Biology subjects

Saha, S.

Publications and source records attributed to Saha, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilising Effects on the Scaffold PR65

Protein phosphatase 2A (PP2A), an important therapeutic target, comprises a scaffold subunit PR65 composed of 15 HEAT (Huntingtin/elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not been fully defined. Here we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the non-functional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force-extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed globally, and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces for only a few repeats of PR65, suggestive of a more localised effect; moreover, subsequent stretch-relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide additional insights how SMAP binding modulates PR65 structure and function.

biophysics↗

bioRxiv2025

Uttroside B, a US FDA-designated Orphan drug, mitigates thedevelopment of hepatocellular carcinoma and its pulmonary metastasis via EGFR/ERK-mediated inhibition of SREBP-1 and STAT-3

Hepatocellular carcinoma (HCC) is a highly aggressive tumor with rapid propensity for extrahepatic metastasis, which critically limits the long-term clinical benefits of conventional chemotherapeutics and decreases the overall survival rate of patients. Our previous findings on the exceptional anti-HCC potential and pharmacological safety of uttroside B (Utt-B) have gained multiple international patents and the compound has been designated as an Orphan Drug against HCC by the US FDA. The current study substantiates the pharmacodynamics of Utt-B and is the first report to date on the anti-metastatic potential of the compound against HCC. Herein, we demonstrate the role of EGFR/ERK signaling axis and their downstream targets SREBP-1 and STAT-3, the key regulators of HCC development and the pulmonary metastasis, respectively, in orchestrating the anti-HCC and anti-metastatic potential of Utt-B. This is evidenced by the abrogation of the cytotoxic and pro-apoptotic effects of Utt-B upon pharmacological inhibition of this signaling axis. Orthotopic xenograft studies validated that Utt-B treatment restricted the development of tumors via the down-regulation of EGFR/ERK axis. Notably, Utt-B diminishes the migratory and invasive properties of liver cancer cells in vitro and impedes the pulmonary metastasis of HCC, in vivo. Taken together, the current findings attest to the exceptional therapeutic potential of Utt-B against primary and metastatic HCC and highlight its potential as a candidate drug to be evaluated in the clinics for the benefit of HCC patients having limited prognosis and therapeutic options.

cancer biology↗

bioRxiv2025

Environmental and Maternal Imprints on Infant Gut Metabolic Programming

Early life is a critical period for immune and metabolic programming, but developmental patterns remain underexplored in populations from low- and middle-income countries. Here, we profiled the microbiome and metabolome of 55 Bangladeshi mother-infant dyads over the first six months of life. Importantly, we observed an increase in microbially-derived bile amidates and N-acyl lipids with age in conjunction with reads matching the bile salt hydrolase/transferase (bsh) gene. While microbial source tracking confirmed maternal fecal seeding, a substantial environmental contribution was also highlighted. Differences in infant fecal metabolic profiles were associated with delivery mode, maternal milk composition, household assets, and household-level water treatment. C-section delivery and untreated drinking water were linked to transient metabolic differences, including increases in bile amidates, N-acyl lipids, and other host-microbe co-metabolic products, including acylcarnitines. Multi-omics analysis revealed specific microbial-metabolite relationships, highlighting how early environmental and maternal living circumstances shape metabolic gut programming through the microbiome.

microbiology↗

bioRxiv2025

Psychrophilic and cryophilic bacterial copiotrophs from a Trans-Himalayan lake-desert ecosystem as biogeothermometers of warming, mitigators of perturbation, and candidates for biodegradation

A Trans-Himalayan lake-desert ecosystem was explored for the low-to-high temperature adaptations of native copiotrophic psychrophiles that have the potentials for rendering substantive carbon remineralization under natural and/or anthropogenically influenced conditions of high organic matter delivery to the environment. Overall 27 species, classified across 15 genera of Actinomycetota, Bacillota, Bacteroidota, and Pseudomonadota, were isolated from the brackish-water and sediment-surface of Tso Moriri (a massive lacustrine body on the Changthang plateau that remains frozen for approximately one third of the year), and the fine talus covering a lake-side rocky mountain. In Luria broth, all the isolates grew at 4{degrees}C and 15{degrees}C; 13 of them grew, while 14 retained 19-99% cells in metabolically-active state, at -10{degrees}C; 23, 11, and none grew at 28{degrees}C, 37{degrees}C, and 42{degrees}C respectively. A Cryobacterium was most heat-susceptible, whereas some -10{degrees}C-growing Arthrobacter, Microbacterium, Paenarthrobacter, Pseudomonas and Streptomyces were most warming-resilient, among the present isolates. Catabolizing different simple/complex organic compounds, all isolates accomplished low/moderate/high growths at 4{degrees}C; 20 performed low growths at -10{degrees}C. Taxonomy/phylogeny did not determine the isolates growth/survival across the temperature-range tested. Lake-water and rock-dust isolates had the widest, while lake-sediment-dwellers had the narrowest, temperature-window for growth/survival. Rock-dust and lake-water isolates had more genes for low and high temperature adaptations, compared to the lake-sediment isolates. In co-cultures, most actinobacterial isolates inhibited the growth of mesophilic bacteria from warmer ecosystems, plus non-actinobacteria and actinobacteria from native/adjoining habitats. The data hold implications for biodegradation in cold/frigid territories, negative feedback control/reversal of warming, and microbiome protection from infiltration amidst climate change. IMPORTANCEPhylogenetically diverse, cold-adapted copiotrophic bacteria were retrieved from a Trans-Himalayan lake-desert ecosystem, and tested for chemoorganoheterotrophic growth at temperatures between -10{degrees}C and 42{degrees}C. Albeit growth dwindled at [≥]28{degrees}C, most of the isolates thrived on simple as well as complex organic substances under freezing condition, which makes them prospective constituents of biodegradation reactors suitable for high-altitude/latitude deployment. In the natural habitats of the psychrophilic isolates, cessation of organotrophic activities at elevated temperatures would stop simple fatty acids and CO2 production, which in turn would preempt methanogenesis, undo greenhouse effect, and reverse environmental thawing (usher cooling). Such negative feedback control of homeostasis is fraught with the danger of microbiome takeover by thermally-better-adapted foreign organisms. Consequently, ecosystem preservation rests on the native microfloras ability to resist external invasion. At 28{degrees}C, majority of the actinobacterial isolates inhibited bacteria from discrete higher-temperature ecosystems; they can, therefore, be viewed as the defenders of the cold/frigid ecosystem.

microbiology↗

bioRxiv2025

Satb1 integrates cohesin mediated genome organization and transcriptional regulation during T cell development

Three-dimensional (3D) genome folding, which is highly cell type-specific, plays a crucial role in orchestrating spatiotemporal gene expression. Although factors such as CTCF have been extensively studied in the hierarchical regulation of 3D chromatin organization, the mechanisms driving dynamic genome folding during T cell fate transitions remain incompletely defined. In this study, we reveal that Satb1, a chromatin organizer enriched in the T cell lineage, co-occupies genomic regions with the cohesin complex and Ctcf in double-positive (DP) thymocytes, where chromatin interactions are notably increased. We show that Satb1 physically interacts with the cohesin subunit Smc1a, and its deletion results in aberrant Smc1a binding and reduced chromatin contacts at sites co-occupied by Satb1 and cohesin. In both DP and immature CD4 single-positive (SP) T cells, Satb1 is essential for proper T cell activation and cytokine signaling. At the Cd3 locus, Satb1 and cohesin collaboratively regulate gene expression, with Satb1 loss leading to disrupted Smc1a occupancy and compromised chromatin interactions. Furthermore, Satb1 shows in vitro properties consistent with liquid-liquid phase separation, and disease-associated mutations impair these properties. Together, our findings uncover a molecular mechanism in which Satb1 facilitates chromatin looping through direct interaction with the cohesin complex and its ability to form nuclear condensates, thereby governing transcriptional regulation during T cell development.

immunology↗

bioRxiv2025

Germline polymorphism in the immunoglobulin kappa and lambda loci explain variation in the expressed light chain antibody repertoire

Variation in antibody (Ab) responses contributes to variable disease outcomes and therapeutic responsiveness, the determinants of which are incompletely understood. This study demonstrates that polymorphisms in immunoglobulin (IG) light chain loci dictate the composition of the Ab repertoire, establishing fundamental baseline differences that preclude functional Ab-mediated responses. Using long-read genomic sequencing of the IG kappa (IGK) and IG lambda (IGL) loci, we comprehensively resolved genetic variation, including novel structural variants, single nucleotide variants, and gene alleles. By integrating these genetic data with Ab repertoire profiling, we found that all forms of IG germline variation contributed to inter-individual gene usage differences for >70% of light chain genes in the repertoire, directly impacting the amino acids of expressed light chain transcripts, including complementarity determining region domains. The genomic locations of usage - associated variants in both intergenic and coding regions indicated that IG polymorphisms modulate gene usage via diverse mechanisms, likely including the modulation of V(D)J recombination, heavy and light chain pairing biases, and transcription/translation. Finally, relative to IGL, IGK was characterized by more extensive linkage disequilibrium and genetic co-regulation of gene usage, illuminating differential regulatory and evolutionary features between the two light chain loci. These results firmly establish the critical contribution of IG light chain polymorphism in Ab repertoire diversity, with important implications for investigating Ab responses in health and disease.

immunology↗

bioRxiv2025

Molecular fingerprints of a convergent mechanism orchestrating diverse ligand recognition and species-specific pharmacology at the complement anaphylatoxin receptors

Complement anaphylatoxin receptors (C3aR and C5aR1) are prototypical G protein-coupled receptors (GPCRs) playing crucial physiological roles in innate immunity by combating pathogenic infections and orchestrating inflammatory responses. They continue to be important therapeutic targets for multiple disorders including autoimmune diseases, acute and chronic inflammation, and allergy-related conditions. Recent structural coverage has provided important insights into their activation and signaling, however, confounding observations in the literature related to ligand efficacy and functional responses, especially in different model systems, present a major challenge for drug discovery efforts. Here, we systematically and comprehensively profile a broad set of natural and synthetic ligands at C3aR and C5aR1 and discover a previously unanticipated level of functional specialization in terms of species-specific pharmacology and receptor activation. Taking a lead from this, we determine seventeen cryo-EM structures of different ligand-receptor-G-protein complexes and uncover distinct orientation of agonists between the human and mouse receptors despite an overlapping positioning in the orthosteric binding pocket. Combined with extensive mutagenesis and functional assays, these structural snapshots allow us to decode and validate a convergent molecular mechanism involving a "Five-Point-Switch" in these receptors that orchestrates the recognition and efficacy of diverse agonists. We also identify species-specific differences at the level of phosphorylation patterns encoded in the carboxyl-terminus of these receptors and directly visualize their impact on {beta}arr binding and activation using cryo-EM structures. Interestingly, we observe that {beta}arrs engage with the mouse C5aR1 using a variation of previously discovered P-X-P-P phosphorylation motif via a "Sliding-Mechanism" and also exhibit distinct oligomeric state for the human vs. mouse receptors. Taken together, this study elucidates functional specialization at the complement anaphylatoxin receptors and underlying molecular mechanisms, offering a previously lacking framework with direct and immediate implications for the development of novel therapeutics.

biochemistry↗

bioRxiv2025

High-Throughput Multiomics Profiling of Model Systems Using the AVITI24 Platform

We present a multiomics platform comprising Teton, a detection assay system, and AVITI24, a dual-flowcell instrument that performs both cellular imaging and sequencing readout. Teton integrates a compartmentalized flowcell for cell culture with methods to measure morphology, RNA, and protein at subcellular resolution. The platform quantifies morphological features through cell painting of 6 cellular components, RNA expression of up to 350 transcripts via sequencing of oligonucleotides hybridized to mRNA, and protein expression of up to 200 targets using antibody-linked oligonucleotide sequencing. The flow cell accommodates >1 million cells in a 10 cm squared open-well format or can be subdivided into 12 or 48 wells to support experiments with multiple conditions or time points. We describe and validate the detection methods of the platform and showcase its capabilities by co-culturing three cancer cell lines and elucidating the cellular pathways triggered by various drug treatments as a function of time. Using multiple time points enables us to capture the dynamics of cellular processes including receptor activation and signaling cascades. The results demonstrate how different cancer cells evade TNF-induced apoptosis by activating compensatory signaling programs that maintain survival despite pro-apoptotic cues. Our model system replicates previously published results and highlights the versatility of the platform in enabling rapid, high-throughput analysis of complex cellular responses in varied biological contexts.

genomics↗

bioRxiv2025

High levels of Cdc42 GTPase underlie an all-or-none decision to fuse

Cdc42 is a Rho-family GTPase conserved across eukaryotes, where it plays essential roles in cell polarization. In single-celled yeast systems, Cdc42 is a key driver of symmetry breaking and polarized growth, forming zones of activity that locally recruit eRectors to organize the cytoskeleton and polarize secretion. Here we show that Cdc42 also functions in cell-cell fusion during Schizosaccharomyces pombe sexual reproduction but concentrates at the fusion site through mechanisms distinct from those proposed in Saccharomyces cerevisiae. Notably, the cdc42-mCherrySW allele (but not the cdc42-sfGFPSW allele), which is functional for cell polarization and has been used across organisms for dynamic studies, exhibits a strong fusion defect. These cells block fusion before cell wall digestion but after actin fusion focus formation, indicating that Cdc42 is required to translate the vesicle cluster into polarized cargo delivery. We trace the defect to instability of Cdc42-mCherrySW and demonstrate that cell fusion requires higher Cdc42 protein levels than mitotic polarized growth. Remarkably, by constructing an allelic series driving Cdc42 expression over a 5-fold range, we discover that polarized growth responds linearly to Cdc42 protein levels, whereas sexual reproduction exhibits a sharp switch-like response. Thus, the topology of the Cdc42 regulatory network is distinct for its polarization and mating functions.

cell biology↗

bioRxiv2025

Lamin A/C Deficiency Drives Genomic Instability and Poor Survival in Small-Cell Lung Cancer through Increased R-loop Accumulation

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [1]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [2]. Here, we investigate LMNAs role in small-cell lung cancer (SCLC), a highly aggressive malignancy characterized by extreme genomic instability [3, 4]. We demonstrate that LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA loss disrupts nuclear pore complex distribution, reducing phenylalanine-glycine (FG)-nucleoporin incorporation and impairing RNA export efficiency. Furthermore, we show that LMNA expression is epigenetically repressed by EZH2 during SCLC differentiation from neuroendocrine (NE) to non-NE states. Clinically, low LMNA levels correlate with significantly worse survival in SCLC patients. These findings uncover a novel role for LMNA in safeguarding genome integrity and shaping tumor heterogeneity, with broad implications for cancer and aging. Significance StatementLamin A/C, a key structural component of the nuclear envelope, is frequently lost or mutated in cancer, laminopathies, and aging-related disorders. Lamin A/C loss is associated with genomic instability, but the underlying mechanisms remain incompletely understood. We demonstrate that LMNA loss drives genomic instability by promoting R-loop accumulation through disrupted nuclear pore dynamics and impaired RNA export. These findings reveal a previously unrecognized link between LMNA loss, nuclear envelope dysfunction, and genome instability. Targeting this pathway could help mitigate genomic instability in aging and laminopathies, while leveraging R-loop accumulation may enhance the efficacy of DNA-damaging therapies in cancer.

cancer biology↗

bioRxiv2025

A novel computational framework to identify translational potential of genetic mouse models in rare genetic obesity

Finding the right animal models has been a critical bottleneck in translational sciences of rare diseases. Traditional methods can be biased, limiting their effectiveness, and do not include cross-analysis with human manifestations of the diseases. This research introduces a novel computational framework that analyzes hundreds of mouse model data and phenotypes simultaneously. This paves the way to make the best disease-specific decisions. We have identified 106 mouse models in genetic obesity with more than 600 different phenotypes. An evaluation of the shared phenotypes across these models unearthed unexpected connections between obesity and 16 other diseases, opening doors for entirely new treatment avenues. This analytical framework has the potential to modernize how we research rare diseases, leading to more effective therapies for a wider range of conditions. Author SummaryA novel computational approach pinpoints ideal mouse models for rare diseases, uncovering hidden connections with other conditions.

bioinformatics↗

bioRxiv2025

Integrative plasma lipidomics and proteomics profiling to decipher potential biomarkers of dilated cardiomyopathy

BackgroundDilated cardiomyopathy (DCM), primarily characterised by left ventricular dilatation and systolic dysfunction, is one of the leading causes of heart failure and requires a critical clinical investigative strategy. However, conventional imaging techniques such as echocardiography and MRI, along with some classical CVD markers (NTproBNP, cTnT), fall short in diagnosing DCM-specific phenotypes. Thus, the need for biochemical markers with enhanced accuracy to DCM is of enormous importance. Lipids and proteins play essential roles in maintaining myocardial function. The homeostatic disruption of such biomolecules might contribute to DCM pathogenesis, thus offering to serve as potential biomarkers for DCM. Moreover, the lack of global lipidomics studies and specific protein markers in DCM patients prompted us to explore the disease pathophysiology through an integrative omics-based analysis coupled with an ML-derived approach. ObjectiveTo identify accurate, precise and specific circulatory lipidomic and proteomic biomarkers of DCM using high-resolution mass spectrometry and machine learning (ML)-based approaches. MethodsHigh-resolution-mass-spectrometry-based lipidomics and proteomics were applied to identify lipid and protein biomarkers in a cohort (n=360) of healthy and DCM patients. Top protein classifiers were further evaluated using single-cell transcriptomics on publicly available datasets from DCM myocardium and validated using ELISA. A biomarker panel was built by the integration of lipidomics and validated proteomics data using machine-learning-based approaches. ResultsA total of 125 lipids and 10 proteins have been primarily discovered with significant alterations in DCM (0.8 [≥] FC [≥] 1.2; padj < 0.05). Using a Boruta-based ML approach, we identified 39 lipids and 10 proteins as primary discriminators between DCM and controls. ELISA validation confirmed the potential of B2M (6.85 {+/-} 2.86 g/ml vs. 4.26 {+/-} 1.25 g/ml; p < 0.0001) and Tetranectin (CLEC3B) (1.99 {+/-} 0.88 g/ml vs. 2.49 {+/-} 0.90 g/ml; p = 0.0006) to emerge as protein biomarkers of DCM. In line with that, the single-cell transcriptomic analysis showed a similar trend of alteration of tetranectin (CLEC3B) in cardiomyocytes and {beta}2microglobulin (B2M) in varied cell types of the myocardium. Further, Integrated ROC analysis combining the top 8 lipid discriminators with B2M and CLEC3B achieved an AUC of 0.995, demonstrating enhanced diagnostic precision compared to the classical CVD marker NTproBNP (0.965). ConclusionThis study offers a system-omics-based perspective on first-global lipidomic and proteomic changes associated with DCM pathophysiology, with a high potential for diagnostic application.

systems biology↗

bioRxiv2025

Discovery and therapeutic exploitation of Master Regulatory miRNAs in Glioblastoma

Glioblastoma is a fatal primary malignant brain tumor, with an average survival of only 15 months despite surgical resection, chemotherapy, and radiation therapy. Due to the concurrent deregulation of numerous genes in glioblastoma, molecular monotherapies have not improved clinical outcomes. Evidence suggests that effectively targeting multiple deregulated molecules is essential for better therapies; however, this is limited by the lack of suitable drugs and the increased toxicity of combination therapies. To address this, we hypothesized that miRNAs, small gene-regulatory RNAs that suppress multiple target genes via sequence complementarity, could be developed to inhibit multiple deregulated genes simultaneously, leading to more effective treatments. We identified master regulatory miRNAs--those that target several deregulated genes in glioblastoma--using PAR-CLIP screenings in glioblastoma cells and analyzed TCGA tumor data to find which targets were deregulated. An algorithm ranked these targets based on their significance in glioblastoma malignancy. We selected two tumor suppressor master regulatory miRNAs, miR-340 and miR-382, and one oncogenic miRNA, miR-17. Validation showed that these miRNAs target critical glioblastoma pathways and significantly inhibit cell growth, survival, invasion, and tumor growth in vivo. We developed an innovative therapeutic delivery approach using Brain Penetrating Nanoparticles in combination with MRI-guided focused ultrasound and microbubbles, resulting in reduced tumor volume and extended survival in glioblastoma-bearing mice. This strategy offers a promising pathway for translating miRNA-based therapies into clinical trials for glioblastoma and other cancers. One Sentence SummaryWe developed and used new computational, experimental, and therapeutic approaches to identify and therapeutically deliver master regulatory miRNAs to inhibit the growth of glioblastoma, the most common and deadly primary brain tumor.

cancer biology↗

bioRxiv2025

Identification of genetic and non-genetic modifiers of genomic imprinting through whole genome screening in humans

Genomic imprinting is required for normal development, and abnormal methylation of differentially methylated regions (iDMRs) controlling the parent of origin-dependent expression of the imprinted genes has been found in constitutional disorders affecting growth, metabolism, neurobehavior, and in cancer. In most of these cases the cause of the imprinting abnormalities is unknown. Also, these studies have generally been performed on a limited number of CpGs, and a systematic investigation of iDMR methylation in the general population is lacking. By analysing the iDMRs in a large number of peripheral blood DNA methylation array datasets of unaffected individuals and patients with rare disorders, we determined the most common iDMR methylation profiles and identified many genetic and non-genetic factors contributing to their variability. We found that methylation variability is not homogeneous within the iDMRs and that the CpGs closer to the ZFP57 binding sites are less susceptible to methylation changes. We demonstrated the methylation polymorphism of three iDMRs and the atypical behaviour of several others, and reported the association of 25 disease- and 47 non-disease-complex traits, including blood cell type composition, as well as 15 mendelian or chromosomal disorders, with iDMR methylation changes in blood DNA. These findings identify several genetic and non-genetic factors associated with genomic imprinting maintenance in humans, which may have a role in the aetiology of the diseases associated with imprinting abnormalities and have clear implications in molecular diagnostics.

genetics↗

bioRxiv2025

Evaluation of Alphafold modeling for elucidation of nanobody-peptide epitope interactions

Models of Ab-antigen complexes can be used to understand interaction mechanisms and for improving affinity. This study evaluates the use of the protein structure prediction algorithm AlphaFold (AF) for exploration of interactions between peptide epitope tags and the smallest functional antibody fragments, nanobodies (Nbs). Although past studies of AF for modeling antibody-target (antigen) interactions suggested modest algorithm performance, those were primarily focused on Ab-protein interactions, while the performance and utility of AF for Nb-peptide interactions, which are generally less complex due to smaller antigens, smaller binding domains, and fewer chains, is less clear. In this study we evaluated the performance of AF for predicting the structures of Nbs bound to experimentally validated, linear, short peptide epitopes (Nb-tag pairs). We expanded the pool of experimental data available for comparison through crystallization and structural determination of a previously reported Nb-tag complex (Nb127). Models of Nb-tag pair structures generated from AF were variable with respect to consistency with experimental data, with good performance in just over half (4 out of 6) of cases. Even among Nb-tag pairs successfully modeled in isolation, efforts to translate modeling to more complex contexts failed, suggesting an underappreciated role of the size and complexity of inputs in AF modeling success. Finally, the model of a Nb-tag pair with minimal previous characterization was used to guide the design of a peptide-electrophile conjugate that undergoes covalent crosslinking with Nb upon binding. These findings highlight the utility of minimized antibody and antigen structures to maximize insights from AF modeling.

biochemistry↗

bioRxiv2025

Topoisomerase IIIb protects from tumorigenesis and immune dysregulation

Topoisomerase III-beta (Top3b) reduces nucleic acid torsional stress and intertwining generated during RNA and DNA metabolism while protecting the genome from pathological R-loops, which otherwise result in DNA breakage and genome instability. By studying Top3b knockout mice (Top3b-KO), we find that the loss of Top3b accelerates the development of spontaneous lymphoid tumors arising in spleens and lymph nodes, the organs with prominent Top3b expression. Aging Top3b-KO mice also display splenomegaly and systemic immune alterations including neutrophilia and lymphopenia suggestive of chronic inflammation. At the molecular level, Top3b deficiency causes genome-wide R-loop accumulation in splenocytes as measured by CUT&Tag sequencing. Increased R-loops is associated with genomic DNA breaks and activation of immune signaling pathways including the IL-6 signaling, interleukin-7 signaling and cGAS-STING. Moreover, knocking-out Top3b promotes the rapid development of syngeneic EL4 T-cell lymphomas. In conclusion, our work implies that, in addition to its role in preserving the nervous system, Top3b protects from tumorigenesis and immune dysregulations. SignificanceTopoisomerase III-beta (TOP3B) is the only dual RNA-DNA topoisomerase. Its inactivation has previously been shown to be critical for neurodevelopmental disorders in humans and R-loop suppression in cell line models. Here, we demonstrate that loss of Top3b accelerates spontaneous lymphomagenesis and promotes syngeneic lymphoma growth in association with innate immune defects and R-loop accumulation in Top3b-KO mice. These findings provide evidence for a previously unappreciated role of Top3b as a tumor suppressor gene and immune regulator.

cancer biology↗

bioRxiv2025

Synergistic RAS-MAPK and AKT Activation in MYC-Driven Tumors via Adjacent PVT1 Rearrangements

MYC-driven (MYC+) cancers are aggressive and often fatal. MYC dysregulation is a key event in these cancers, but overexpression of MYC alone is not always enough to cause cancer. Plasmocytoma Variant Translocation 1 (PVT1), a long non-coding RNA (lncRNA) adjacent to MYC on chromosome 8 is a rearrangement hotspot in many MYC+ cancers. In addition to being co-amplified with MYC, the genomic rearrangement at PVT1 involves translocation, which has had obscure functional consequences. We report that translocation at the PVT1 locus cause asymmetric enrichment of 5-PVT1 and loss of 3-PVT1. Despite being classified as a non-coding RNA, the retained 5 region of PVT1 generates a circular RNA (CircPVT1) that codes for the novel peptide we call Firefox (FFX). FFX augments AKT signaling and synergistically activates MYC and mTORC1 in these cells. Further, the 3 end of PVT1, which is lost during the translocation, codes for a tumor-suppressing micropeptide we named as Honeybadger (HNB). We demonstrate that HNB interacts with KRAS and disrupts the activation of KRAS effectors. Loss of HNB leads to activation of RAS/MAPK signaling pathway, and enhances MYC stability by promoting phosphorylation of MYC at Ser62. These findings identify PVT1 as a critical node that synchronizes MYC, AKT, and RAS-MAPK activities in cancer. Our study thus identifies a key mechanism by which rearrangements at the PVT1 locus activate additional oncogenic pathways that synergize with MYC to exacerbate the aggressiveness of MYC+ cancers. This newfound understanding explains the poor prognosis associated with MYC+ cancers and offers potential therapeutic targets that could be leveraged in treatment strategies for these cancers.

cancer biology↗

bioRxiv2025

Virus-against-virus dominant-negative interference strategy targeting a viral CC chemokine prevents cytomegalovirus-related neurodevelopmental pathogenesis

BackgroundCongenital cytomegalovirus (CMV) infections are one leading cause of human neurodevelopmental disorders. Increasing evidence for the pathogenic involvement of brain immune alterations was obtained in the recent years. Host and virus-encoded chemokines might play important roles in CMV-related neuropathogenesis by regulating leukocyte trafficking and microglia recruitment in the CMV-infected brains, and by interfering with key neurodevelopmental steps. In a rat model of CMV infection of the fetal brain in utero that leads to detrimental neurologic and other severe phenotypes postnatally, we reported on the early alteration of microglia and on the infiltration of the infected brains by lymphoid and myeloid cells. Particularly, expression of the r129 gene encoding the viral chemokine RCK3 was detected as early as 24h post-infection; together with the previously reported chemotaxis properties exerted by RCK3 on lymphocytes and on macrophages in vitro, this suggested that RCK3 might be involved in the brain immune cell alterations seen in the CMV-infected developing brains, and in the related neuropathogenesis. MethodsInfection of the rat fetal brain was done by intracerebroventricular injections in utero of either rat CMV encoding wild-type (wt) RCK3 (RCMV-wt), or a mutant CMV counterpart encoding RCK3 with a deletion in its chemokine domain (RCMV-r129{Delta}NT). As RCMV-r129{Delta}NT had shown dominant-negative effects on the chemotaxis properties of RCK3 in vitro, simultaneous and successive co-infection rescue assays were also performed. The detrimental postnatal phenotypes in vivo and the epileptiform activity ex vivo usually detected after infection of the rat developing brain with RCMV-wt were monitored in the RCMV-r129{Delta}NT condition and in co-infection assays. ResultsIn sharp contrast with RCMV-wt whose infection of the fetal brain led to decreased postnatal survival, impaired sensorimotor development, hindlimb hyperextension and epileptic seizures in neonatal pups, RCMV-r129{Delta}NT infection was not associated with any severe postnatal phenotype in vivo. Consistently, the epileptiform activity recorded in most neocortical slices from RCMV-wt-infected pups was not detected in any slice from RCMV-r129{Delta}NT-infected pups. Simultaneous co-infection assays led to dramatic prevention against the postnatal phenotypes in vivo and the altered network activity ex vivo, revealing a dose-dependent rescuing effect exerted by RCMV-r129{Delta}NT on RCMV-wt. Importantly, successful rescue was also obtained when the mutant RCMV-r129{Delta}NT was inoculated in the fetal brains either before or after infection with RCMV-wt. SignificanceOur data demonstrate the crucial neuropathogenic role of RCK3 CMV chemokine in vivo. The dramatic success and apparent safety of the dominant-negative RCK3 rescue assays in vivo provide a proof-of-principle for the beneficial use of a virus-against-virus approach against CMV-related pathogenesis.

neuroscience↗