Biology subjects

Sharma, A.

Publications and source records attributed to Sharma, A..

At least 145 records · Page 8Linked to original sources

bioRxiv2025

ATaRVa: Analysis of Tandem Repeat Variation from Long Read Sequencing data

Tandem Repeats (TRs) are contiguous repetitions of DNA motifs whose variations modulate key cellular processes and traits. Their expansions are causally linked to over 70 neurological disorders in humans. Historically, genotyping TRs has been challenging due to their low sequence complexity and allele lengths exceeding typical short-read sequencing capabilities. While long-read sequencing improves TR genotyping by spanning large expansions and sequencing native DNA molecules, existing long-read genotypers often are inaccurate, computationally inefficient, and show platform-specific biases. Here, we present ATaRVa, a genotyper that achieves superior accuracy across both PacBio and Oxford Nanopore platforms, while running [~]15x faster than existing tools. Beyond genotyping, ATaRVa features sequence-level motif decomposition and methylation profiling. Evaluated across diverse datasets and genome-wide catalogs, ATaRVa especially excels in low coverage datasets, and accurately identifies known pathogenic expansions in clinical samples. Importantly, by quantifying uninterrupted target motifs in a population scale analysis, the tool systematically resolves false positives caused by benign, interrupted alleles. In addition, we present VisuaMiTRa, an auxiliary interface that enables interactive visualization of motif-level decomposition and base-level methylation of the TR alleles. By combining high computational efficiency with sequence-level structural awareness, ATaRVa provides a highly scalable framework for TR analysis in both population cohorts and clinical settings.

genomics↗

bioRxiv2025

Carboxyl Terminal Domain Missense Mutations Alter Distinct Gating Properties of the Cardiac Sodium Channel

Voltage-gated sodium channels undergo reversible voltage/time-dependent transitions from closed to open and inactivated states. The voltage setpoints and efficiency of cardiac sodium channel Nav1.5 state transitions are crucial for tuning the initiation and conduction of myocardial action potentials. The channels cytoplasmic carboxyl-terminal domain (CTD) regulates gating by intramolecular interactions and by serving as a hub for the binding of accessory proteins. We have investigated the roles of the CTD in intrinsic and FGF homologous factor (FHF)-modulated Nav1.5 gating through structure-guided CTD subdomain mutagenesis. The EF-hand module within the CTD was found to exert the most profound effects on channel gating, strongly influencing voltage-dependence of inactivation and activation, accelerating inactivation from the closed state, decelerating inactivation from the open state, minimizing persistent sodium current, and serving as the binding domain for FHF proteins. Nav1.5D1788K bearing a missense mutation in the EF-hand motif displayed a depolarizing shift in voltage dependence of activation and generated greatly enhanced persistent sodium current without altering the voltage dependence of channel inactivation. Reciprocally, Nav1.5L1861A bearing a different missense mutation in the EF-hand underwent closed-state inactivation at more negative membrane potential and at an accelerated rate, but did not display other phenotypes associated with CTD deletion. Nav1.5V1776A/T1778A bearing mutations in the juxtamembrane region between the EF-hand and the channel pore helices displayed wild-type intrinsic gating properties, while FHF modulation of inactivation gating was impaired. Our channel physiology studies together with prior structural data suggest that the voltage and rate of channel inactivation from the closed state are governed by an intramolecular hydrophobic interaction of the CTD EF-hand with the cytoplasmic inactivation loop helix and the extension of this binding interface upon FHF-induced restructuring of the juxtamembrane region, while a distinct CTD intramolecular electrostatic interaction modulates voltage-dependent activation and minimizes persistent sodium current.

physiology↗

bioRxiv2025

SF3B1K700E Drives Oncogenic Alternative Splicing of Cell Cycle Checkpoint Genes in Cancer

Pre-mRNA splicing plays a crucial role in maintaining cellular homeostasis, with strict regulation required for processes such as cell cycle progression. SF3B1, a core component of the spliceosome, has emerged as a key player in alternative splicing regulation and is frequently mutated in cancer. Among these mutations, SF3B1K700E disrupts normal splicing patterns and deregulates cell cycle control. Here we profiled K562 erythroleukaemia cells expressing either wild-type or SF3B1K700E by RNA-seq and uncovered 763 high-confidence splicing alterations enriched for G2/M regulators, including ARPP19, ENSA, STAG2 and ECT2. Notably, increased inclusion of ARPP19 exon 2 produces the ARPP19-long isoform, which sustains PP2A-B55 inhibition and promotes mitotic progression. A core subset of the K700E-linked splicing changes re-appeared after siRNA-mediated SF3B1 depletion in HeLa cells, underscoring a mutation-dependent spliceosomal signature that transcends cell type. Pharmacological inhibition of DYRK1A (INDY) or broad serine/threonine phosphatases (okadaic acid) shifted ARPP19 exon 2 inclusion in the same direction as SF3B1K700E, pointing to a kinase-phosphatase signaling axis that influences these splice events. Functionally, ectopic expression of ARPP19-long accelerated mitotic exit, and high ARPP19-long abundance associated with poorer overall survival in the TCGA-AML cohort. Our findings highlight a connection between SF3B1-dependent splicing, cell cycle progression, and tumorigenesis, offering new insights into the molecular mechanisms underlying cancer-associated splicing dysregulation.

molecular biology↗

bioRxiv2025

Phylogeny-driven design of broadly protective sarbecovirus receptor-binding domain nanoparticle vaccines

Vaccines against emerging SARS-CoV-2 variants and sarbecoviruses with pandemic potential must elicit a robust humoral immune response in a population imprinted with the SARS-CoV-2 spike (S) protein. Here, we designed protein nanoparticle (NP) vaccines co-displaying the SARS-CoV-2 BA.5, SARS-CoV-1, and BtKY72 receptor-binding domains (RBDs) with or without the Wuhan-Hu-1 (Wu) RBD. We show that these vaccines elicit cross-reactive and broadly neutralizing plasma antibody responses against SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune animals. Immunization with multivalent RBD-NPs overcomes immune imprinting and elicits neutralizing antibodies and memory B cells specific for the BA.5, SARS-CoV-1, and BtKY72 RBDs in mRNA-1273-vaccinated non-human primates. Multivalent RBD-NPs outperform a monovalent Wu RBD-NP vaccine by providing superior protection in mice and non-human primates challenged with the vaccine-mismatched SARS-CoV-2 XBB.1.5 or the pre-emergent RsSHC014. These data support the use of multivalent RBD-NP vaccines for SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune populations.

immunology↗

bioRxiv2025

γδ T Cells Target and Ablate Senescent Cells in Aging and Alleviate Pulmonary Fibrosis

A variety of physiological and pathological stimuli elicit the cellular senescence response. Immune cells are known to execute surveillance of infected, cancerous, and senescent cells, and yet senescent cells accumulate with age and drive inflammation and age-related disease. Understanding the roles of different immune cells in senescent cell surveillance could enable the development of immunotherapies against biological aging and age-related disease. Here, we report the role of human gamma delta ({gamma}{delta}) T cells in eliminating senescent cells. Human donor V{gamma}9v{delta}2 T cells selectively remove senescent cells of different cell types and modes of induction while sparing healthy cells, with parallel findings in mouse cells. We find that senescent cells express high levels of multiple {gamma}{delta} T cell ligands, including cell-surface BTN3A1. Individually blocking NKG2D or {gamma}{delta} TCR of {gamma}{delta} T cells only partially reduces V{gamma}9v{delta}2 T cell cytotoxicity, evidencing their versatility in senescence removal. {gamma}{delta} T cells expand in response to the induction of a mouse model of idiopathic pulmonary fibrosis (IPF), accompanied by the emergence of senescent cells, and colocalize with senescent cells in lung tissue from patients with IPF. Finally, we show that adoptive cell transfer of {gamma}{delta} T cells into an IPF mouse model reduces the number of p21-expressing senescent cells in affected lung tissue and improves outcomes. {gamma}{delta} T cells or modalities that activate their surveillance activity present a potent approach for removing senescent cells and their attendant contribution to aging and disease.

immunology↗

bioRxiv2025

CRIS: A Centralized Resource for High-Quality RNA Structure and Interaction Data in the AI Era

As interest in RNA-based therapeutics expands, there is a growing demand for RNA structure elucidation and RNA-RNA interactions in both academic and clinical settings. Despite rapid advances in methods for RNA structure determination, the field faces persistent challenges in data reproducibility, quality control, and accessibility, largely due to inconsistencies in data processing and analysis workflows. Concurrently, methodological improvements have generated increasingly complex datasets, which necessitate a standardized framework. Here, we present the Crosslinking-based RNA Interactomes and Structuromes (CRIS) database, a comprehensive resource designed to address these limitations. Among existing experimental and computational approaches for RNA structure characterization, crosslinking-based technologies offer superior reliability, high throughput, and high resolution. CRIS provides rigorously curated datasets, standardized workflows, and user-friendly tools, together with built-in quality metrics and detailed visualization guidance to ensure reproducibility and transparency while pairing seamlessly with existing experimental pipelines. By delivering high-complexity RNA datasets alongside accessible computational tools, CRIS serves as a standardized reference for both new and existing data, facilitating investigation through comparative analyses and providing a training resource for deep learning-based computational exploration. This will enable integration into machine learning workflows for large scale, novel RNA structure discovery.

bioinformatics↗

bioRxiv2025

A distinct subset of stem-cell memory is poised for the cytotoxicity program in CD4+ T cells in humans.

The peripheral CD4+ T-cells with cytolytic potential (CD4-CTLs) have been detected in several diseases, including infectious diseases and various cancers. They form the component of protective immune response and are shown to be enriched in the effector memory expressing CD45RA (TEMRA) in humans. However, the lack of understanding about their lineage, molecular characters, and cytolytic potential in comparison to CD8-CTLs has restricted their utility. Thus, in this study, by parallelly analysing the CD4-CTLs and CD8-CTLs, we demonstrate that they are indistinguishable for the cytolytic program, with both showing similar gene expression profile and T cell antigen-receptor (TCR) clonal expansion in humans. Further, using an integrative multi-omics analysis combining the transcriptome, TCR repertoire, and open chromatin profile of CD4+ naive (CD4-TN) and memory T-cell subsets, we discovered a distinct stem-cell memory subset (TSCM-CTL) that is pre-committed to cytolytic program that shared significant TCR clonotypes with expanded CD4-CTL-effectors. Further, through an in vitro differentiation model, we developed CD4+ T-cells with cytolytic potential (iCD4-CTLs) from CD4-TN cells that showed gradual and progressive acquisition of cytolytic program, exhibiting progressive chromatin accessibility at cytotoxicity-associated genes. Of particular interest was the property of iCD4-CTLs that co-expressed longevity-associated genes along with cytotoxicity-associated genes, hence generating long-lived CD4-CTL-effectors. Overall, the process of generation of iCD4-CTLs from CD4-TN, deciphered the molecular signatures of early commitment to the cytotoxicity program. Together, our study advocates for exploring both CD4-CTLs and CD8-CTLs for vaccine development, vaccine efficacy testing, as well as immunotherapies and cell-based therapies for precision medicine. TeaserIntegrative multi-omics analysis of CD4+ T cell subsets revealed the cytolytic potential of CD4-TEMRA cells in comparison to CD8-TEMRA and their origin from stem-cell memory.

immunology↗

bioRxiv2025

Spatial patterns of diversity in forest birds of peninsular India

Biodiversity is structured via complex interactions among ecological, geological, and climatic processes. Regions with high heterogeneity in climate and topography are known to harbor fine scale patterns in diversity which are often overlooked in global scale analyses. Here, we investigate spatial phylogenetic patterns of forest bird diversity across peninsular India, a region with high topographic and climatic heterogeneity. Using a comprehensive global bird phylogeny, we employ metrics such as Phylogenetic Diversity (PD), Phylogenetic Endemism (PE), and their relative forms (RPD, RPE) to quantify evolutionary history and endemism of forest birds in peninsular India. We examine the roles of contemporary climate, historic climatic stability, and topography in shaping these patterns. Our results reveal a strong gradient in diversity, with the southern Western Ghats acting as a major hotspot for both taxonomic and phylogenetic diversity and endemism. We detect distinct ecological and phylogenetic community structures across the peninsula, likely shaped by regional species pools and biogeographic barriers. Areas with greater topographic complexity, higher precipitation, and greater historic climatic stability were found to support high diversity. Our study provides critical insights for biogeography in this understudied yet highly biodiverse region for forest birds.

ecology↗

bioRxiv2025

Dexamethasone-Induced p57-Mediated Quiescence Drives Chemotherapy Resistance in Sonic Hedgehog Medulloblastoma

Medulloblastoma (MB), the most common malignant pediatric brain tumor, remains difficult to cure upon relapse, with only 12.4% of patients surviving five years post-recurrence. While specific quiescent tumor cell populations are known to contribute to treatment-resistance, the molecular mechanisms that maintain quiescence remain poorly defined. Here, we identify the cell cycle inhibitor p57 as a regulator of quiescence and chemotherapy resistance in Sonic Hedgehog (SHH) MB. Nuclear p57 was enriched in Sox2+ and Nestin+ stem-like MB cells compared to proliferative Atoh1+ cells. Inducing p57 expression in SHH MB cells led to a six-fold increase in G0-phase cells and conferred resistance to the frontline chemotherapeutic vincristine. Clinically, dexamethasone is a glucocorticoid given to nearly all MB patients to manage cerebral edema and is administered with wide variability in timing and dosing. We show that dexamethasone significantly increased nuclear p57 levels and expanded the G0 population in both Ptch1+/- and Ptch1+/-;Trp53-/- SHH MB mouse models. Pre-treatment with dexamethasone reduced vincristine sensitivity in SHH MB cells. Together, our findings reveal a clinically relevant and previously unrecognized mechanism of treatment resistance, whereby dexamethasone, despite its benefits in managing edema, may inadvertently contribute to tumor persistence or recurrence by driving a quiescent, drug-resistant state. Addressing the lack of standardization in steroid use or targeting p57 may improve treatment response and reduce recurrence in patients diagnosed with SHH MB.

cancer biology↗

bioRxiv2025

MTBseq-nf: Enabling Scalable Tuberculosis Genomics "Big Data" Analysis through a User-Friendly Nextflow Wrapper for MTBseq pipeline

The MTBseq pipeline, published in 2018, was designed to address bioinformatics challenges in tu- berculosis research using whole-genome sequencing data. It was the first publicly available pipeline on GitHub to perform full analysis of whole-genome sequencing (WGS) data for Mycobacterium tuberculosis encompassing quality control through mapping, variant calling for lineage classifica- tion, drug resistance prediction, and phylogenetic inference. However, the pipelines architecture is not optimal for analyses on high-performance computing or cloud computing environments, which often involve large datasets. To optimize the pipeline, we created MTBseq-nf, a Nextflow wrapper which offers shorter execution times through parallelization along with multiple other key improvements. The MTBseq-nf wrapper, as opposed to the linear, batched analysis of samples in the TBfull step of MTBseq pipeline, can execute multiple instances of the same step in parallel and therefore makes full use of the provided computational resources. For evaluation of scalability and reproducibility, we used 90 M. tuberculosis genomes (European Nucleotide Archive - ENA- accession PRJEB7727) for the benchmarking analysis on a dedicated computational server. In our experiments the execution time of MTBseq-nf parallel analysis mode is at least twice as fast as the standard MTBseq pipeline for more than 20 samples. Furthermore, the MTBseq-nf wrapper facilitates reproducibility using the nf-core, bioconda, and biocontainers projects for platform independence. The proposed MTBseq-nf wrapper pipeline is, user-friendly, optimized for hardware efficiency, scalable for larger datasets, and exhibits improved reproducibility.

bioinformatics↗

bioRxiv2025

Mosquito sex under lock and key

Mosquitoes, the worlds deadliest animal, exemplify single-mating systems where females mate only once in their lifetime, making mate choice critically important for reproductive success and mosquito control. Despite this importance, the mechanisms of female mating control and what prevents additional matings remain poorly understood. To address this gap, we developed a dual-color fluorescent sperm system in invasive Aedes aegypti mosquitoes and quantified mating patterns, confirming that 86-96% of females mate only once. Using behavioral tracking of mating pairs, deep learning, and quantitative analysis at increasing resolution, we discovered that females actively control mating initiation through a previously undescribed behavior: genital tip elongation. This female response is triggered by rapidly evolving male genital structures, creating a precise lock-and-key mechanism that determines mating success. Comparative analysis revealed that Aedes albopictus, separated from Aedes aegypti by 35 million years of evolution, employs a similar female-controlled system. However, Aedes albopictus males uniquely bypass female control when attempting cross-species matings with Aedes aegypti females but not with conspecific females. This "lock-picking" ability, combined with the known sterility of cross-species matings, may explain how Aedes albopictus competitively displaces Aedes aegypti populations in overlapping territories. Our findings redefine mosquito reproduction as a female-controlled process and establish a quantitative framework for investigating the molecular and neurobiological mechanisms underlying mating control and species competition in these globally important disease vectors.

animal behavior and cognition↗

bioRxiv2025

Circadian rhythms and the light-dark cycle interact to regulate amyloid plaque accumulation and tau phosphorylation in 5xFAD mice

BackgroundCircadian disruption has long been appreciated as a downstream consequence of Alzheimers Disease in humans. However, an upstream role for behavioral circadian disruption in regulating AD pathology remains an open question. MethodsTo determine the role of the central circadian clock in the suprachiasmatic nucleus (SCN) in regulating amyloid pathology, we generated mice harboring deletion of the critical clock gene Bmal1 in GABAergic neurons using VGAT-iCre, which is expressed in >95% of SCN cells, and crossed this line to the 5xFAD amyloid mouse model. To examine the role of the light-dark cycle in this process, we aged these mice in either regular 12:12 light-dark (LD) or constant darkness (DD) conditions. Transcriptional, behavioral, and physiological rhythms were examined in VGAT-iCre; Bmal1fl/fl; 5xFAD (VGAT-BMAL1KO;5xFAD) mice under varying light conditions. Amyloid plaque deposition, peri-plaque tau phosphorylation, glial activation, and transcriptomic changes were examined. ResultsVGAT-BMAL1KO;5xFAD mice showed loss of SCN BMAL1 expression and severe disruption of behavioral rhythms in both LD and DD, with loss of day-night rhythms in consolidated sleep and blunting of rhythmic clock gene expression in the brain. Surprisingly, VGAT-BMAL1KO;5xFAD mice kept under LD showed reduced total plaque accumulation and peri-plaque tau phosphorylation, compared to Cre-negative controls. These changes were gated by the light-dark cycle, as they were absent in VGAT-BMAL1KO;5xFAD mice kept in DD conditions. Total plaque accumulation was also reduced in control 5xFAD mice kept in DD as compared to LD, suggesting a general effect of light-dark cycle on amyloid aggregation. Expression of murine presenilin 1 (Psen1), as well as amyloidogenic cleavage of amyloid precursor protein, were suppressed in VGAT-BMAL1KO;5xFAD specifically under LD conditions. ConclusionsIn 5xFAD mice, the central circadian clock and the light-dark cycle interact to regulate amyloid pathology. Disruption of the central clock in the presence of a light-dark cycle may reduce APP cleavage and plaque formation. These results call into question the proposed simple positive feedback loop between circadian rhythm disruption and Alzheimers Disease pathology.

neuroscience↗

bioRxiv2025

Natural product-mediated reaction hijacking mechanism validates Plasmodium aspartyl-tRNA synthetase as an antimalarial drug target

Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5'-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2 phosphorylation. We show that DACM targets multiple amino acyl tRNA synthetase (aaRS) targets, including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition.

biochemistry↗

bioRxiv2025

Downregulation of KRT proteins in the Neuroendocrine System of Amyotrophic Lateral Sclerosis with PT150 as a Multi-Target Neuroprotectant

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron degeneration. Although ALS biomarkers exist, they lack specificity and early-onset. ALS also has a 40% misdiagnosis rate. This paper proposes the use of a Graph Neural Network (GNN) to diagnose ALS, new ALS-specific biomarkers, and a therapeutic based on this group. Gene expression and proteomic datasets were sourced. A kNN data representation was used to train a GNN for disease diagnosis. Achieving 98% accuracy between ALS and control, and 85% between ALS patients and ALS mimics (the specific diseases within this class were not disclosed), outperforming existing approaches. Integrated Gradients then quantified each genes importance in the models decision. KRTAP3-1 and KRTAP9-8 were substantial in the models classification process due to involvement in cytoskeletal organization. In parallel, statistical proteomi analysis revealed the significance of KRT6A, KRT6B, and KRT4 due to involvement in cytoskeletal organization, tissue development, and cellular differentiation, all related to ALS. These keratin proteins were expressed in the pituitary gland, suggesting that their downregulation contributes to hormonal dysregulation in ALS. Once Glucocorticoid Receptor (GR) was found to be the reason for keratin downregulation, molecular docking identified PT150 as a therapeutic, binding with and inhibiting GR and Cyclooxygenase 2, also reducing neuroinflammation in ALS.

systems biology↗

bioRxiv2025

Sp1 mechanotransduction regulates breast cancer cell invasion in response to multiple tumor-mimicking extracellular matrix cues

Breast cancer progression is marked by extracellular matrix (ECM) remodeling, including increased stiffness, faster stress relaxation, and elevated collagen levels. In vitro experiments have revealed a role for each of these factors to individually promote malignant behavior, but their combined effects remain unclear. To address this, we developed alginate-collagen hydrogels with independently tunable stiffness, stress relaxation, and collagen density. We show that these combined tumor-mimicking ECM cues reinforced invasive morphologies and promoted spheroid invasion in breast cancer and mammary epithelial cells. High stiffness and low collagen density in slow-relaxing matrices led to the greatest cell migration speed and displacement. RNA-seq revealed Sp1 target gene enrichment in response to both individual and combined ECM cues, with a greater enrichment observed under multiple cues. Notably, high expression of Sp1 target genes upregulated by fast stress relaxation correlated with poor patient survival. Mechanistically, we found that phosphorylated-Sp1 (T453) was increasingly located in the nucleus in stiff and/or fast relaxing matrices, which was regulated by PI3K and ERK1/2 signaling, as well as actomyosin contractility. This study emphasizes how multiple ECM cues in complex microenvironments reinforce malignant traits and supports an emerging role for Sp1 as a mechanoresponsive transcription factor.

bioengineering↗

bioRxiv2025

Inhibition of PIKfyve kinase induces senescent cell death by suppressing lysosomal exocytosis and leads to improved outcomes in a mouse model of idiopathic pulmonary fibrosis

Cellular senescence is a pivotal hallmark of aging, which limits lifespan and contributes to the development of age-related diseases. Efforts to identify senolytics - drugs that selectively eliminate senescent cells, have so far yielded candidates with limited translational potential. Here, we characterize the senescent cell surface proteomic landscape and identify proteins that are abnormally present on the plasma membrane of senescent cells. Many of these proteins are lysosomal enzymes, pointing to lysosomal exocytosis as a likely mechanism that leads to their persistent display on the cell surface. Blocking lysosomal exocytosis via PIKfyve kinase inhibition with a small molecule drug apilimod results in selective killing of senescent cells in vitro, while this treatment does not affect quiescent and proliferating cells. Furthermore, apilimod can be safely administered in vivo and effectively removes senescent cells and reduces tissue remodeling in a bleomycin mouse model of pulmonary fibrosis. We conclude that apilimod is an effective and well-tolerated senolytic that may be useful for the treatment of senescence-associated diseases of aging.

molecular biology↗

bioRxiv2025

Hyper-responsiveness of cancer stem cells to microenvironmental cues controls metastasis and therapy response through YAP/TAZ/TEAD

Cancer stem cells (CSCs) are key drivers of metastasis and therapy resistance but have been challenging to visualize and study in situ. Using a fluorescent CSC reporter, we observed very different population dynamics for CSCs and nonCSCs during metastatic lung colonization in breast cancer models. CSC expansive self-renewal drives early lesion formation before switching to a maintenance mode of balanced self-renewal and differentiation, whereupon nonCSC proliferation takes over as the main driver of metastatic expansion. Mechanistic analyses showed that CSCs are hyper-responsive to microenvironmental cues such as cell crowding and nutrient availability, suggesting a novel role for CSCs as sensors and early responders to fluctuating local conditions in the tumor. Incoming signals converge on YAP/TAZ/TEAD, with heightened CSC sensitivity and response supported by elevated receptor expression and increased chromatin accessibility around enhancers with TEAD binding sites. Targeting inputs to the YAP/TAZ/TEAD node reversed chemotherapy-induced enrichment of CSCs in lung metastases. HighlightsO_LIDifferent population dynamics for breast cancer stem cells (CSCs) and their differentiated progeny in early metastatic colonization C_LIO_LICSCs are hyper-responsive to microenvironmental cues and serve as sensors of local conditions for the tumor C_LIO_LIMany microenvironmental inputs converge on YAP/TAZ to regulate self-renewal vs differentiation fate decisions in the CSC C_LIO_LITargeting YAP/TAZ input pathways blocks chemotherapy-induced enrichment of CSCs C_LI

cancer biology↗

bioRxiv2025

Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

BackgroundSignaling by estrogen-receptor alpha (ER) plays a major role in breast cancer initiation Investigations of the mechanism of DNA damage mediated by ER signaling are carried out in breast cancer cell lines due to the lack of ER+ normal human breast epithelial cells lines (HBEC). Defining the mechanisms by which ER induces DNA damage and initiates tumorigenesis requires normal HBECs that express ER, demonstrate estrogenic responses, and are amenable to long term propagation in culture. MethodsWe utilized lentiviral expression of an inducible ER construct to generate four HBEC lines (HBEC-ESR1). We studied these cells for ER-dependent responses using a luciferase reporter, endogenous gene expression and proliferation assays. RNA-Seq was performed to characterize the ER-mediated transcriptomic patterns in the four HBEC lines. ER mediated DNA double strand breaks (DSBs) were analyzed using {gamma}H2AX immunofluorescence. ResultsExpression and functional activation of ER were observed in all HBEC-ESR1 lines, whereas proliferation in response to 17{beta}-estradiol (E2) was observed in 3 of the cell lines. Proliferative responses were due to intrinsic signaling within the HBECs as conditioned media from the cells failed to cause proliferation. A total of 682 genes were differentially expressed at 24h following treatment with 10nM E2 with 43% of these genes were also observed in ER+ breast cancer cell lines (MCF7 or T47D). Gene-set enrichment analysis identified differential expression of genes in ER signaling pathways and DNA repair pathways in E2-treated cells. E2-induced ER signaling also increased {gamma}H2AX foci in 3 of the 4 cell lines. Levels of DSBs were increased by inhibition of the non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways. DSBs were also increased in MCF10A-ESR1 cells heterozygous for the BRCA1185delAG mutation causing a truncated protein. ConclusionsInducible expression of ER in immortalized HBECs recapitulate transcriptional, replicative and DNA damage responses. Increased DSBs in MCF10A-ESR1 cells with heterozygous mutation of BRCA1 indicate haploinsufficiency and the potential for increased genetic instability due to ER signaling.

cancer biology↗