Biology subjects

Beard, S.

Publications and source records attributed to Beard, S..

6 recordsLinked to original sources

bioRxiv2026

Genomic alterations enable BRCA1 methylation loss and promoter bypass to drive resistance in high-grade serous ovarian cancer

BRCA1 promoter methylation predicts sensitivity to PARP inhibitors in high-grade serous ovarian cancer, yet therapeutic resistance is common and mechanistically unresolved. Using long-read direct DNA sequencing of patient-derived xenografts and cell lines, we resolved BRCA1 methylation at single-molecule resolution with structural and transcriptomic analyses. We revealed two convergent PARP inhibitor and platinum resistance mechanisms, validated in patient tumors. First, focal, allele-specific loss of BRCA1 methylation arose through local cis-acting genomic alterations, instead of global epigenetic reprogramming. Engineered in cis sequence alterations near the methylated BRCA1 promoter were sufficient to induce methylation loss, restore homologous recombination, and confer resistance. Similar associations were observed across the genome, suggesting this mechanism extends beyond BRCA1. Second, BRCA1 expression was restored despite intact promoter methylation via structural variant-mediated promoter bypass or alternative transcription initiation. Together, these findings redefine BRCA1 methylation loss as a locus-restricted process and reveal multiple routes by which tumors escape PARP inhibitor therapy. Statement of SignificanceWe show that high-grade serous ovarian cancers can restore BRCA1 expression after therapy through multiple genomic mechanisms, including local methylation loss and promoter bypass, thereby re-establishing homologous recombination and driving PARP inhibitor resistance. These findings challenge reliance on BRCA1 methylation alone as a predictive biomarker and support rational combination therapies for more durable responses.

cancer biology↗

bioRxiv2025

First-trimester exposure to anti-cytomegalovirus medications: a study of the effects of valaciclovir, letermovir and maribavir on early placental development

Congenital cytomegalovirus (CMV) infection is the leading preventable cause of childhood disability, including sensorineural hearing loss and cerebral palsy in high-income countries. Early maternal treatment with valaciclovir has been shown to reduce fetal infection. However, newer, more potent anti-CMV medications such as letermovir and maribavir are not routinely used in pregnancy due to limited safety data. This study aimed to assess the effect of aciclovir (the active metabolite of valaciclovir), letermovir, and maribavir on cell viability, functionality, and key cell signalling pathways in first-trimester human placental tissue. First-trimester placental tissue was obtained from individuals undergoing surgical abortion for psychosocial indications at 9-13 weeks of gestation. The effect of anti-CMV medications on primary cytotrophoblast cell survival was measured using a standard cell viability assay. The effects of anti-CMV medication exposure on Wnt and epithelial-to-mesenchymal transition (EMT) signalling pathways were evaluated in placental explant cultures. Their impact on cellular proliferation and migration was assessed using placental outgrowth cultures. Exposure to physiological or supraphysiological levels of aciclovir, letermovir, or maribavir for 48 hours did not significantly alter cytotrophoblast cell viability compared to vehicle-treated controls. Aciclovir did not significantly impact Wnt and EMT signalling gene expression in first-trimester placental explants; however, letermovir and maribavir induced significant gene dysregulation. None of the three anti-CMV medications altered first-trimester trophoblast proliferation or migration. These results provide reassuring data supporting the safety of maternal valaciclovir therapy in the first trimester. They may also inform the selection of newer anti-CMV medications for future clinical trials for use in pregnancy.

developmental biology↗

bioRxiv2025

Amniotic fluid extracellular vesicle properties evolve with gestational age and reflect fetal development

Amniotic fluid (AF) is a valuable source of extracellular vesicles (EVs) derived from the fetoplacental unit. Preclinical and clinical studies have highlighted promising applications of AF-EVs and their role in cellular communication, yet our understanding of AF-EV physiology is limited. This study aimed to examine the physiological importance of AF-EVs in fetal development from the second trimester to term gestation. We obtained AF samples from routine second-trimester amniocentesis and prelabour Caesarean section at term. We isolated EVs using a combination of differential centrifugation, filtration, and ultracentrifugation and characterised them using nanoparticle tracking analysis, cryoelectron microscopy, and Western blotting. The differential EV proteome was analysed using label-free proteomics. We assessed the second trimester and term AF-EV properties through an enrichment analysis. The EV size and protein enrichment difference revealed a gestational-age-dependent variation in the predominant EV subtype. Second-trimester-derived EVs were enriched in ectosomes, while term EVs contained a significant proportion of exosomes. We identified several morphologies of AF-EVs, including unilamellar, multilamellar, multicompartmental and granular-centred EVs, across gestations. Proteomics analysis of AF-EVs identified 4137 proteins with high confidence, of which 1099 exhibited significant differential expression between the two groups. Second-trimester-enriched AF-EV proteins represented molecule assembly processes, metabolism and organogenesis. At term, AF-EV proteins corresponded to impending newborn functions such as immunity and digestion. In conclusion, we provide compelling evidence that EV biogenesis and secretion in the fetoplacental unit undergo significant alterations across gestation, revealing a complex and dynamic physiology and intercellular communication that adapts to the needs of the developing fetus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/649052v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@3358c4org.highwire.dtl.DTLVardef@108ec96org.highwire.dtl.DTLVardef@e49b91org.highwire.dtl.DTLVardef@a1888b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO This figure summarises the studys main findings, including the shift in the predominant EV subtype, the different organs and biofluids represented by the enriched proteins at each gestation, and the main biological pathways. C_FIG

developmental biology↗

bioRxiv2025

Demethylating agents drive PARP inhibitor resistance in ovarian carcinomas with BRCA1 gene silencing

DNA methyltransferase 1 inhibitor (DNMT1i) therapy is a promising option for increasing immune response as part of combination cancer therapy. High-grade serous ovarian carcinoma (HGSOC) is a highly aggressive cancer with poor survival outcomes, where DNMT1i therapy is being increasingly explored. HGSOC with epigenetically silenced BRCA1 has been shown to respond to PARP inhibitor (PARPi) treatment - a core targeted therapy for HGSOC. However, loss of silencing of even a single BRCA1 allele causes PARPi and platinum chemotherapy resistance. We tested whether BRCA1 silencing was robust to DNMT1i therapy, or would be reversed, thus driving PARPi resistance. We previously generated two homozygously silenced BRCA1 HGSOC cell lines: WEHI-CS62 and an OVCAR8 derivative. DNMT1i treatment caused sustained BRCA1 promoter methylation loss, gene re-expression and PARPi resistance in both of these silenced BRCA1 lines, but not in mutated BRCA1/2 or RAD51C lines. Methylation arrays confirmed transient global CpG methylation losses following DNMT1i. CRISPR deletion of the re-expressed BRCA1 copy in WEHI-CS62 restored silencing and PARPi sensitivity. Furthermore, DNMT1i treatment of a silenced BRCA1 PDX caused heterogeneous BRCA1 promoter methylation loss. In summary, DNMT1 inhibitors caused sustained reduction of BRCA1 promoter methylation in HGSOC cells. This resulted in BRCA1 re-expression and PARP inhibitor resistance, presenting a significant risk for up to 17% of HGSOC patients with BRCA1 gene silencing who could benefit from PARP inhibitor therapy. We conclude that DNA demethylation therapy should be avoided for HGSOC patients with epigenetically silenced BRCA1.

cancer biology↗

bioRxiv2025

Plasmidome-defensome interactions drive adaptation of 'Fervidacidithiobacillus caldus' in natural and industrial extreme acidic environments

Plasmids are major drivers of microbial evolution, enabling horizontal gene transfer (HGT) and facilitating adaptation through the dissemination of relevant functional genes and traits. However, little is known about plasmid diversity and function in extremophiles. Fervidacidithiobacillus caldus, a meso-thermo-acidophilic sulfur oxidizer, is a key player in sulfur cycling in natural and industrially engineered acidic environments. Here, we present a comprehensive analysis of the plasmidome, and associated anti-mobile genetic element (anti-MGE) defense systems (defensome), across genomes of this species and metagenomes from diverse natural and industrial settings harboring F. caldus. We identified >30 distinct plasmids, representing five consistent replication-mobilization families. Plasmids ranged in size between 2.5-65 kb, with gene content and plasmid modularity scaling with element size and copy numbers inversely correlating with size. Plasmids carried variable numbers of hypothetical proteins and transposases, with annotated cargo genes reflecting functional differentiation by habitat. Defensome profiling revealed over 50 anti-MGE systems in sequenced F. caldus isolates, including diverse restriction-modification systems, CRISPR-Cas types IV-A and V-F, and widespread abortive infection and composite defense systems such as Wadjet, Gabija, and Zorya. In environmental populations, an inverse relationship was observed between defensome complexity and plasmidome abundance and diversity, underscoring a pivotal role of the host defensome in modulating persistence, compatibility, and overall plasmid diversity across F. caldus populations. Yet, other plasmids appeared decoupled from both host abundance and defensome complexity, suggesting potential host shifts, environmental persistence, or differential replication under suboptimal growth conditions for the host. Altogether, these findings reveal a modular, adaptive plasmidome shaped by selective pressures and host-plasmid-defensome interactions and positions plasmids as key contributors to adaptation, gene flow, and functional innovation in this extreme acidophile. ImportancePlasmids are key vehicles of gene exchange and adaptation in bacteria, yet their roles in extremophilic systems remain poorly understood. This study provides the first integrated view of the plasmidome and defense systems in Fervidacidithiobacillus caldus, a sulfur-oxidizing acidophile relevant to both natural biogeochemical cycling and industrial bioleaching. We uncover a rich plasmid diversity structured into modular families with variable cargo and backbone features and reveal their coexistence with complex anti-MGE defense repertoires. By combining genomic and metagenomic approaches, we expose principles of plasmid compatibility, persistence, and habitat-specific adaptation. These insights expand current knowledge of mobile genetic elements in extreme environments and provide a foundation for plasmid-based vector design and synthetic biology in acidophiles, with direct implications for biomining and environmental remediation in extreme environments.

genomics↗

bioRxiv2024

Generation of a PARPi-sensitive homozygous BRCA1-methylated OVCAR8 cell line using targeted CRISPR gene editing

Up to 17% of high grade serous ovarian carcinomas (HGSOC) harbour BRCA1 promoter methylation (meBRCA1), making them susceptible to treatment with targeted PARP inhibitor (PARPi) therapy. Unfortunately, meBRCA1 loss can be acquired following PARPi or platinum chemotherapy, resulting in BRCA1 re-expression and PARPi resistance. Our understanding of meBRCA1 stability in HGSOC is currently limited, in part due to a paucity of pre-clinical models with homozygous meBRCA1. Herein, we describe the generation of a several OVCAR8 cell line derivatives containing landing pad constructs, for future functional studies, and representing various BRCA1 states, including a homozygous meBRCA1 variant. Our PARPi resistant OVCAR8 has two methylated BRCA1 copies and one unmethylated copy, enabling BRCA1 expression. CRISPR-Cas9 gene editing was used to delete copies of the BRCA1 gene in landing pad-containing clones of this cell line (A6 and H4). We produced one variant with deletion of all BRCA1 copies (H4-53), and another with two copies deleted and only a single methylated gene copy remaining (A6-30 - validated further using nanopore long-read sequencing). These both lacked BRCA1 gene expression and were sensitive to PARPi treatment. The A6-30 line was transplanted into immunocompromised mice to generate a xenograft model that retained homozygous meBRCA1 and demonstrated some response to PARPi in vivo. Thus, using CRISPR gene editing we have created several novel isogenic HGSOC cell line models, including one with homozygous meBRCA1, that will support future studies of meBRCA1 stability and PARPi resistance.

cancer biology↗