Biology subjects

Zhang, A.

Publications and source records attributed to Zhang, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Genetic determinants of Brevundimonas-mediated attenuation of plant pattern-triggered immunity

Root-associated commensal bacteria from diverse taxonomic groups can attenuate plant pattern-triggered immunity (PTI), yet the bacterial mechanisms underlying immune attenuation remain poorly understood. Brevundimonas sp. MF374 is a root-associated Alphaproteobacterium previously identified as a strong attenuator of flg22-triggered immunity. Here, to identify the genetic determinants underlying this phenotype, we conducted transposon mutagenesis and screened 4,032 MF374 mutants for defects in immune attenuation. Deletion and complementation of candidate loci identified tonB1 (BLR19_RS15045) as the strongest determinant of immune attenuation, with ppiD (BLR19_RS02880) making a weaker contribution. A panel of GUS reporter assays designed to distinguish among different mechanisms of attenuation showed that intact MF374 cells directly reduced the immune-eliciting activity of flg22 in a tonB1-dependent manner. TonB1 also contributed to attenuation of responses triggered by a distinct flg22 sequence and the endogenous plant elicitor AtPEP1. Together, these findings implicate TonB-dependent transport in attenuation of flg22-induced immunity by MF374 and reveal a previously uncharacterized mechanism of immune attenuation by a root-associated commensal.

microbiology↗

bioRxiv2026

AltORF-encoded microprotein recruits primary-ORF-encoded dehydrogenase to cell poles upon heat shock

Alternative open reading frames (altORFs) initiating from in-frame or out-of-frame start sites within annotated protein-coding genes represent a largely unexplored source of protein-coding potential and remain poorly understood in terms of regulation, function, and impact on physiology. Here we investigate Escherichia coli GndA, encoded by an out-of-frame altORF within gnd, which encodes 6-phosphogluconate dehydrogenase. We observe that the GndA protein is induced by heat shock, consistent with the initial identification in a quantitative membrane proteomics screen, but is unstable, while the levels of the stable Gnd dehydrogenase are unchanged. Using translational fusion assays and in vivo RNA structure probing, we show that an internal complementary sequence upstream of gndA acts as a thermo-responsive secondary RNA structure controlling GndA translation. Mutagenesis of either gnd or gndA reduce heat shock survival of E. coli indicating both gene products are important for resistance. GndA fractionates with membranes consistent with in silico predictions that the protein forms an amphipathic -helix. In contrast, Gnd is predominantly cytoplasmic but shows GndA-dependent relocalization to cell poles upon heat shock. Together, our results demonstrate that an altORF-encoded microprotein is required for stress-dependent relocalization of its primary ORF partner, revealing a functional interplay between proteins encoded by overlapping ORFs in bacterial stress adaptation.

microbiology↗

bioRxiv2026

Robust and accessible in situ sequencing with an open-source automated sequencer

Targeted in situ sequencing underlies many spatial transcriptomic, barcoded connectomic, and multiplexed perturbation techniques. Each experiment requires many cycles of chemical reactions and imaging that are time-consuming and error-prone to perform manually, and few commercial platforms can sequence highly diverse RNA barcodes in situ at cellular resolution across large tissue sections. Here we present Seq-o-Matic, an open-source automated in situ sequencer built entirely from commercially available components, which integrates fluidics, heated incubation, and imaging of up to three continuous 18 mm x 50 mm areas on standard microscope slides. Relative to manual sequencing, Seq-o-Matic reduced hands-on time 6- to 17.5-fold and total run time about 2.6-fold. Across three runs spanning two mouse strains and a neonatal marmoset, sequencing quality, measured by signal retention, channel specificity, and phasing, was similar to that of four manually sequenced mouse brains. In four mouse hemispheres sequenced in separate runs (two C57BL/6J, one NOD, and one WSB), consensus cell types were present in every brain and corresponded to those of a manually sequenced C57BL/6J brain. Within cortical excitatory neurons, the two C57BL/6J brains were more similar to each other than were brains of different strains, suggesting that technical variation between runs was small relative to strain differences. Seq-o-Matic thus makes consistent, high-throughput in situ sequencing accessible to laboratories without specialized in situ sequencing expertise.

genomics↗

bioRxiv2026

Combining an autoactivated cGAS mutant with membrane-tethered IL-12 via mRNA lipid nanoparticles promotes systemic tumor control

Local tumor therapies rarely induce systemic immunity capable of controlling metastatic disease. Here, we engineer an mRNA lipid nanoparticle (LNP) platform to coordinate innate immune sensing and T cell activation in situ through co-delivery of an autoactivated cyclic GMP-AMP synthase mutant (cGASR241E) and membrane-tethered IL-12 (IL-12TM). cGASR241E mRNA LNPs autonomously generated cyclic GMP-AMP in vivo, activating host STING signaling and initiating antitumor responses. Co-administration with IL-12TM mRNA LNPs promoted localized cytokine production, reduced regulatory T cells, and restored effector function in tumor-infiltrating T cells. This combination elicited systemic antitumor immunity and suppressed distal tumors across multiple mouse models. The therapeutic efficacy required CD8+ T cells and IFNg; signaling in host, supporting a model in which innate DNA sensing is coupled to IL-12-mediated T cell functional reinforcement. In addition, the combination enhanced responsiveness to PD-L1 blockade. Together, these findings establish a clinically translatable strategy to integrate cGAS-STING activation with cytokine-driven T cell programming to drive systemic tumor control.

cancer biology↗

bioRxiv2026

Soil invertebrate biomass estimation from image-derived body area and contour length

1. Soil invertebrate biomass is closely linked to ecosystem processes, yet its routine quantification remains constrained by the effort required to weigh large numbers of small individuals. Length-mass relationships provide an indirect alternative, but body length can be difficult to standardise and extract automatically from images of curved, coiled or contracted organisms. Projected body area may provide a more tractable image-derived predictor, but its performance across diverse soil-faunal taxa and at the community scale remains insufficiently evaluated. 2. Here, we developed a framework using projected body area and contour length to estimate fresh and dry mass from measurements of 1,792 soil invertebrates representing 15 higher-level taxa. We fitted taxon-specific models for six well-represented groups and general models across taxa, and evaluated predictive performance using leave-one-out cross-validation. We then assessed cross-dataset transferability by comparing our data with those of Lemoine et al. (2026) and pooled the two datasets to derive combined models. Finally, we tested whether individual predictions could be scaled to independently measured community biomass. 3. Taxon-specific models generally reduced prediction error relative to general models, with mean absolute percentage error (MAPE) values of 6.9-37.3% for fresh mass and 7.6-30.2% for dry mass. General models retained MAPEs of 27.2% and 22.4%, respectively. Comparisons of our dataset with that of Lemoine et al. (2026) showed no detectable differences in six of eight taxon-by-mass slopes. Although intercepts frequently differed, no overall dataset effect was detected after accounting for taxa. Pooling both datasets broadened model development, while taxon-specific models generally retained lower error than the combined general model. Finally, community-level predictions tracked measured biomass across all model sets (R2 = 0.87-0.99), but absolute error varied among models and faunal groups (MAPE = 9.6-52.5%). 4. Body area and contour length extracted from images therefore provide a practical basis for scaling soil invertebrate biomass estimation from individuals to communities. Integration with automated imaging and segmentation tools such as flatbug, BIODISCOVER and CollembolAI could extend soil invertebrate monitoring at larger scales beyond abundance and taxonomic composition to biomass, while providing scalable inputs for analyses of food webs and general ecosystem ecology.

ecology↗

bioRxiv2026

Cross-Tissue Inflammatory Convergence Supports a Putative Muscle-Brain Axis in Alzheimer's Disease

Background Reduced muscle strength is associated with cognitive decline and Alzheimer's disease (AD), suggesting that skeletal-muscle aging and AD-related neurodegeneration may share biological mechanisms. Vascular dysfunction, immune activation, and extracellular-matrix remodeling have been implicated in both tissues, but the extent of shared cellular and molecular programs remains unclear. We investigated cross-tissue transcriptional programs in aging skeletal muscle and AD-related brain tissue and evaluated whether related signals are detectable in peripheral blood. Methods We analyzed independent, unpaired human transcriptomic datasets from skeletal muscle (23 donors), the SEA-AD middle temporal gyrus atlas (84 donors), and peripheral blood. Donor-aware models identified age-associated changes in muscle and transcriptomic changes across continuous pseudoprogression score (CPS) in brain. Cross-tissue comparisons focused on 30 brain donors within an early CPS window. Shared tissue-derived programs were assessed in peripheral blood mononuclear cells and independently tested in frailty bulk-blood and Alzheimer's Disease Neuroimaging Initiative (ADNI) whole-blood datasets. CellOracle simulations were used to prioritize candidate transcriptional regulators. Results Aging muscle showed increased inflammatory, extracellular-matrix, and stromal communication programs. During early AD-related brain progression, vascular and immune populations showed increasing inflammatory and matrix-associated activity together with shifts in intercellular communication. Cross-tissue analysis identified six concordantly enriched pathways comprising 109 leading-edge genes in endothelial populations and four pathways comprising 51 genes in myeloid populations. Related peripheral-blood signatures varied across cohorts and cell types. However, in independent frailty and ADNI mild cognitive impairment and AD datasets, 23 of 30 predefined tissue-derived pathway-subset tests showed significant positive enrichment after false-discovery-rate correction, whereas individual gene-level bulk-blood associations did not survive correction. CellOracle analyses prioritized ETS2 and BHLHE40 as exploratory endothelial regulators whose simulated perturbation consistently reduced selected shared program scores across tissues. Conclusions Aging skeletal muscle and AD-related brain tissue exhibit convergent endothelial and myeloid inflammatory programs, with related pathway-level signals detectable in peripheral blood. These cross-tissue programs and candidate endothelial regulators provide testable hypotheses for mechanisms linking systemic aging, muscle dysfunction, and AD-related neurodegeneration.

neuroscience↗

bioRxiv2026

ProxiNet transfers spatially learned cellular proximity to dissociated single-cell transcriptomes

Spatial transcriptomics reveals cellular organization within intact tissues, whereas dissociated single-cell RNA sequencing provides broad transcriptomic coverage but loses information about cellular proximity and neighborhood structure. Here, we developed ProxiNet, a spatially supervised framework that learns transcriptomic signatures of pairwise cellular proximity from spatial reference datasets and transfers these relationships to dissociated single-cell transcriptomes. ProxiNet predicted cellular proximity across brain regions and spatial technologies, including zero-shot cross-technology transfer, and gradient-based attribution identified genes and broader transcriptional programs associated with proximity predictions. In spatial datasets with known coordinates, ProxiNet-derived cellular neighborhoods recovered reproducible tissue organization and anatomical structure, providing independent spatial validation of the inferred proximity relationships. Applying the spatially calibrated model to dissociated scRNA-seq revealed heterogeneous cellular neighborhoods with distinct cell-type compositions and candidate communication programs. In an Alzheimer dataset, ProxiNet further identified age-associated remodeling of inferred neighborhoods, including an AD-associated neighborhood at 8 months characterized by distinct astrocyte and neuronal transcriptional states and candidate intercellular communication programs. Together, these results establish pairwise cellular proximity as an interpretable and transferable representation for extending spatially learned tissue organization to dissociated single-cell transcriptomes.

bioinformatics↗

bioRxiv2026

Disease relevance and replicability of deep learning gene expression prediction

Recent deep learning (DL) models predict average gene expression levels from DNA sequences with high overall correlation to measured values. We examine these DL models through the lens of disease research. The seemingly high overall performance of DL models is largely due to capturing whether genes are "On" or "Off", and to a lesser extent, disease-relevant expression level changes for "On" genes. Indeed, the more bimodal the gene expression distribution, the better the reported performance. We track the extent of this issue across tissues, molecular systems, and cancers. Compounding the model evaluation issue, we find inconsistencies between the published code and reported performance, highlighting the importance of versioning and publishing performance evaluation code. These findings indicate that the high reported performance of popular DL models falls unexpectedly short in disease applications, and that the problem of personalized genomic prediction remains far from solved in a disease context.

genomics↗

bioRxiv2026

Reliability-guided meta-control in intuitive physics

Intuitive physical reasoning is an important part of daily life, but the computations underlying it remain debated. Some prominent accounts propose intuitive physics relies on mental simulation, with people evolving mental scenes forward through step-by-step. However, representing every intermediate mental state in detail can be computationally costly. We suggest that intuitive physical reasoning combines mental simulation with learned predictive abstractions, and that the trade-off between these two is determined by the prospective reliability of an abstraction. We implemented the abstraction part of our proposal as a recurrent neural network model that learns a predictive abstraction by directly predicting the object's future position from a short visual history. This network bypasses intermediate states, while also estimating its own prediction error. Our network produced simplified linear trajectories that shortcut ground-truth physical trajectories (in line with human behavior), and its error estimates were associated with human pupil size during a physical prediction task. We further developed a meta-control model that used the network's error estimate to arbitrate between abstraction (direct prediction by the network) and simulation (running a mental simulation). The meta-control model captures key patterns in human response time and accuracy, and its predicted abstraction and simulation phases corresponded to distinct eye-movement signatures, over and above an earlier blended model that uses a hand-designed abstraction. Our results suggest that efficient physical reasoning can emerge from an interaction between learned abstractions, estimates of how reliable these abstractions are, and selective engagement of detailed simulation based on reliability estimates.

animal behavior and cognition↗

bioRxiv2026

Molecular Editing in Protein Degradation: From a Dual NSD2/GSPT1 Degrader to an NSD2-Selective PROTAC and a GSPT1-Selective Molecular Glue

Both NSD2 and GSPT1 represent compelling targets for anti-cancer drug discovery. Herein, we carried out single-atom modification of LLC0424, an NSD2 degrader with GSPT1 neosubstrate engagement, to yield two structurally analogous molecules with divergent modes of action: 424-ND, an NSD2-selective degrader, and 424-GD, a GSPT1-selective degrader. 424-ND drove selective NSD2 degradation and suppressed androgen receptor (AR) signaling in prostate cancer cells. 424-GD induced selective degradation of GSPT1 and upregulated the integrated stress response markers ATF4 and ATF3. Biolayer interferometry revealed distinct ternary complex cooperativity profiles in the presence of NSD2 or GSPT1, which directly correlate with the observed biased degradation activity. Molecular dynamics and metadynamics simulations showed that the compounds adopt distinct low-energy conformational ensembles with different spatial orientations, likely underlying their divergent cooperativity and selectivity. These findings demonstrate that minimal structural alteration permits precision control over degrader target selectivity, while furnishing selective chemical probes for NSD2 and GSPT1.

cancer biology↗

bioRxiv2026

Salinity is associated with habitat use and mitochondrial lineage diversity in halophilic Ephydra flies (Diptera: Ephydridae)

Brine flies of the genus Ephydra (Diptera: Ephydridae) are ubiquitous extremophile animals whose larval forms are able to thrive in high-stress environments, including hypersaline and high carbonate waters. Despite their historical and ecological importance, little is known about their evolutionary history. Here, we focus on an understudied population of Ephydra living in former industrial salt evaporation ponds of the San Francisco Bay Area (SFBA), a region which has undergone significant environmental changes in the past century. We observed and collected Ephydra specimens and took salinity measurements of waters they inhabited during a 6-month period in 2024. We found that E. millbrae and E. gracilis demonstrate strong salinity preferences. We also analyzed DNA from Ephydra spp. specimens from distant sites and a timespan of ~40 years, generating the most comprehensive gene genealogy of the group to date based on a single mitochondrial locus. Our analysis of phylogenetic signals suggests that salinity envelopes are influenced by evolutionary relationships in Ephydra. This study provides new information about the natural history of extremophile flies in the SFBA and fills in current gaps in our knowledge about the evolutionary history of the genus more broadly.

evolutionary biology↗

bioRxiv2026

Integrated morphological, multi-omics, and functional profiling reveals microglial plasticity driven by AD risk genes

Microglia, the resident macrophages of the central nervous system, are highly dynamic cells essential for brain homeostasis. While genome-wide association studies (GWAS) strongly implicate microglial dysfunction in Alzheimer's disease (AD), the mechanistic links coordinating their diverse transcriptional, morphological, and functional states remain poorly understood. Using human induced pluripotent stem cell-derived microglia (iMicroglia) and single-cell RNA sequencing, we identified six distinct transcriptional profiles and mapped them to specific morphological phenotypes via targeted immunofluorescence, establishing a link between microglial morphology and molecular identity. Transcriptomic and morphological profiling further demonstrated profound microglial plasticity, revealing distinct, stimulus-specific responses to AD-relevant pathologies, including Tau PFF, amyloid-beta, and apoptotic neurons. To assess how AD risk variants perturb these states, we performed high-efficiency CRISPR-Cas9 ribonucleoprotein (RNP) knockouts of specific AD GWAS genes. Bulk RNA-seq profiling revealed extensive transcriptional remodeling following genetic perturbation. Crucially, we show that depletion of these AD risk genes disrupts baseline morpho-transcriptomic coupling and fundamentally alters microglial phagocytic capacity. Together, this study reveals how AD GWAS genes may drive microglia into dysfunctional states characterized by altered morphology, distinct multi-omic signatures, and impaired functions.

neuroscience↗

bioRxiv2026

Selective collateralization of transcriptomically distinct neurons organizes visual-stream output from the primary visual cortex

Parallel visual streams segregate information into pathways specialized for distinct computations, but how this segregation is achieved by anatomical segregation of primary visual cortex output remains unclear. This problem is complicated because individual neurons frequently send broadcasting projections to multiple cortical areas, and the target choice depends on both topographical location and molecular identity. Here we developed axonal BARseq2 to jointly map gene expression and high-resolution axonal projections from 1,448 neurons spanning the mouse primary visual cortex (VISp). Axonal BARseq2 recapitulated projection patterns observed by bulk tracing and single-neuron reconstruction, and recovered transcriptomic identities consistent with reference snRNA-seq datasets. Retinotopy strongly predicted projections to individual cortical targets, particularly for areas proximal to VISp, but explained little of which areas are frequently co-innervated. Instead, co-innervation patterns defined three preferential output pathways that largely corresponded to the ventral stream and two subdivisions of the dorsal stream. These pathways were associated with fine-grained transcriptional identities of L4/5 intra-telencephalic neurons, which were further validated with an external MERFISH dataset. Thus, VISp output is organized by two distinct rules: retinotopy constrains where neurons project, whereas cell-type-associated collateralization constrains which targets are co-innervated. This selective broadcasting, in which single neurons reach many higher visual areas in cell-type-specific combinations, could provide an anatomical substrate for visual-stream segregation at the level of VISp output in mice.

neuroscience↗

bioRxiv2026

Neogenin-1 marks myeloid-primed fetal hematopoietic stem cells that undergo progressive lineage-restriction with age

During aging, hematopoietic stem cells (HSCs) increasingly shift from balanced to myeloid-biased differentiation, resulting in reduced lymphoid output and impaired adaptive immunity. The question of whether this lineage bias is established in a subset of HSCs during early development or primarily emerges with aging warrants further investigation. Here, we investigate whether myeloid-biased HSCs (my-HSCs) are established at the fetal liver stage by specifically examining Neogenin-1 (NEO1), a previously defined marker of my-HSCs. We identify two distinct populations of Hoxb5+ HSCs in the fetal liver: NEO1+ and NEO1-, with NEO1+ HSCs exhibiting transcriptional and functional characteristics consistent with my-HSCs. With age, my-HSC-associated transcriptional programs become increasingly reinforced across the Hoxb5+ pHSC compartment, with NEO1+ cells showing early enrichment of this program and both NEO1+ and NEO1- cells acquiring broader myeloid-biased features in aging. These findings suggest that lineage programming can begin early in development and is further shaped by age-related changes, potentially contributing to the functional decline observed in the aging hematopoietic system.

developmental biology↗

bioRxiv2026

Genome evolution at the extreme of angiosperm miniaturization

Eukaryotic genomes vary by several orders of magnitude, yet this vast variation bears little relation to the complexity of the organisms they encode. Thus, how genome evolution accompanies changes in organismal complexity remains unresolved. A central obstacle is that major differences in body plan usually occur among deeply divergent lineages, entangling body plan evolution with the genomic divergence accumulated over long independent histories. Duckweeds offer a rare system in which successive body plan reductions can be traced within a single plant family. Across this trajectory, body size declined by nearly an order of magnitude and roots were progressively lost, culminating in the extreme of angiosperm miniaturization. Yet genome size increased nearly sixfold. Here, using a new chromosome-scale genome of Wolffia globosa and comparative genomics across nested evolutionary scales, we show that genome size, gene number, and functional repertoire followed distinct trajectories during miniaturization. Genome expansion was driven largely by transposable element accumulation, whereas the number of protein-coding genes remained stable. Aquatic adaptation itself promotes functional simplification, but establishes only a baseline. Duckweeds pushed this streamlining much further through additional contraction of developmental, structural, and biotic defense functions, alongside selective expansion of functions associated with growth and abiotic adaptation. This remodeling accumulated across successive evolutionary transitions through continued contraction of the same gene families and, more commonly, contraction of different families affecting the same biological processes. Organismal complexity may therefore reflect not simply the size of a genome or its functional repertoire, but how that repertoire is selectively reconfigured through evolution.

evolutionary biology↗

bioRxiv2026

Endocrine-Adapted Pituitary Macrophages Regulate Gonadotropin Secretion through CXCL5-CXCR2-MAPK Signaling

Chronic inflammation disrupts hormonal balance in the Hypothalamic-Pituitary-Gonadal (HPG) axis, contributing to reproductive disorders. While immune cells in the hypothalamus and ovaries have been extensively studied, their impact on the pituitary remains largely unexplored. Our research identifies pituitary macrophages (PitMacs) as the dominant pituitary immune cell population with a role in regulating reproductive gonadotropin secretion both in vitro and in vivo. Using a targeted AAV-based depletion strategy, we demonstrate that a reduction of PitMacs decreases serum gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), in female mice. PitMacs are transcriptomically distinct from other tissue-resident macrophages and harbor a unique translational program that reflects the pituitarys endocrine identity, including active translation of growth hormone (Gh) and prolactin (Prl). Cytokine profiling identified CXCL5 and IFN-{gamma} as key PitMac-derived mediators of gonadotropin regulation. Mechanistically, CXCL5 signals through CXCR2 to activate the MAPK pathway, converging with Gonadotropin-Releasing Hormone (GnRH) signaling in a time-dependent manner to regulate LH secretion and GnRH receptor surface expression. These findings establish PitMacs as essential endocrine-immune integrators, opening new avenues for understanding inflammation-driven reproductive disorders. One Sentence SummaryPituitary macrophages are unique hormone-producing immune cells that regulate hormone secretion via cytokine signaling.

immunology↗

bioRxiv2026

Stereoselective Covalent Inhibitor of the Ovarian Cancer-Driving Transcription Factor PAX8

Transcription factors remain among the most challenging therapeutic targets in part because they lack well-defined ligandable binding pockets. We recently showed that aberrantly reactive cysteines in transcription factors can be directly targeted with electrophilic small molecules to induce selective transcription factor destabilization and degradation. Here, we extend this strategy to the lineage-defining oncogenic transcription factor PAX8, a critical driver of ovarian cancer. Screening of a chemically diverse library of more than 3,000 cysteine-reactive compounds against an endogenously HiBiT-tagged PAX8 reporter identified a sulfinyl aziridine chemotype that selectively reduced PAX8 abundance. Structure-activity and stereochemical analyses revealed highly enantio- and diastereoselective activity, identifying KL6-159A as the lead compound. Quantitative proteomics demonstrated selective loss of PAX8, while cellular thermal shift analysis and chemoproteomic profiling established direct covalent engagement of PAX8 at cysteine C57. Mutation of C57 completely abolished KL6-159A-induced PAX8 depletion, demonstrating that this residue is essential for compound activity. Transcriptomic profiling revealed broad suppression of the PAX8 transcriptional program, with FOXM1 emerging as the most significantly downregulated regulatory network together with numerous established PAX8 target genes. Collectively, these studies establish direct covalent engagement, transcriptional inhibition, and destabilization of PAX8 and further demonstrate the generality of covalent chemoproteomic approaches for drugging previously intractable transcription factors.

biochemistry↗

bioRxiv2026

Suppression of RIPK3 by EZH2 contributes to cigarette smoking-induced chemoresistance in lung cancer

Lung cancer remains the leading cause of cancer-related mortality worldwide, with cigarette smoking (CS) representing its primary risk factor. In addition to promoting tumorigenesis, chronic CS exposure contributes to chemotherapy resistance, although the underlying mechanisms remain poorly understood. Here, we established a long-term CS exposure model by repeatedly treating Lewis lung carcinoma (LLC) cells with cigarette smoke extract (CSE). After 4 months of exposure, CSE-treated cells exhibited enhanced proliferation, migration, and resistance to chemotherapy-induced cell death. Mechanistically, chronic CSE exposure suppressed receptor-interacting protein kinase 3 (RIPK3) expression by upregulating the epigenetic regulator enhancer of zeste homolog 2 (EZH2), which promoted repressive histone methylation at the RIPK3 promoter. Loss of RIPK3 impaired chemotherapy-induced cell death primarily by inhibiting ferroptosis rather than necroptosis. Importantly, genetic depletion or pharmacological inhibition of EZH2 restored RIPK3 expression and sensitized lung cancer cells to gemcitabine treatment both in vitro and in vivo. Furthermore, analysis of human lung cancer datasets revealed an inverse correlation between EZH2 and RIPK3 expression, with RIPK3 levels progressively decreasing with smoking history. Collectively, these findings identify the EZH2/RIPK3 axis as a critical mediator of smoking-associated chemoresistance and uncover a previously unrecognized role for RIPK3 in ferroptosis regulation. Targeting EZH2-mediated RIPK3 suppression may represent a promising therapeutic strategy to overcome chemoresistance in lung cancer patients with a history of smoking.

cancer biology↗