Biology subjects

Ganesan, S.

Publications and source records attributed to Ganesan, S..

At least 19 recordsLinked to original sources

bioRxiv2026

A dsRNA Virus Converts Early Endosomes into Stable Replication Organelles Through Maturation Arrest and Retrograde Transport

Infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus (IPNV) are major animal pathogens that impose a heavy burden on the poultry and aquaculture industries. Yet, major aspects of their unique replication program remain unknown. IBDV is unusual among double-stranded RNA viruses in that it replicates in association with host endosomes. We previously demonstrated this association to be orchestrated by the viral protein VP3 and the early endosomal lipid phosphatidylinositol 3-phosphate [PtdIns(3)P], suggesting an essential role for VP3 in forming birnaviruses specialized replication compartments. Here, combining high-resolution fluorescence and correlative light-electron microscopy with diverse molecular and biochemical analyses, we define how the IBDV protein VP3 targets and remodels host endosomes, and establish that endosome-associated replication is conserved in members of the Birnaviridae family. We show that IBDV VP3 targets early endosomes via the PtdIns(3)P effector Early Endosomal Antigen 1 (EEA1) to arrest their maturation and prompt their association with the Golgi complex via ESCPE-1-mediated retrograde transport. We further show that IPNV also exploits endosomal membranes for replication in a PtdIns(3)P dependent manner, although for this virus VP3 engages endosomes through an EEA1-independent mechanism yet to be determined. This work uncovers how birnaviruses exploit a single viral protein to orchestrate endosome remodeling and intracellular trafficking to establish specialized replication compartments while revealing both conserved and host-adapted features of birnaviruses replication.

cell biology↗

bioRxiv2026

Identifying Putative Pathogenic Non-Coding Variants in Unresolved Rare Disease Patients Using Topologically Associated Domains

Unresolved rare disease is a major public health challenge affecting ~300 million people worldwide. At least 50% of these individuals remain genetically unresolved after applying exome sequencing and/or whole genome sequencing. One source of these missing diagnoses is the presence of rare variants within the non-coding genome that are detected but not interpreted by whole genome sequencing. In order to systematically evaluate candidate pathogenic non-coding variants, we created the Genomic Analysis of Variants in Unresolved Rare Disease (GAVURD) system. GAVURD leverages trio whole genome sequencing alignment data to produce a short list of putative pathogenic non-coding variants for a given proband. GAVURD uses best practices for de novo and rare inherited variant identification, links variants to human disease genes harnessing topologically associated domain (TAD) data, and rank prioritizes variants based on phenotypic overlap. As a proof-of-concept, we applied GAVURD to ten probands with unresolved rare disease and implicated six potentially causal non-coding variants based on a confluence of evidence supportive of pathogenicity. The GAVURD system serves an important role in prioritizing candidate non-coding causal variants for unresolved rare disease that can serve as the high value and informed focus of additional functional follow-up studies.

genomics↗

bioRxiv2026

A Comparative Chemical-Genetic Screen Reveals Divergent Roles for Host ALK2 Signaling Across Intracellular Bacterial Niches

Diverse vacuolar architectures present unique signaling challenges for intracellular bacteria, yet whether distinct niches share host requirements remains unclear. Because host kinases coordinate these complex cellular responses, we performed a comparative chemical-genetic screen of Coxiella burnetii, Chlamydia trachomatis, and Salmonella enterica. The screen revealed that C. burnetii is uniquely dependent on host kinase pathways. Deconvolution of these inhibitor profiles identified the bone morphogenetic protein (BMP) type I receptor, ALK2, as a critical regulatory checkpoint. While C. burnetii activates ALK2-mediated phospho-SMAD1/5/9 signaling to support replication, C. trachomatis actively suppresses this cascade. Mechanistically, ALK2 functions upstream of TFEB nuclear translocation and LAMP1 recruitment, driving the lysosomal biogenesis required to sustain the phagolysosome-like niche of C. burnetii while disrupting the non-lysosomal inclusion of C. trachomatis. Consequently, targeting ALK2 switches replication outcomes, demonstrating how distinct niches implement opposing strategies to exploit a single host signaling node.

microbiology↗

bioRxiv2026

Cross-talk between intracellular pathogen infection and IFNγ signaling drives enhanced pro-inflammatory responses

Many intracellular pathogens have evolved to evade immune responses and establish a secure niche inside host cells. One such stealth pathogen is the obligate intracellular bacterium Coxiella burnetii, the causative agent of Q-fever. Coxiella translocates an array of bacterial proteins ( effectors) into the host cell through a type IVB secretion system (T4BSS) that mediates suppression of pathogen sensing and innate immunity. Yet, at a systemic level, immunocompetent hosts often restrict pathogens through Th1-mediated and cell-autonomous immunity through the expression of immune-inducible genes. However, the expression and regulation of chemokines, particularly, the CXC-ligands (CXCL9,-10,-11) that are considered biomarkers of Q-fever, is poorly understood. We observed minimal to no CXCL10 transcript levels during Coxiella infection. However, Coxiella-infected cells robustly augmented IFN{gamma}-activated expression of CXCL10 in both phagocytic and non-phagocytic cells, and this process was dependent on viability and T4BSS in epithelial cells. This phenomenon extends to other highly pro-inflammatory cytokines and other pathogens including Salmonella, Mycobacteria (H37Ra) and Toxoplasma. Synergistic increase in CXCL10 expression in Coxiella-infected, IFN{gamma}-activated cells requires ISRE and NF-{kappa}B transcriptional elements in the promoter, and the transcription factors STAT1, STAT3 and IRF9. Inhibition of STAT3 by small molecule inhibitors potently decreased the excess promoter activity of CXCL10. In addition, treatment of Coxiella-infected cells with IFN{gamma} is associated with decreased expression of SOCS1, a negative regulator of the IFN{gamma} signaling axis and relatively higher detection of extracellular bacteria. Altogether, these data demonstrate that intracellular pathogens including those conventionally considered to be "immunologically silent", robustly synergize with IFN{gamma} signaling, with STAT3 activation emerging to be a nodal point for promoting both persistent infection as well as synergism in the expression of immune genes. Author summaryAcute host immune response is often associated with production of soluble messenger molecules called cytokines/chemokines which direct the migration, recruitment and activation of leukocytes and serve as biomarkers in infectious and inflammatory diseases. The regulation of expression of these molecules and their influence on the infection process is not well-understood. In particular, interferon-gamma (IFN{gamma}), a potent pro-inflammatory cytokine produced by activated T and NK cells, activates signaling pathways involved in host defense and inflammation in macrophages and other cell types. We observed that infection with many intracellular bacterial/parasitic pathogens that employ sophisticated immune evasion strategies, synergize with IFN{gamma} signaling and significantly amplify the levels of pro-inflammatory mediators implicating the origin of adverse immune pathologies. We investigated the mechanistic basis of this seemingly counter-intuitive phenomenon, underlying host and bacterial factors involved in distinct cell types, and identified the small molecule-targetable-transcription factor STAT3 as a host determinant in promoting excess cytokine synthesis.

immunology↗

bioRxiv2026

A Genome-wide Visual Screen Identifies Lysophosphatidylcholine as Counter Spatial Regulator of DAG and Sterols in Yeast

Membrane lipids are heterogeneously distributed across the bilayers of cellular membranes. Cytosolic-facing pools of diacylglycerol (DAG) in the yeast Saccharomyces cerevisiae are enriched at both ends of the endomembrane system from the vacuolar membrane to the polarized plasma membrane (PM) of buds. However, how this distribution is maintained remains unknown. Using a genome-wide DAG biosensor screen in yeast, we identify regulators of DAG spatial distribution, enriched in proteins involved in vesicle or lipid transport and in phospholipid or sterol metabolism. A subset of mutants exhibited DAG mislocalization predominantly to the PM, with the most severe phenotype linked to a mutant of a predicted lipase we have named Drl1 (DAG redistribution lipase 1). Reversion of this phenotype required both enzymatic activity and the presence of an intrinsically disordered carboxy-terminal domain. Lipidomic analysis revealed that loss of Drl1 increased cellular lysophosphatidylcholine (LysoPC) levels. Remarkably, we find that supplementing cells with a non-metabolizable LysoPC analogue replicated the mutant DAG phenotype, implicating LysoPC as a novel spatial regulator of DAG. High-resolution imaging suggests that LysoPC reduces the PM sterol pool resulting in DAG expansion into new PM territories. More globally, our work expands the known interplay between various lipids and their co-regulation to maintain accurate membrane properties.

cell biology↗

bioRxiv2025

Monoallelic expression characterizes a distinct molecular and clinical group of breast tumors

In diploid cells, allelic imbalance occurs when gene alleles are expressed at different levels. To investigate the allelic imbalance landscape in tumor samples, we developed Interval-Based Allelic Imbalance Detection (IB-Aid), a quantitative framework that uses interval arithmetic to robustly distinguish monoallelic from biallelic gene expression by computing confidence intervals based on sequencing measurement uncertainty. We applied this approach to The Cancer Genome Atlas Breast Invasive Carcinoma tumor samples and through unsupervised gene enrichment analyses, identified a group of patients with a distinct monoallelic gene expression signature. Notably, these patients were enriched in Black/African American patients and had tumors which had not been previously classified into any established molecular subtype. Clinically, these tumors were associated with poor overall survival, with survival outcomes comparable to the aggressive basal subtype. These findings suggest a potential link between allelic imbalance and breast cancer development and point to genetic and epigenetic mechanisms that drive allelic imbalance as novel biomarkers for prognosis and design of targeted treatment strategies.

cancer biology↗

bioRxiv2025

Environmental Regulation of Toxin Production in Bacillus anthracis

Pathogenic Bacillus anthracis strains carry two plasmids - pX01, which encodes a tripartite protein exotoxin complex (PA, LF, and EF); and pX02, which encodes a poly-D-gamma-glutamic acid capsule. A multidomain transcription factor, AtxA, regulates the expression of these virulence genes. AtxA has two DNA-binding Helix-Turn-Helix (HTH) domains, two phosphoenolpyruvate:carbohydrate phosphotransferase system regulatory domains (PRD1 and PRD2), and a putative EIIB domain (a component of PTS sugar transport EII-complexes). Previous studies showed that glucose and CO2 increase AtxA-dependent toxin gene transcription, along with histidine phosphorylation of PRD1 and PRD2. Our transcriptional profiling of virulence factors, PA secretion, and fluorescent reporter strain analyses confirms a synergistic effect of glucose and CO2 on AtxA-dependent toxin production. Deletion of AtxA ({Delta}atxA) significantly reduced glucose uptake in bacteria, suggesting that AtxA may act within the glucose-PTS system. Mutation analysis of the EIIB domain of AtxA identified the cysteine at position 402 as essential for the transcriptional activity of AtxA. Deletion of glucose PTS permease PtsG ({Delta}ptsG) significantly reduced the expression of PA, LF, and EF. Loss of PtsG also caused attenuation in a mouse model of infection. Intracellular imaging using FLIM confirms a physical interaction of PtsG and AtxA through EIIB domain of AtxA. Using phosphomimetic and phosphoablative mutants of AtxA, we confirmed that the physical interaction of PtsG and AtxA is essential for AtxA activity. Finally, the synergy between glucose and CO2 was targeted by deleting pyruvate carboxylase Pyc ({Delta}pyc), which regulates anaplerosis. This deletion confirms that Pyc stimulates the level of phosphoenolpyruvate (PEP) and increases the phosphorelay in glucose-PTS to enhance AtxA activity. Therefore, we propose that a histidine-phosphorelay from PEP regulates AtxA via PTS enzymatic activity, impacting AtxA activity through physical interaction of AtxA and PtsG. Finally, we propose AtxA as an integral component of the glucose-PTS, where transcriptional activity of AtxA is regulated by environmental signals including glucose and CO2.

molecular biology↗

bioRxiv2025

Exhausted-like effector CD8 T cells mediate immune-stromal interactions at mucosal Chronic Graft-versus-Host Disease onset

Chronic graft-versus-host disease (cGVHD) frequently follows allogeneic hematopoietic stem cell transplantation and impacts the mucosa. This atlas study highlights the role of exhausted-like effector CD8+ T cells (TEXEF) in oral mucosal cGVHD pathogenesis. Biopsies from the oral mucosa were collected at oral cGVHD onset or at six months post-transplant and analyzed using single-cell RNA sequencing and other modalities. At cGVHD onset, in addition to changes in the myeloid compartment, two distinct populations of CD8+T cells with a TEXEF phenotype were prevalent: an inflammatory CD8T cell cluster (CD8T_4) expressing CCL4, CD69, and TNFSF9, and an exhausted CD8T cell cluster (CD8T_5) characterized by the expression of CXCL13, HIF1A, and HLA-DRA, both correlated with clinical disease severity. Pseudo-time analysis suggests a transition from the inflammatory pre-exhausted CD8T_4 cluster to the cytolytic, terminally exhausted CD8T_5 cluster. The CXCL9-CXCR3 interactions among CD8+ T cells, fibroblasts and myeloid cells are implicated in tissue damage. Onset of cGVHD despite frequent oral mucosal FOXP3 regulatory CD4T cells suggests potential impaired Treg function or CD8+T cell dysfunction. These findings highlight TEXEF cells as key contributors to oral cGVHD pathology and potential therapeutic targets for mitigating sustained tissue injury in cGVHD.

immunology↗

bioRxiv2025

Sorafenib, a clinically approved kinase inhibitor attenuates Streptococcus pneumoniae pathogenesis in vivo by targeting serine/threonine kinase StkP

Streptococcus pneumoniae is a respiratory commensal bacterium responsible for over one million annual fatalities globally, particularly among children under five years of age. The rapid emergence of macrolide-resistant strains led the WHO in 2024 to designate S. pneumoniae as a priority pathogen, underscoring the need for alternative strategies such as anti-virulence therapy. Here, we repurposed the FDA-approved cancer drug, sorafenib identified by in silico screening of compounds targeting the bacterial Serine/Threonine kinase protein StkP, an essential regulator of cell division and peptidoglycan synthesis conserved across many bacterial pathogens. Sorafenib interacts with the StkP-kinase domain and exhibited broad-spectrum activity against diverse pneumococcal serotypes including multi-drug-resistant clinical isolates. Ectopic expression of StkP in both wild-type and isogenic mutant strains conferred partial resistance to sorafenib, confirming on-target activity. Scanning electron microscopy revealed aberrant cell-wall morphology, and differential viability staining demonstrated increased membrane permeability. Consistently, sorafenib-treated bacteria showed significantly higher complement C3 deposition and consequent killing in human serum. In human lung epithelial cells, sorafenib reduced bacterial adherence and invasion without detectable host cytotoxicity. Serial passaging at sub-microbicidal concentrations indicated a low propensity for resistance development in vitro. In vivo, sorafenib administration reduced mortality and lung bacterial load in a mouse model of pneumonia. Taken together, our data identify StkP as target of sorafenib in S. pneumoniae and justify its continued preclinical development for therapeutic intervention.

microbiology↗

bioRxiv2025

Fibroblast depletion reveals mammalian epithelial resilience across neonatal and adult stages

Regenerative organs, like the skin, depend on niche-stem cell interactions that sustain continuous cellular turnover. In cell culture, skin fibroblasts promote epidermal stem cell proliferation and differentiation. Yet, it remains elusive how fibroblasts regulate epidermal stem cell behaviors and differentiation in skin in vivo. Here, we asked how fibroblast depletion may impact epidermal stem cell proliferation in the context of adult homeostasis. Surprisingly, we find that significant depletion of fibroblast density does not affect epidermal stem cell proliferative capacity during adult stages in vivo. We next probed earlier neonatal stages when skin is actively remodeling but found no change in epidermal stem cell proliferative capacity following fibroblast depletion. These results demonstrate that across different ages, epidermal stem cell proliferative capacity can persist in the face of a largely reduced fibroblast population. Interestingly, neonatal fibroblast depletion does not significantly reduce their secreted collagen I density but affects basement membrane mechanics and epidermal stem cell delamination. Despite these changes, the skin continues to maintain its protective barrier function. Thus, our work demonstrates the skin regenerative program employs robust compensatory mechanisms in the face of fibroblast depletion to maintain functional capacity.

cell biology↗

bioRxiv2025

A novel AAA+ ATPase required for sporulation and stress response in Bacillus anthracis

AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase BAS PrkA in B. anthracis Sterne 34F2 which has 88 % protein homology to Bacillus subtilis PrkA. Conserved domain analysis confirms BAS PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on Alpha Fold3 predicted structure, we classified BAS PrkA as part of Clade III of the AAA+ superfamily. Contrary to the reported enzymatic activity in B. subtilis PrkA, we observed that BAS PrkA has negligible protease and kinase activity under in-vitro conditions. Nonetheless, BAS PrkA plays a significant role in regulating sporulation. It is temporally expressed during Stages II to VI during sporulation. A null mutant of BAS PrkA exhibits severe sporulation defects, with reduced spore viability, and down regulation of genes related to spore-coat formation. These phenotypes were restored in a complementation strain expressing BAS PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions. Analysis of the BAS PrkA interactome revealed enrichment of two proteins, ProA and EzrA, that are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BAS PrkA plays a critical role in sporulation and osmotic stress response in B. anthracis.

microbiology↗

bioRxiv2025

The intracellular vacuolar pathogen Coxiella burnetii promotes exocytosis in a type IVB secretion system dependent manner

Coxiella burnetii is an obligate intracellular bacterial pathogen that causes a zoonotic disease known as Q fever. Upon internalization into host cells, Coxiella extensively remodels host vesicle trafficking and replicates within acidic, lysosome-derived, spacious vacuoles by secreting a suite of effectors through the type IVB secretion system (T4BSS), and hijacking the host cell machinery. However, how fundamental lysosomal functions, such as exocytosis, are subverted during Coxiella infection has not been well understood. In this study, we aimed to investigate the regulation and role of exocytosis in the context of Coxiella infection. Biochemical and fluorescence-based imaging approaches indicated that C. burnetii infection promotes release of extracellular vesicles (EVs). Increase in extracellular levels of endolysosomal proteins (LAMP1 and cathepsin D) and surface LAMP1 expression were identified in both phagocytic and non-phagocytic cells, during later stages of infection. Interestingly, infection-induced exocytosis was dependent on the activity of the bacterial T4BSS. Modulating the activity of TRPML1, a host calcium channel that induces exocytosis by regulating the release of calcium from lysosomes into the cytosol, using synthetic agonist/antagonist, led to a decrease in intracellular C. burnetii replication, suggesting a complex, tightly regulated role of TRPML1 during infection. Together, this study demonstrates that infection by the lysosome-adapted pathogen Coxiella burnetii activates exocytosis in a temporal, host and bacterial factors-dependent manner.

cell biology↗

bioRxiv2025

Phenotypic analysis of 11,125 trio exomes in neurodevelopmental disorders

Genomic sequencing is widely used to identify causative genetic changes in neurodevelopmental disorders, such as autism, intellectual disability, and epilepsy. Most neurodevelopmental disorders also present with diverse clinical features, and delineating the interaction between causative genetic changes and phenotypic features is a key prerequisite for developing personalized therapies. However, assessing clinical features at a scale that parallels genomic sequencing remains challenging. Here, we standardize phenotypic information across 11,125 patient-parent trios with exome sequencing data using biomedical ontologies, analyzing 674,767 phenotypic terms. We find that individuals with de novo variants in 69 out of 261 neurodevelopmental genes exhibit statistically significant clinical similarities with distinct phenotypic fingerprints. We also observe that phenotypic relatedness follows a gradient, spanning from highly similar to dissimilar phenotypes, with intra-gene similarities suggesting clinically distinct subgroups for seven neurodevelopmental genes. For most genetic etiologies, only a small subset of highly phenotypically similar individuals carried de novo variants in the same gene, highlighting the heterogeneous and complex clinical landscape of neurodevelopmental disorders. Our study provides a large-scale overview of the dynamic relationship between genotypes and phenotypes in neurodevelopmental disorders, underscoring how the inherent complexity of these conditions can be deciphered through approaches that integrate genomic and phenotypic data.

genetics↗

bioRxiv2025

Single-cell mapping of regulatory DNA:Protein interactions

Gene expression is coordinated by a multitude of transcription factors (TFs), whose binding to the genome is directed through multiple interconnected epigenetic signals, including chromatin accessibility and histone modifications. These complex networks have been shown to be disrupted during aging, disease, and cancer. However, profiling these networks across diverse cell types and states has been limited due to the technical constraints of existing methods for mapping DNA:Protein interactions in single cells. As a result, a critical gap remains in understanding where TFs or other chromatin remodelers bind to DNA and how these interactions are perturbed in pathological contexts. To address this challenge, we developed a transformative single-cell immuno-tethering DNA:Protein mapping technology. By coupling a species-specific antibody-binding nanobody to a cytosine base editing enzyme, this approach enables profiling of even weak or transient factor binding to DNA, a task that was previously unachievable in single cells. Thus, our Docking & Deamination followed by sequencing (D&D-seq) technique induces cytosine-to-uracil edits in genomic regions bound by the target protein, offering a novel means to capture DNA:Protein interactions with unprecedented resolution. Importantly, this technique can be seamlessly incorporated into common single-cell multiomics workflows, enabling multimodal analysis of gene regulation in single cells. We tested the ability of D&D-seq to record TF binding both in bulk and at the single-cell level by profiling CTCF and GATA family members, obtaining high specificity and efficiency, with clear identification of TF footprint and signal retention in the targeted cell subpopulations. Furthermore, the deamination reaction showed minimal off-target activity, with high concordance to bulk ChIP-seq reference data. Applied to primary human peripheral blood mononuclear cells (PBMCs), D&D-seq successfully identified CTCF binding sites and enabled integration with advanced machine-learning algorithms for predicting 3D chromatin structure. Furthermore, we integrated D&D-seq with single-cell genotyping to assess the impact of IDH2 mutations on CTCF binding in a human clonal hematopoiesis sample, uncovering altered binding and chromatin co-accessibility patterns in mutant cells. Altogether, D&D-seq represents an important technological advance enabling the direct mapping of TF or chromatin remodeler binding to the DNA in primary human samples, opening new avenues for understanding chromatin and transcriptional regulation in health and disease.

genomics↗

bioRxiv2024

High-precision neurofeedback-guided meditation training optimises real-world self-guided meditation practice for well-being

Meditation can benefit well-being and mental health, but novices often struggle to effectively recognize and disengage from mental processes during meditation due to limited awareness, potentially diminishing meditations benefits. We investigated whether personalised high-precision neurofeedback (NF) can improve disengagement from mental activity during meditation and enhance meditations outcomes. In a single-blind, controlled, longitudinal paradigm, 40 novice meditators underwent two consecutive days of meditation training with intermittent visual feedback from either their own (N=20) or a matched participants (N=20; control group) posterior cingulate cortex (PCC) activity measured using 7 Tesla functional magnetic resonance imaging. During training, the experimental group showed stronger functional decoupling of PCC from dorsolateral prefrontal cortex, indicating better control over disengagement from mental processes during meditation. This led to greater improvements in emotional well-being and mindful awareness of mental processes during a week of real-world self-guided meditation. We provide compelling evidence supporting the utility of high-precision NF-guided meditation training to optimise real-world meditation practice for well-being.

neuroscience↗

bioRxiv2024

Kupffer cell and recruited macrophage heterogeneity orchestrate granuloma maturation and hepatic immunity in visceral leishmaniasis

In murine models of visceral leishmaniasis (VL), parasitization of resident Kupffer cells (resKCs) is responsible for early growth of Leishmania infantum in the liver, which leads to granuloma formation and eventual parasite control. We employed the chronic VL model, and revealed an open niche established by KCs death and their migration outside of the sinusoids, resulting in their gradual replacement by monocyte-derived KCs (moKCs). While early granulomas were composed of resKCs, late granulomas were found outside of the sinusoids and contained resKC-derived macrophages, and monocyte-derived macrophages (momacs). ResKCs and moKCs within the sinusoids were identified as homeostatic/regulatory cells, while resKC-derived macrophages and momacs within late granulomas were pro-inflammatory. Despite the infection being largely confined to the resKC-derived macrophages, in the absence of monocyte recruitment, parasite control was strongly compromised. Macrophage heterogeneity, involving migration and reprogramming of resKCs, along with recruitment of monocyte-derived cells, is a hallmark of granuloma maturation and hepatic immunity in VL.

immunology↗

bioRxiv2024

Immune Checkpoint Blockade Delays Cancer and Extends Survival in Murine DNA Polymerase Mutator Syndromes

Mutations in polymerases Pold1 and Pole exonuclease domains in humans are associated with increased cancer incidence, elevated tumor mutation burden (TMB) and response to immune checkpoint blockade (ICB). Although ICB is approved for treatment of several cancers, not all tumors with elevated TMB respond. Here we generated Pold1 and Pole proofreading mutator mice and show that ICB treatment of mice with high TMB tumors did not improve survival as only a subset of tumors responded. Similarly, introducing the mutator alleles into mice with Kras/p53 lung cancer did not improve survival, however, passaging mutator tumor cells in vitro without immune editing caused rejection in immune-competent hosts, demonstrating the efficiency by which cells with antigenic mutations are eliminated. Finally, ICB treatment of mutator mice earlier, before observable tumors delayed cancer onset, improved survival, and selected for tumors without aneuploidy, suggesting the use of ICB in individuals at high risk for cancer prevention. HighlightsO_LIGermline somatic and conditional Pold1 and Pole exonuclease domain mutations in mice produce a mutator phenotype. C_LIO_LISpontaneous cancers arise in mutator mice that have genomic features comparable to human tumors with these mutations. C_LIO_LIICB treatment of mutator mice with tumors did not improve survival as only a subset of tumors respond. C_LIO_LIIntroduction of the mutator alleles into an autochthonous mouse lung cancer model also did not produce immunogenic tumors, whereas passaging mutator tumor cells in vitro caused immune rejection indicating efficient selection against antigenic mutations in vivo. C_LIO_LIProphylactic ICB treatment delayed cancer onset, improved survival, and selected for tumors with no aneuploidy. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/597960v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@6cd159org.highwire.dtl.DTLVardef@243fd1org.highwire.dtl.DTLVardef@1d9da6borg.highwire.dtl.DTLVardef@192be51_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

RESPIRATION DEFECTS LIMIT SERINE SYNTHESIS REQUIRED FOR LUNG CANCER GROWTH AND SURVIVAL

Mitochondrial function is important for both energetic and anabolic metabolism. Pathogenic mitochondrial DNA (mtDNA) mutations directly impact these functions, resulting in the detrimental consequences seen in human mitochondrial diseases. The role of pathogenic mtDNA mutations in human cancers is less clear; while pathogenic mtDNA mutations are observed in some cancer types, they are almost absent in others. We report here that the proofreading mutant DNA polymerase gamma (PolGD256A) induced a high mtDNA mutation burden in non-small-cell lung cancer (NSCLC), and promoted the accumulation of defective mitochondria, which is responsible for decreased tumor cell proliferation and viability and increased cancer survival. In NSCLC cells, pathogenic mtDNA mutations increased glycolysis and caused dependence on glucose. The glucose dependency sustained mitochondrial energetics but at the cost of a decreased NAD+/NADH ratio that inhibited de novo serine synthesis. Insufficient serine synthesis, in turn, impaired the downstream synthesis of GSH and nucleotides, leading to impaired tumor growth that increased cancer survival. Unlike tumors with intact mitochondrial function, NSCLC with pathogenic mtDNA mutations were sensitive to dietary serine and glycine deprivation. Thus, mitochondrial function in NSCLC is required specifically to sustain sufficient serine synthesis for nucleotide production and redox homeostasis to support tumor growth, explaining why these cancers preserve functional mtDNA. In briefHigh mtDNA mutation burden in non-small-cell lung cancer (NSCLC) leads to the accumulation of respiration-defective mitochondria and dependency on glucose and glycolytic metabolism. Defective respiratory metabolism causes a massive accumulation of cytosolic nicotinamide adenine dinucleotide + hydrogen (NADH), which impedes serine synthesis and, thereby, glutathione (GSH) and nucleotide synthesis, leading to impaired tumor growth and increased survival. HighlightsO_LIProofreading mutations in Polymerase gamma led to a high burden of mitochondrial DNA mutations, promoting the accumulation of mitochondria with respiratory defects in NSCLC. C_LIO_LIDefective respiration led to reduced proliferation and viability of NSCLC cells increasing survival to cancer. C_LIO_LIDefective respiration caused glucose dependency to fuel elevated glycolysis. C_LIO_LIAltered glucose metabolism is associated with high NADH that limits serine synthesis, leading to impaired GSH and nucleotide production. C_LIO_LIMitochondrial respiration defects sensitize NSCLC to dietary serine/glycine starvation, further increasing survival. C_LI O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/596339v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@3dac07org.highwire.dtl.DTLVardef@e93401org.highwire.dtl.DTLVardef@555c48org.highwire.dtl.DTLVardef@1d7d31_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗