic Aging to Alter Gene Expression Trajectories

To understand how early-life environmental exposures shape health and disease risk across the lifecourse, the TaRGET II Consortium exposed mice to diverse toxicants from pre-conception through weaning, and followed individual animals into adulthood, generating over 800 epigenomic and transcriptomic profiles. These profiles revealed that early-life exposures induced persistent epigenomic reprogramming and significantly disrupted the adult transcriptome. Notably, despite their diverse mechanisms of action, the exposure signatures of the xenoestrogen BPA, obesogen TBT, dioxin TCDD, and air pollutant PM2.5, were all largely comprised of genes normally differentially expressed during liver aging. Epigenetic histone modifications at enhancers--and, to a lesser extent, promoters--emerged as key targets for this reprogramming. Despite differing mechanisms of action, these four toxicants imparted similar "fingerprints" on the adult liver, characterized by direction-and cell type-specific polarization of the transcriptome. Hepatocyte genes that typically increase with age, particularly those in metabolic pathways, were downregulated, while conversely, non-parenchymal cell genes that typically decrease with age, such as those involved in extracellular matrix production, were upregulated. A similar signature of anti-correlation with programmed aging aging was also found in the transcriptome of patients with liver disease and hepatocellular carcinoma (HCC), and was effective at distinguishing healthy from diseased human livers. These findings demonstrate that the plasticity of epigenomic aging is vulnerable to early-life environmental exposures, which can reprogram the epigenome with lasting impacts on the transcriptome, and disease risk, later in life.

genomics↗

bioRxiv2025

Bypassing cisplatin resistance in Nrf2 hyperactivated head and neck cancer through effective PI3Kinase targeting

BackgroundFor patients with head and neck squamous cell carcinoma (HNSCC), failure of definitive radiation combined with cisplatin nearly universally results in death. Although hyperactivation of the Nrf2 pathway can drive radiation and cisplatin resistance along with suppressed anti-tumor immunity, treatment-refractory HNSCC tumors may retain sensitivity to targeted agents secondary to synergistic lethality with other oncogenic drivers (e.g., NOTCH1 mutations). PurposeWe evaluated the efficacy of PI3K inhibitors (PI3Ki) in bypassing Nrf2-mediated cisplatin resistance in HNSCC. MethodsWe measured transcriptomic, metabolomic and signaling changes driven by PI3Kis in cisplatin-resistant HNSCCs in vitro and tested efficacy in vivo in subcutaneous, orthotopic and metastatic xenograft models using immunodeficient and humanized murine models of HNSCC coupled with spatial transcriptomics. ResultsThe PI3K pathway is activated in Nrf2-driven cisplatin-resistant HNSCC and is suitable for blockade as demonstrated in an in vivo shRNA screen. The PI3Ki gedatolisib inhibits cisplatin-resistant HNSCC proliferation, induces G2M arrest and potentiates cisplatin effectiveness through activation of autophagy, senescence and disruption of fatty acid metabolism. Gedatolisib suppresses HNSCC tumor growth in orthotopic and metastatic settings and demonstrates profound anti-tumor activity in humanized murine models of HNSCC, coupled with a reduction in hypoxia-rich regions and reduced infiltration by regulatory T lymphocytes. ConclusionOur findings emphasize the critical role of the PI3K-AKT-mTOR pathway in cisplatin-resistant HNSCC and highlight the therapeutic potential of PI3K inhibitors. Gedatolisib induced metabolic regulation and substantial re-sensitization of resistant cells to cisplatin, positioning it as a promising candidate for combination therapies aimed at overcoming primary chemo-radiation failure in HNSCC. Statement of translational relevanceCisplatin resistance, whether intrinsic or acquired, translates to treatment failure and nearly universal death in head and neck squamous cell carcinoma (HNSCC). However, the development of effective systemic regimens for cisplatin-resistant HNSCC has not yet been successful. Here, we present, for the first time, a mechanistic, biomarker-informed strategy for effective targeting of the PI3Kinase pathway in cisplatin-resistant HNSCC with substantial anti-tumor activity in both orthotopic and metastatic models, which may be capable of bypassing or reversing cisplatin resistance in this disease.

cancer biology↗

bioRxiv2024

Tobacco smoke exposure is a driver of altered oxidative stress response and immunity in head and neck cancer

PurposeExposomes are critical drivers of carcinogenesis. However, how they modulate tumor behavior remains unclear. Extensive clinical data link cigarette smoke as a key exposome that promotes aggressive tumors, higher rates of metastasis, reduced response to chemoradiotherapy, and suppressed anti-tumor immunity. We sought to determine whether smoke itself can modulate aggressive tumor behavior in head and neck squamous cell carcinoma (HNSCC) through reprogramming the cellular reductive state. Experimental designUsing established human and murine HNSCC cell lines and syngeneic mouse models, we utilized conventional western blotting, steady state and flux metabolomics, RNA sequencing, quantitative proteomics and flow cytometry to analyze the impact of smoke exposure on HNSCC tumor biology. ResultsCigarette smoke persistently activated Nrf2 target genes essential for maintenance of the cellular reductive state and survival under conditions of increased oxidative stress in HNSCC regardless of HPV status. In contrast to e-cigarette vapor, conventional cigarette smoke mobilizes cellular metabolism toward oxidative stress adaptation, resulting in development of cross-resistance to cisplatin. In parallel, smoke exposure modulates both expression of PDL1 and the secretory phenotype of HNSCC cells through activation of NF-{kappa}B resulting in an altered tumor immune microenvironment (TIME) in syngeneic mouse models and altered PBMC differentiation that includes downregulated expression of antigen presentation and costimulatory genes in myeloid cells. ConclusionCigarette smoke exposome is a potent activator of the Nrf2 pathway and is a likely primary trigger for the tripartite phenotype of aggressive HNSCC consisting of: 1) reduced chemotherapy sensitivity, 2) enhanced metastatic potential and 3) suppressed anti-tumor immunity. Statement of significanceThe smoke exposome drives aggressive tumor behavior, treatment resistance and suppressed immunity through coordinated metabolic reprogramming. Successfully targeting this adaptation is critical to improving survival in smokers with head and neck cancer.

cancer biology↗

bioRxiv2024

Let-7 restrains an oncogenic epigenetic circuit in alveolar type 2 cells to prevent ectopic formation of fibrogenic AT2 intermediates and pulmonary fibrosis

Analysis of lung alveolar type 2 (AT2) progenitor stem cells has highlighted fundamental mechanisms that direct their differentiation into alveolar type 1 cells (AT1s) in lung repair and disease. However, microRNA (miRNA) mediated post-transcriptional mechanisms which govern this nexus remain understudied. We show here that the let-7 miRNA family serves a homeostatic role in governance of AT2 quiescence, specifically by preventing the uncontrolled accumulation of AT2 transitional cells and by promoting AT1 differentiation. Using mice and organoid models, we demonstrate genetic ablation of let-7a1/let-7f1/let-7d cluster (let-7afd) in AT2 cells prevents AT1 differentiation and results in accumulation of AT2 transitional cells in progressive pulmonary fibrosis. Integration of AGO2-eCLIP with RNA-sequencing from AT2 cells uncovered the induction of direct targets of let-7 in an oncogene feed-forward regulatory network including BACH1/EZH2/MYC which drives an aberrant fibrotic cascade. Additional analyses using CUT&RUN-sequencing revealed an epigenetic role of let-7 in induction of chromatin histone acetylation and methylation and maladaptive AT2 cell reprogramming. This study identifies let-7 as a key gatekeeper of post-transcriptional and epigenetic chromatin signals to prevent AT2-driven pulmonary fibrosis.

cell biology↗

bioRxiv2024

Nerve injury downregulates δ-opioid and cannabinoid CB1 receptor genes through REST in primary sensory neurons

The transcription repressor REST in the dorsal root ganglion (DRG) is upregulated by peripheral nerve injury and promotes the development of chronic pain. However, the genes targeted by REST in neuropathic pain development remain unclear. The expression levels of 4 opioid receptor (Oprm1, Oprd1, Oprl1, Oprk1) and the cannabinoid CB1 receptor (Cnr1) genes in the DRG regulate nociception. In this study, we determined the role of REST in the control of their expression in the DRG induced by spared nerve injury (SNI) in both male and female mice. Transcriptomic analyses of male mouse DRGs followed by quantitative reverse transcription polymerase chain reaction analyses of both male and female mouse DRGs showed that SNI upregulated expression of Rest and downregulated mRNA levels of all 4 opioid receptor and Cnr1 genes, but Oprm1 was upregulated in female mice. Analysis of publicly available bioinformatic data suggested that REST binds to the promoter regions of Oprm1 and Cnr1. Chromatin immunoprecipitation analyses indicated differing levels of REST at these promoters in male and female mice. Full-length Rest conditional knockout in primary sensory neurons reduced SNI-induced pain hypersensitivity and rescued the SNI-induced reduction in the expression of Oprd1 and Cnr1 in the DRG in both male and female mice. Our results suggest that nerve injury represses the transcription of Oprd1 and Cnr1 via REST in primary sensory neurons and that REST is a potential therapeutic target for neuropathic pain.

neuroscience↗