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Results for “systems biology”

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bioRxiv2026

Elucidating the functional domain architecture of ArCS1, a biomineralizing myosin chitin synthase: I. The role of lipids

In molluscs, chitin synthases are essential for biologically controlled biomineralization, with some variants possessing a myosin motor domain that may link polymer synthesis to the cytoskeleton. Experimentally, we established a reliable workflow for expressing ArCS1_E22TM in Dictyostelium discoideum and developed effective purification methods to reconstitute ArCS1_E22TM in nanodiscs using MSPs and specific lipid composition. MSP1D1deltaH5 proved optimal for nanodisc formation, yielding homogeneous, monodisperse discs (~8.2 nm). Lipids were refined to POPC:POPE:POPG (3:1:1) with 20% cholesterol, improving nanodisc quality and uniformity as observed by negative-stain EM. The full-length ArCS1 and its subdomains were modeled using AlphaFold3; the myosin motor, glycosyltransferase, and transmembrane regions are well-defined internally but loosely constrained relative to one another, suggesting flexible linking and conformational coupling. Modelling with Mg2+ and oleic acid as ligands and comparative analyses with bacterial cellulose synthase and yeast chitin synthase 1 provided insights into substrate binding and a potential mechanism for chitin polymerization and translocation. This research establishes a standard procedure for comprehensive structural analyses of recombinant molluscan chitin synthase in near-native or biomimetic membranes. This sets the stage for high-resolution cryo-electron microscopy to determine the first experimentally resolved structure of a molluscan chitin synthase and to provide insight into the enzyme's architecture and the regulatory mechanisms of biomineralization.

molecular biology↗

bioRxiv2026

Dissecting the TMEM132A-EGFR Dependency to Unlock Translational Therapeutic Opportunities for Pan-Solid Tumor

Solid tumors remain refractory to conventional treatments, yet cell surface proteins, by virtue of their extracellular accessibility and critical roles in tumor signaling, represent an attractive class of targets for precision-targeted therapy. Here, we report that TMEM132A is an essential and previously unrecognized pan-cancer target. TMEM132A interacts directly with EGFR and stabilizes its expression, thereby tethering EGFR at the plasma membrane and sustaining constitutive activation of lipid synthesis. Mechanistically, the TMEM132A-EGFR axis promotes lipogenesis by facilitating SREBP nuclear translocation, which in turn upregulates ACLY and ACSS2 expression to drive acetyl-CoA production and downstream lipid biosynthesis, ultimately disrupting lipid droplet homeostasis. To therapeutically target this axis, we developed a nanobody, LFNanoT132A#3, which effectively blocks the TMEM132A-EGFR interaction, abrogates downstream signaling activation, and potently inhibits proliferation across multiple solid tumor types. Notably, LFNanoT132A also exerts robust antitumor activity against H1975 xenografts, a model resistant to first- and second- generation EGFR inhibitors, underscoring its potential to overcome conventional drug resistance. Our findings establish TMEM132A#3 as a critical node in membrane-tethered oncogenic signaling and metabolic rewiring, and position LFNanoT132A#3 as a promising therapeutic candidate for precision cancer therapy.

cancer biology↗

bioRxiv2026

DIFFERENTIAL PHOTOSYNTHETIC RESPONSES TO GLUFOSINATE AMMONIUM IN TWO GRASS WEEDS: Lolium multiflorum AND Echinochloa crus-galli.

Background: Weed control is one of the main challenges in agriculture today, particularly due to the increasing occurrence of herbicide-resistant populations. Among the most problematic species are Lolium multiflorum (L.) and Echinochloa crus-galli (L.) Beauv., for which glyphosate-resistant populations have been reported. In this context, glufosinate ammonium has emerged as an alternative for their control; however, its efficacy may vary depending on species and photosynthetic metabolism. Objective: The objective of this study was to evaluate the differential sensitivity of ryegrass (C3) and barnyardgrass (C4) to ammonium glufosinate by analyzing physiological responses associated with leaf senescence and photosystem II activity. Methods: Visual injury, chlorophyll fluorescence, and ammonium accumulation were assessed. Results: Results revealed a differential response between species. Barnyardgrass exhibited earlier symptom onset and a greater reduction in the quantum yield of photosystem II ({Phi}PSII), whereas ryegrass showed a slower senescence process. These differences indicate a higher sensitivity of barnyardgrass to glufosinate ammonium, possibly associated with its C4 photosynthetic metabolism. Conclusions: It is concluded that the effectiveness of glufosinate ammonium depends on the type of photosynthetic metabolism and on the ability of each species to cope with herbicide-induced oxidative stress. This information contributes to optimizing glufosinate ammonium use and to the development of management strategies aimed at delaying the evolution of herbicide resistance.

plant biology↗

bioRxiv2026

POU2AF2/OCA-T1 coactivates POU2F2 and defines a lineage-specific dependency in diffuse large B-cell lymphoma

Lineage-restricted transcriptional programs establish cell identity and can create selective dependencies in cancer. Here, we identify POU2AF2, encoding the transcriptional co-activator OCA-T1, as a critical lineage-specific dependency in a subset of diffuse large B-cell lymphoma (DLBCL). Pan-cancer dependency analyses and patient cohorts reveal elevated POU2AF2 expression in genetically aggressive DLBCL, where its depletion markedly suppresses tumor growth in vitro and in vivo. Mechanistically, POU2AF2 cooperates with the B-cell lineage-defining transcription factor POU2F2 (OCT2) to activate lymphocyte activation gene programs through direct chromatin engagement, thereby sustaining malignant transcriptional networks. We further identified a key epigenetic regulatory axis composed of the lineage-specific transcription factor TCF3 and the histone methyltransferase SET1A-COMPASS that drives POU2AF2 expression downstream of B-cell receptor signaling. Single-cell transcriptomic analysis reveals that POU2AF2 marks and sustains an innate-like B1 B-cell population in vivo, a candidate cell of origin for lymphoma. Together, these findings define a lineage-restricted POU2AF2/POU2F2 transcriptional module, controlled by a TCF3/SET1A epigenetic network, that sustains both innate-like B-cell identity and malignant fitness in DLBCL. Our study uncovers a previously unrecognized lineage-specific transcriptional dependency and highlights POU2AF2 and its associated regulatory circuitry as potential therapeutic targets in aggressive B-cell malignancies.

cell biology↗

bioRxiv2026

Dynamic coupling of cell fate specification and cell sorting during mouse preimplantation development

During preimplantation development in mice, cells of the inner cell mass undergo a cell fate decision to become either Epiblast (Epi) or Primitive Endoderm (PrE) cells. Cell fate patterns during this stage range from an alternating pattern at the beginning to the separation of Epi and PrE at the end. Several mechanisms guiding this decision and pattern formation have been proposed, including intra- and intercellular signalling, cell division and cell sorting. The current understanding is that signalling generates the cell fates and subsequent sorting introduces the spatial cell fate separation. We used agent-based modelling to investigate whether cell differentiation and cell sorting can act concurrently and how their relative contributions to pattern formation may change over time. Comparing our model to experimental data for mouse blastocysts and ICM organoids, we find two mechanistic regimes that can produce the experimentally observed spatial separation: (i) simultaneous long-range intercellular signalling and cell sorting, and (ii) a gradual transition from short-range signalling to cell sorting, in which the timing is mediated via reducing cell fate plasticity. While the second agrees better with existing experimental evidence for late blastocysts, the first might still be relevant for early and mid blastocysts. Together, our results refine the sequential view of Epi/PrE patterning by showing that fate specification and cell sorting can be dynamically coupled, with their relative contributions changing over the course of blastocyst development.

developmental biology↗

bioRxiv2026

Arterial Elastin Abundance, Rather Than Orthologue Origin, Modulates Medial Arterial Calcification in Matrix Gla Protein-Deficient Mice

Abstract Calcific deposits in the arterial media have been associated with a number of metabolic and genetic disorders including diabetes, chronic kidney disease and generalized arterial calcification of infancy. While medial calcification and physiologic hard tissue mineralization in the skeleton are both regulated by several common determinants, emerging data suggest that there might be fundamental differences in the mechanisms underlying these two processes. Objective: We previously demonstrated that elastin haploinsufficiency delays medial calcification in MGP-deficient mice. Here, using mice in which a human ELN transgene rescues mouse elastin deficiency, we investigated whether the origin and abundance of arterial elastin differentially affect the initiation and progression of medial calcification. Approach and Results: We pursued a transgenic approach to alter the arterial elastin scaffold in MGP-deficient mice. Our analyses of a humanized MGP-deficient model with 40% reduction of medial elastin content showed a complete absence of the early-stage vascular calcification. Additionally, we showed that mouse and human elastin orthologues affect vascular calcification in a comparable manner. Conclusion: Arterial elastin abundance, rather than orthologue origin, modulates the initiation and progression of medial calcification in MGP-deficient mice. A further reduction in arterial elastin beyond that achieved by elastin haploinsufficiency profoundly delays mineral deposition and maturation, whereas restoration of elastin abundance through transgenic human ELN expression restores arterial calcification.

cell biology↗

bioRxiv2026

Highly plastic macrophage niches orchestrate acquired quiescence and reactivation in breast-cancer bone metastasis

Recurrence and metastasis remain major causes of cancer mortality, sustained by therapy-resistant micrometastatic cells. Bone is a frequent site of breast-cancer relapse, yet the cues that reawaken disseminated cells remain poorly defined. We identify a previously unrecognized, highly plastic CXCL16 macrophage population that integrates tumor-associated macrophage programs found in distant metastatic sites such as lung and brain with non-tumor disease-associated traits in bone marrow. These CXCL16 macrophages establish a transient niche that restrains disseminated cancer-cell proliferation. Single-cell transcriptomics delineate functional remodeling of myeloid niches within the bone metastatic microenvironment: a CXCL16 macrophage niche that transiently constrains metastatic growth, and G-CSF macrophage and neutrophil niches that reignite tumor outgrowth. In primary tumors, cancer-associated fibroblasts (CAFs) aberrantly secrete G-CSF in response to cancer-cell signals, expanding G-CSF-receptor-positive subset of cancer cells with high metastatic potential. In advanced human bone metastases, CXCL16 macrophages localize to CAF-rich stroma but are excluded from cancer-cell clusters, indicating immune evasion. Together, these findings uncover CAF-bone-marrow cross-talk as a therapeutic target linking stromal inflammation, immune remodeling, and metastatic progression.

cancer biology↗

bioRxiv2026

miR-34/449 miRNAs regulate choroid plexus ciliogenesis to control cerebrospinal fluid production

A developmental increase in cerebrospinal fluid (CSF) production during development is essential for neuronal growth and ventricular expansion. A key regulator of CSF production is the specialized sensory multicilia of the choroid plexus (ChP), which mediate non-canonical Sonic hedgehog (Shh) signaling to suppress water channel and ion transporter expression, thereby limiting CSF production. ChP multicilia progressively shortens during development, attenuating Shh signaling and promoting CSF production. Here, we identify miR-34/449 miRNAs as essential regulators of ChP multiciliogenesis. Whereas mutations in canonical ciliogenesis genes elevate CSF production and contribute to hydrocephaly, deletion of miR-34/449 reduces CSF volume and causes microcephaly. Loss of miR-34/449 miRNAs causes excessive basal body amplification, defective basal body docking, and failure of developmental multiciliary shortening. Consequently, miR-34/449-deficient ChP cilia remain abnormally long and fail to attenuate Shh signaling, resulting in sustained repression of water channel and ion transporter expression and reduced CSF production. Mechanistically, miR-34/449 miRNAs directly target Gmnc, a master transcriptional regulator of multiciliogenesis, to restrain basal body amplification and promote basal body docking. Together, our findings identify miR-34/449 miRNAs as critical regulators of ChP multiciliogenesis and establish the developmental remodeling of ChP multicilia as a mechanism to couple Shh signaling dynamics to developmental control of CSF production.

developmental biology↗

bioRxiv2026

VITAL-3D: Volumetric Single-Cell Quantification Reveals Microenvironment-Dependent Drug Responses in Breast Cancer

Preclinical drug evaluation relies heavily on two-dimensional (2D) monolayer assays, which fail to recapitulate the structural and functional complexity of the tumor microenvironment and may therefore misrepresent therapeutic efficacy. Here, we present VITAL (Volumetric Imaging-based Toxicity and Live Analysis), a high-throughput imaging platform that enables direct single-cell quantification of proliferation and cell death in both 2D and three-dimensional (3D) extracellular matrix (ECM) cultures using a 96-well format. By combining volumetric imaging with automated single-cell analysis, VITAL enables dynamic assessment of drug responses beyond conventional viability assays and EC measurements. Using breast cancer cell lines treated with anticancer agents, we systematically compared drug responses between 2D and 3D microenvironments. Although EC values were often comparable between culture formats, growth kinetics and concentrations required to induce complete growth arrest or net cell loss differed substantially in 3D cultures. In particular, drug concentrations required to induce net cell loss were consistently higher in 3D, revealing microenvironment-dependent survival responses that were not captured by EC alone. Furthermore, clinically expected subtype-specific responses, including tamoxifen sensitivity in ER-positive cells and olaparib sensitivity in BRCA1-mutant cells, were more accurately resolved under 3D culture conditions and extended treatment durations. Together, these findings demonstrate that growth-based, single-cell quantification provides a more comprehensive assessment of therapeutic efficacy than conventional endpoint measurements and establish VITAL as a scalable platform for physiologically relevant preclinical drug screening.

cancer biology↗

bioRxiv2026

β4-integrins safeguard nuclear mechanics to suppress prostate cancer progression

Prostate cancer (PCa) progression is accompanied by profound alterations in cell-extracellular matrix (ECM) adhesion, nuclear architecture and mechanical adaptability, yet the molecular mechanisms linking these processes remain poorly understood. Hemidesmosomes (HDs), formed by 6{beta}4-integrins, anchor epithelial cells to the basement membrane and couple extracellular forces to the intermediate filament (IF) cytoskeleton. Here, we identify a previously unrecognized tumor-suppressive function of {beta}4-integrins in preserving nuclear integrity in prostate epithelial cells. Loss of {beta}4-integrins disrupted the cytokeratin-5 network and its coupling to the nucleus, leading to nuclear softening, lamin remodeling, reduced heterochromatin content and enhanced confined migration. Unexpectedly, proximity-labeling proteomics revealed that {beta}4-integrins engage nuclear pore complex (NPC) components in an 6-independent manner, particularly upon HD disassembly. Selected interactions were validated using proximity ligation and co-immunoprecipitation assays. {beta}4-integrin loss was associated with enlarged nuclear pores and aberrant nucleocytoplasmic transport, including nuclear accumulation of YAP1. Consistent with these findings, reduced {beta}4-integrin expression in a large PCa tissue cohort correlated with altered nuclear morphology, adverse clinicopathological features, metastatic progression, and poor patient survival. Collectively, our study establishes {beta}4-integrins as a critical molecular link between cell-ECM adhesion, nuclear mechanics and genome integrity.

cancer biology↗

bioRxiv2026

Antibody co-administration robustly improves proton therapy with radiosensitizing nanoparticles: a mathematical modeling study

Radiosensitizing nanoparticles represent a promising approach for enhancing the efficacy of proton radiotherapy; however, their performance is constrained by restricted penetration into tumor tissue, resulting in preferential perivascular accumulation. Here, we develop a spatially distributed mathematical model of a growing tumor undergoing proton therapy with intravenously administered radiosensitizing nanoparticles to investigate treatment optimization strategies. Using physiologically plausible parameter ranges informed by our own experimental measurements and published data, we demonstrate that co-administration of targeted nanoparticles with antibodies binding to the same tumor receptors can overcome transport-induced localization and promote a more uniform intratumoral redistribution of nanoparticles before irradiation. Population-level simulations across heterogeneous parameter sets suggest that moderate antibody doses consistently prolong tumor regrowth time, whereas higher antibody doses produce a pronounced and robust increase in tumor cure probability under a single high-dose irradiation regimen representative of preclinical settings. A key conceptual result of our analysis is the asymmetric risk associated with antibody co-administration. In contrast to antibody--drug conjugates, for which excessive dosing of unconjugated antibodies may severely compromise therapeutic efficacy, co-administration of antibodies with nanoparticle-based radiosensitizers constitutes a "safe-by-design" strategy with respect to tumor cell kill in the modeled single high-dose irradiation setting: although excessive antibody doses may yield suboptimal outcomes, they cannot reduce tumor cell kill below that achieved with targeted nanoparticles administered without antibodies. These findings identify antibody-mediated spatial redistribution of radiosensitizing nanoparticles as a favorable strategy that is expected to provide robust therapeutic benefit despite substantial variability in tumor characteristics.

cancer biology↗

bioRxiv2026

A century of soybean breeding increased photosynthetic capacity but not NPQ relaxation

Accelerating photoprotective regulation to improve carbon assimilation is a promising strategy to increase crop productivity. Although rapid non-photochemical quenching (NPQ) relaxation has been validated as a target through metabolic engineering, it remains unclear whether conventional breeding has improved this trait. Here, we investigated whether more than a century of soybean breeding enhanced NPQ relaxation alongside light-saturated carbon assimilation and seed traits. We evaluated a historical panel of 24 soybean genotypes across vegetative and reproductive developmental stages by integrating NPQ relaxation, gas exchange parameters, xanthophyll-cycle pigment profiles, expression of key photoprotective genes (VDE, PsbS, and ZEP), seed number and seed weight. NPQ relaxation parameters were not consistently associated with genotype release year, seed number, or seed weight at either developmental stage. The only exception was the amplitude of the rapidly relaxing NPQ component (AqE), which was negatively correlated with all three variables during the reproductive stage. In contrast, genotype release year was positively associated with maximum net CO2 assimilation rate (Amax), maximum carboxylation rate of Rubisco (Vcmax), maximum electron transport rate (Jmax), seed number, and seed weight, while Amax and Vcmax were positively correlated with seed number and seed weight. These findings indicate that the greater photosynthetic capacity of modern genotypes was not accompanied by faster photoprotective response. Thus, photoprotective regulation has not kept pace with gains in photosynthetic capacity under field conditions. We conclude that rapid NPQ relaxation remains an important target for synchronizing photoprotection with the high photosynthetic capacity of modern soybean lines.

plant biology↗

bioRxiv2026

LINC00536 regulates transcriptional repressor TRPS1 in breast cancer

Metastatic breast cancer with complex molecular mechanisms of progression accounts for most cancer related deaths in women. To improve diagnosis and drug development, it is important to identify novel biomarkers and critical molecular pathways involved in tumor initiation and progression. Here, we profiled and analyzed the expression of long non-coding RNAs (lncRNAs) from three distinct stages of tumor initiation and progression (hyperplasia, adenoma, and carcinoma). We performed RNAseq on tumor and mammary epithelial cells derived from ROSAmT/mG tumor and non-tumor mice. We identified 1913 differentially expressed protein coding genes and 324 lncRNAs in breast cancer cells of all stages compared with normal mammary epithelial cells. Pearson correlation analysis correlated 93 differentially expressed lncRNAs with protein coding genes, providing a comprehensive lncRNA-protein coding genes co-expression network. Among them, we focused on Gm19303 which was paired with the differentially expressed protein coding gene, transcriptional repressor GATA binding 1 (Trps1), and identified its human counterpart as LINC00536. Both LINC00536 and TRPS1 are only overexpressed in breast cancer and correlate with poor prognosis of patient from the TCGA and GTEx databases. Single cell RNAseq data from the Atlas of Human breast cancers further confirmed that TRPS1 is upregulated in human breast cancer compared to normal human mammary tissue with highest expression in ER+ subgroup. In summary, our study explored the potential role of lncRNAs in breast cancer initiation and progression. *Implications statement: Our findings imply that human LINC00536/TRPS1 serves as a novel and early biomarker of cancer progression and a potential therapeutic target for breast cancer.

cancer biology↗

bioRxiv2026

Hypothermic Conditions Impair GnRH Pulse Generator Activity and Gametogenesis

Mammalian reproductive function is driven by arcuate kisspeptin neurons, pacemakers of gonadotropin secretion. During energy shortages, animals reallocate resources from reproduction to survival; however, the underlying neural mechanisms remain elusive. Here we used fiber photometry to chronically monitor synchronized episodes of arcuate kisspeptin neuron activity (SEskiss) in adult mice under various energy-saving conditions. In both sexes, SEskiss frequency was markedly suppressed during fasting-induced torpor and pharmacologically induced hypothermia, whereas hypometabolism alone had no discernible effect. A Q neuron-induced hypothermic state (QIH) robustly suppressed SEskiss, leading to impaired gamete maturation, whereas warming the body temperature during QIH fully restored SEskiss frequency. These findings demonstrate that hypothermia, rather than hypometabolism, is the primary driver of suppression of the hypothalamic reproductive axis during energy-saving conditions. This study provides insights into how thermal signals act as critical gatekeepers in the mammalian reproductive system.

neuroscience↗

bioRxiv2026

Rapid repurposing of microvillar content drives a flagellate-to-amoeboid switch in the closest relative of animals

Animal cells extensively remodel their cytoskeleton during differentiation and can notably switch between two major motility modes: flagellum-based swimming and actin-based crawling. We previously showed that choanoflagellates, the closest living relatives of animals and classically viewed as obligate flagellated swimmers, can retract their collar complex and adopt an amoeboid form within seconds under spatial confinement, independently of regulated gene expression. Here, using live imaging, ultrastructural expansion microscopy, and cryo-electron tomography in Salpingoeca rosetta, we identify rapid, cell-wide cytoskeletal remodeling as the ultrastructural basis of this switch. Unconfined choanoflagellates lack a detectable actin cortex but display an apical flagellum and cortical microtubules, with F-actin being largely restricted to microvilli. Confinement triggers calcium release from intracellular stores, which induces microvillar retraction and absorption of microvillar material into the cell body, including actin, ezrin-radixin-moesin 1, and plasma membrane. Remodeling of the internalized F-actin and repurposing of associated proteins supports de novo actin cortex formation, which is necessary for amoeboid motility. In parallel, cortical microtubules are disassembled, and the reabsorbed microvillar plasma membrane increases the surface area of the cell body, allowing the cell to flatten under confinement. Cryo-electron tomography reveals stepwise actin reorganization from internalized microvillar bundles to a cortical contractile meshwork combining bundles and scattered filaments. This work reveals considerable ultrastructural plasticity in the cytoskeletal architecture of choanoflagellates and supports an ancestral role for microvilli as reservoirs of membrane and cytoskeleton to potentiate cell phenotypic transitions.

evolutionary biology↗

bioRxiv2026

Inheritance of a Single Edited CD46 Allele Is Associated with Reduced Ex Vivo Susceptibility to Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV) remains an economically important pathogen of cattle despite widespread vaccination. A homozygous CD46-edited Gir heifer (Ginger) was previously shown to have significantly reduced susceptibility to BVDV. The edited allele contains an in-frame six amino acid substitution within the virus-binding domain of the BVDV entry receptor CD46, replacing residues G82QVLAL with A82LPTFS. Here, we investigated whether reduced BVDV susceptibility is maintained when the edited allele is inherited in the heterozygous state. Ginger was artificially inseminated with semen from an unedited Gir bull and produced a healthy heterozygous CD46-edited bull calf (Giraldo). Whole-genome sequencing confirmed the inheritance and structural integrity of Giraldo's edited allele. Compared with Ginger, Giraldo exhibited similarly reduced ex vivo BVDV susceptibility across primary fibroblasts, lymphocytes, and monocytes, despite inheriting a wild-type CD46 allele from the sire. Allele-specific CD46 RNA expression analysis demonstrated expression of both the edited and wild-type CD46 alleles. Thus, the reduced-susceptibility phenotype was not attributable to transcriptional silencing of the wild-type allele. Lentiviral complementation studies in CD46-knockout Madin-Darby bovine kidney (MDBK) cells further demonstrated that this wild-type CD46 allele was competent to support BVDV infection when expressed independently. Together, these findings indicate that the CD46 A82LPTFS allele can confer reduced BVDV susceptibility in the heterozygous state despite expression of a functional wild-type CD46 allele. This result suggests the potential to more rapidly disseminate reduced BVDV susceptibility through conventional breeding using homozygous CD46-edited sires.

molecular biology↗

bioRxiv2026

Glutaminase contributes to MYC-induced cell-autonomous autophagy and to RasV12-dependent non-autonomous autophagy in the Drosophila wing disc epithelium

MYC-driven metabolic reprogramming supports rapid cell growth but also creates metabolic demands that require adaptive mechanisms to maintain cellular homeostasis. Here, combining clonal analysis in Drosophila wing imaginal discs with studies in Schneider S2 cells, we identify glutamine metabolism as a component of Myc-induced autophagy. Myc increased the expression of genes involved in glutamine utilization, including glutaminase (GLS), and enhanced ammonia production, a metabolic by-product of glutaminolysis. Genetic depletion of GLS in clones suppressed the accumulation of Myc-induced Atg8a-positive structures and reduced autophagic flux, demonstrating that glutaminase contributes to the autophagic response elicited by Myc. Exogenous NHCl was sufficient to induce Atg8a-positive structures and partially restored their accumulation following GLS depletion, supporting ammonia as a downstream contributor to this response. Mechanistically, Myc-induced autophagy in clones required the core autophagy factor Atg5 but was not suppressed by depletion of Rheb or Atg1, consistent with an autophagic program that can operate independently of canonical TOR-Atg1 signaling. We further found that Myc activity is required for RasV12-driven epithelial overgrowth and that RasV12 cells induce a pronounced non-cell-autonomous accumulation of Atg8a-positive structures in wild-type cells surrounding RasV12 clones. Depletion of either Myc or GLS in RasV12 cells strongly reduced this neighboring autophagic response, linking Myc-dependent glutamine metabolism in transformed cells to autophagy in the surrounding tissue. Together, our findings identify GLS-dependent glutamine metabolism as a previously unrecognized component of Myc-induced autophagy and extend this relationship to Ras-transformed epithelia, where Myc and Gls contribute to non-cell-autonomous autophagic responses in neighboring cells.

cell biology↗

bioRxiv2026

PGM3 inhibition rewires RUVBL2-dependent DNA repair and induces a BRCAness-like state in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) exhibits profound metabolic rewiring and strong resistance to DNA-damaging therapies, yet how metabolic pathways regulate genome maintenance remains poorly understood. The hexosamine biosynthetic pathway (HBP) integrates nutrient availability with protein glycosylation through production of UDP-GlcNAc, but its role in DNA damage response (DDR) regulation is unclear. Here we show that inhibition of the HBP enzyme phosphoglucomutase-3 (PGM3) reduces DNA repair capacity in pancreatic cancer cells. Transcriptomic and functional analyses reveal that the selective PGM3 inhibitor FR054 amplifies gemcitabine-induced replication stress, disrupts ATR-CHK1 and ATM-CHK2 checkpoint signaling, and selectively impairs homologous recombination. Glycoproteomic profiling identifies the AAA+ ATPase RUVBL2 as a key metabolic-DDR node. Gemcitabine increases RUVBL2 O-GlcNAcylation, with Thr81 identified as a modified residue within the Walker A nucleotide-binding motif. Structural modelling predicts that Thr81 O-GlcNAcylation stabilizes the RUVBL1-RUVBL2 complex without compromising ATP-Mg engagement. PGM3 inhibition and Thr81 mutation similarly reduced ATR and ATM abundance and promoted persistent DNA damage, supporting a role for RUVBL2 Thr81 O-GlcNAcylation in sustaining checkpoint signalling and genome stability. Consequently, PGM3 inhibition induces a BRCAness-like state that sensitizes pancreatic cancer cells to PARP inhibition, both in vitro and in vivo, as well as to ionizing radiation. These findings reveal a nutrient-sensitive mechanism linking protein glycosylation to genome maintenance and identify HBP-dependent DNA repair as a potentially actionable vulnerability in pancreatic cancer.

cancer biology↗