Cell Death & Differentiation

Infiltration of conventional immune cells has been ascribed as the fundamental drivers of innate immune signaling in the damaged myocardium. However, the emerging intrinsic immunoregulatory potential of cardiomyocytes still remains poorly understood. Interferon gamma (IFN{gamma}) is a pleiotropic cytokine with context-dependent detrimental as well protective role in regulating cardiac inflammatory circuits. The prevailing view of IFN{gamma} as a prime pro-inflammatory cytokine has been challenged due to its paradoxical actions both as an inducer as well as negative regulator of inflammation, but the players involved in these converse processes remains enigmatic. Here we show that cardiomyocytes exhibit a cell-autonomous immunocompetent response upregulating innate inflammatory signaling upon type I and type II IFN stimulus. Notably, hiPSC-derived cardiomyocytes display a robust increase in guanylate binding protein 5 (GBP5), one of the major IFN{gamma}-induced GTPase involved in inflammasome signaling, followed by upregulation of AIM2/CASP1 pathway whereas NLRP3 levels remain unaltered by IFN{gamma} stimulation. GBP5 knockdown and overexpression studies in hiPSC-derived cardiomyocytes identify GBP5/TGF{beta} axis as a non-canonical anti-inflammatory feedback regulation on the IFN{gamma}-induced inflammatory cascade.

Autosomal recessive Stargardt disease type 1 (STGD1) is the most prevalent inherited juvenile macular degeneration, ultimately leading to irreversible blindness through photoreceptor cell loss, yet the underlying cell death mechanisms remain poorly defined. Receptor-interacting protein kinase 3 (RIPK3) is a well-established mediator of necroptosis and, under certain circumstances, apoptosis downstream of TNF family ligands, and it was found to be progressively upregulated in the retina of a STGD1 abca4-/-rdh8-/- (DKO) mouse model coinciding with the onset and progression of photoreceptor degeneration. Despite elevated RIPK3 expression, necroptosis was not detectable in this model, as evidenced by the absence of phosphorylated MLKL and unaltered photoreceptor degeneration in abca4-/- rdh8-/-mlkl-/- mice. Intriguingly, genetic ablation of Ripk3 exacerbated photoreceptor loss in DKO mice in both chronic (age-dependent) and acute (light-induced) retinal degeneration paradigms. This detrimental effect was partially ameliorated by pan-caspase inhibition in the acute degeneration model, indicating caspase-dependent apoptosis as the primary executioner. Mechanistically, we demonstrated that RIPK3 suppressed extrinsic apoptosis by attenuating caspase-8 activation downstream of TNF family ligands. Collectively, our findings reveal a non-canonical, protective role of RIPK3 in photoreceptors, as a brake on apoptotic signaling rather than a necroptotic executor, in the context of STGD1. These findings redefine the role of RIPK3 in retinal degeneration and emphasize the contextual plasticity of cell death regulators in neurodegenerative diseases.

Inflammasome assembly rapidly triggers caspase-1 activation to initiate pyroptosis, an inflammatory cell death characterized by the release of cytosolic contents, including interleukin (IL)-1{beta}/. Here, we report that inflammasome activation drives necrotic cell death independent of gasdermin D (GSDMD) and GSDME, which are essential executors of pyroptosis by forming a pore on the plasma membrane and increasing membrane permeability. NLRP3 inflammasome activation induced necrotic cell death, coupled with IL-1{beta}/ release in Gsdmd-/-Gsdme-/- macrophages. Mechanistically, the oligomerization of ninjurin-1 (NINJ1) was caused by inflammasome activation even in the absence of GSDMD and GSDME. Concordantly, glycine, an inhibitor of NINJ1, blocked plasma membrane permeabilization triggered by inflammasome activation in Gsdmd-/-Gsdme-/- macrophages, but not in WT macrophages. The dimerizer-mediated ASC oligomerization promoted NINJ1-mNeonGreen cluster formation in the absence of GSDMD and GSDME. Moreover, NINJ1 deficiency prevented membrane permeabilization initiated by ASC oligomerization in Gsdmd-/-Gsdme-/- immortalized bone marrow-derived macrophages (iBMDM). Blocking of phosphatidylserine (PtdSer) exposure, a feature of inflammasome-driven necrotic cell death, by Xkr8 deficiency inhibited plasma membrane permeabilization in Gsdmd-/-Gsdme-/- iBMDM. These results suggest that inflammasome-triggered activation of caspase-1 itself drives inflammatory necrotic cell death independent of gasdermins.

Cellular senescence exerts powerful non-cell autonomous effects through the senescencelzlassociated secretory phenotype (SASP). This SASP comprises soluble factors and extracellular vesicles (EVs). Although soluble SASP components can induce senescence in neigbouring cells, the specific contribution of EVs to paracrine senescence is poorly defined. Here, we show that EVs released by senescent tumor cells are necessary and sufficient to propagate senescence. Conditioned media from bleomycinlzlinduced senescent A549 cells triggered a permanent growth arrest with morphological changes and upregulation of senescence markers in recipient tumor cells. Pharmacological inhibition of EV biogenesis using GW4869 or genetic downregulation of the EV secretion mediator RAB27A markedly attenuates paracrine senescence without affecting soluble SASP factor secretion or the senescent state of producer cells. Proteomic characterization reveals that senescent EVs exhibit a distinct molecular signature enriched for extracellular components and processes related to wound healing and hemostasis. Importantly, purified senescent EVs, devoid of soluble SASP factors, fully recapitulated paracrine senescence induction. These findings identify senescent EVs as key autonomous SASP effectors and highlight vesicular pathways as potential therapeutic targets in cancer and therapylzlinduced senescence.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron degeneration associated with genomic instability and mitochondrial dysfunction, although the mechanistic relationship between these hallmarks remains unclear. To determine whether nuclear DNA damage alone induces mitochondrial dysfunction, we exploited the AID-DIvA system, selectively generating DNA double-strand breaks (DSBs) in nuclear DNA. DSB induction caused early impairment of mitochondrial bioenergetics, including reduced basal respiration, ATP-linked respiration, and maximal respiratory capacity, preceding more pronounced mitochondrial alterations, after prolonged damage. Resolution of DSBs restored mitochondrial function, demonstrating a direct and reversible link between nuclear genome instability and mitochondrial dysfunction. Mechanistically, persistent activation of the DNA damage response (DDR) triggered PARP1-dependent NAD{square} depletion, while PARP1 inhibition rescued mitochondrial respiration and ATP synthesis. We next investigated the consequences of DDR activation triggered by the expression of 102 (G4C2) repeats in an inducible cell model of C9ORF72-linked ALS. In these cells, DDR activation preceded mitochondrial dysfunction, recapitulating the sequence observed in AID-DIvA cells. Mitochondrial defects included impaired oxidative phosphorylation and reduced ATP production without increased mitochondrial ROS, suggesting that DNA damage signalling acts upstream of mitochondrial dysfunction. In support of this hypothesis, inhibition of ATM as well as nicotinamide riboside-mediated replenishment of cellular NAD+significantly restored mitochondrial functions. Collectively, our findings identify nuclear DNA damage as a trigger of mitochondrial dysfunction and uncover a pathogenic DDR-mitochondria crosstalk mediated by persistent DNA damage signalling. These results support a bidirectional relationship between genome instability and mitochondrial dysfunction and highlight mitochondrial and DNA damage response modulators as potential therapeutic targets for ALS and related neurodegenerative disorders.

Inflammasomes lead to activation of inflammatory caspases, which induce pyroptosis and an inflammatory immune response to control microbial infections. Inflammasomes are tightly regulated to avoid lethal sepsis and chronic autoimmune conditions. However, posttranslational regulation of inflammatory caspases remains poorly defined. We constructed 375 individual ubiquitin ligase knockout lines by CRISPR-Cas9, performed an unbiased screening, and identified Muscle Excess 3B (MEX3B), an RNA-binding protein and ubiquitin ligase, as a positive regulator of the caspase-4 inflammasome. Genetic depletion of MEX3B inhibited not only the caspase-4 but also NLRP3 and NLRC4 inflammasomes, regarding caspase activation, pyroptosis, and secretion of inflammasome-dependent cytokines, in human cells and murine primary macrophages. This MEX3B function required its RNA-binding, but not ubiquitin ligase activity. These results suggest that MEX3B is a pan-inflammasome regulator and a potential therapeutic target for inflammation.

Precancerous oncogenic activation in a target organ often induces senescence, a tumor-suppressive response known as oncogene-induced senescence (OIS). Clinical observations indicate a strong association of metabolic syndrome (MetS) with the precancerous and early-stage cancers. Notably, cells displaying OIS are characterized by a senescence-associated secretory phenotype (SASP), in which they secrete factors, including inflammatory cytokines. Thus, SASP from cells displaying OIS may trigger host MetS, which likely underpins its association with cancers, such as colorectal cancer (CRC). Here, we tested this hypothesis and show that, in Drosophila, the activated RasG12V oncogene, which is frequently implicated in human CRC, induces OIS in imaginal disc epithelium and systemically triggers host larval MetS via the conserved cytokine Upd1/IL6. Thus, the larval host with RasG12V-induced epithelial OIS displays MetS, characterized by obesity, increased lipid and glycogen accumulation in the fat body, and altered insulin signaling, marked by transition from hyperinsulinemia to insulin resistance--all at a precancerous stage. Further, we also noted hyperphagia and increased expression of insulin-like peptides (dILP2/3/5) in the brain of larvae displaying RasG12V-induced OIS. Notably, RasG12V-induced OIS is systemically relayed, leading to activation of a senescence-like program in the distant fat body. Genetic suppression of upd1 or pharmacological intervention with the senomorphic agent, Metformin, attenuated fat body senescence and mitigated MetS-associated phenotypes. Our findings thus identify a causal relationship between OIS and host MetS, suggesting its utility as an early biomarker for detecting cancers such as CRC and its potential as a prophylactic target.

Recent evidence indicates that mitochondria, through the activity of the E3 Ub ligase MARCH5, are critical for de novo peroxisome biogenesis. Here we report that peroxisome biogenesis factor Pex26 is a MARCH5 client protein. In peroxisome-containing cells, MARCH5 interacts with Pex26 and facilitates the transfer of newly synthesized Pex26 from the OMM to peroxisomes. MARCH5 also controls peroxisomal delivery of other candidate peroxins in peroxisome-containing cells. On the other hand, in peroxisome-deficient cells, the turnover rate of Pex26 is dramatically increased, and MARCH5 targets this protein for p97-dependent proteasomal degradation. Both activities are mediated by MARCH5-dependent Pex26 ubiquitination. Knockout of Pex26 induces the accumulation of cells containing Tom20-positive, Catalase-deficient pre-peroxisomes. Further supporting the critical role of MARCH5 in peroxisome biogenesis, these structures are absent in Pex26/MARCH5 double knockout cells. The data support the model, where in peroxisome-containing cells, MARCH5 acts as a peroxisome biogenesis factor, while with defective peroxisome biogenesis, as in Zellweger syndrome cells, it protects mitochondria from potentially toxic accumulation of peroxins on the OMM.

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

Skeletal muscles regenerate following injury, owing not only to myogenic stem cells, but also to non-myogenic cells such as fibro-adipogenic progenitors (FAPs). Quiescent in healthy muscles, FAPs transiently proliferate in response to tissue-damage, to support myogenic repair. Aside from their pro-myogenic role in healthy muscles, FAPs are also the origin of intramuscular fibro-adipose tissue that infiltrate muscles with chronic inflammation and degeneration in various muscle pathologies. Here, we investigate how the Fat1 Cadherin, previously identified as a regulator of embryonic muscle morphogenesis, influences FAP biology during damage-induced muscle regeneration. Fat1 expression is transiently induced in FAPs and myogenic cells after muscle damage. We found that mesenchyme-specific Fat1 ablation leads to increased fibro-adipogenic infiltrations following glycerol injury, while minimally affecting myogenic repair. Using an inducible Pdgfra-cre/ERT line, we further demonstrated that Fat1 restricts FAP adipogenic differentiation through both cell-autonomous and non-cell-autonomous mechanisms. These findings identify Fat1 as a novel regulator of FAP biology, essential for limiting FAP differentiation and the development of fibro-fatty infiltrations after muscle injury.

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

Viral infections are one of the main environmental factors triggering type 1 diabetes (T1D). Pancreatic alpha cells are more resistant than beta cells to diabetogenic viruses, partially explaining their survival in T1D. Similarly, bats have enhanced viral resistance, suggesting putative convergent evolution in antiviral mechanisms. Herein, we compared global gene expression in bat fibroblasts under basal conditions or exposed to double-stranded RNA to human alpha and beta cells and found that alpha cells exhibit greater similarity than beta cells to the antiviral responses of bat cells, as well as stronger intrinsic resistance to viral infection. Interferon-stimulated gene 15 (ISG15), a key regulator of antiviral responses in humans and bats, has higher expression in alpha compared to beta cells in five single-cell RNASeq datasets from human islet cells and in human induced pluripotent stem cell (hiPSC)-derived alpha-like cells. ISG15 knockdown in human insulin-producing EndoC-{beta}H1 cells and human islets increases apoptosis under basal conditions and after IFN exposure, exacerbates IFN responses and increases cell death and viral production after infection with the diabetogenic virus coxsackievirus B1, while its overexpression protects EndoC-{beta}H1 cells from the virus. Collectively, the present results demonstrate that alpha cells but not beta cells have similarities with the virus resistance gene program present in bats and identify ISG15 as an important factor for islet cells to cope with viral and diabetogenic stresses.

Biallelic mutations in the sorbitol dehydrogenase (SORD) gene have been identified as one of the most common causes of autosomal-recessive Charcot-Marie-Tooth disease type 2 (CMT2) and distal hereditary neuropathy, collectively referred to as SORD deficiency. These mutations result in loss of sorbitol dehydrogenase activity, a key enzyme in the polyol pathway that metabolizes glucose, leading to marked accumulation of sorbitol in patient-derived fibroblasts. However, the mechanisms by which SORD dysfunction drives axonal degeneration remain poorly understood, and robust in vitro models of human SORD-deficient motor neurons (MNs) are still lacking. To address this gap, we established a human in vitro model of SORD deficiency by generating induced pluripotent stem cells (iPSCs) from fibroblasts affected individual carrying biallelic SORD mutations (SORDc.757delG/c.316_425+165del), and unaffected heterozygous carriers (SORDc.757delG/wt and SORDwt/c.316_425+165del). These iPSCs were subsequently differentiated into motor neuron progenitors (MNPs) and MNs. Comprehensive analysis of SORD-deficient human cells--including fibroblasts, MNPs, and MNs--revealed pronounced structural and functional abnormalities in the mitochondrial compartment, characterized by mitochondrial fragmentation and increased proton leak. Importantly, fibroblasts derived from two additional unrelated patients carrying the SORD mutation (SORDc.757delG/ c.757delG) further confirmed that SORD deficiency is associated with a mitochondrial phenotype. At the molecular level, SORD deficiency led to upregulation of aldose reductase (AR), another key enzyme of the polyol pathway, resulting in disruption of cellular redox homeostasis and increased oxidative stress. Consistent with these alterations, MNs derived from CMT2/SORD patients exhibited clear neurodegenerative features, including severe defects in neurite branching and synaptic architecture, ultimately impairing neuronal connectivity. Notably, pharmacological inhibition of AR effectively rescued both mitochondrial dysfunction and neuronal structural defects, supporting the targeting of AR as a promising therapeutic strategy for polyol pathway-associated neuropathies as CMT2/SORD and diabetic neuropathy.

The mitochondrial membrane protein phosphoglycerate mutase 5 (PGAM5) is a protein of interest in the complex transition from hepatic steatosis to hepatocellular carcinoma. PGAM5 is a serine/threonine/histidine phosphatase that plays a role in mitochondrial biogenesis, mitophagy, and multiple cell death pathways. Increased expression of PGAM5 in hepatocellular carcinoma is correlated with reduced patient survival. In this study, we demonstrate that loss of PGAM5 alters the bioenergetic landscape of liver cancer by promoting mitochondrial oxidant injury and suppressing the glycerophospholipid and lysophospholipid pathways, leading to accumulation of the bioactive phospholipid lysophosphatidylcholine. Additionally, PGAM5 deletion downregulates fatty acid biosynthesis, resulting in reduced cellular diacylglycerol concentrations through two probable mechanisms: attenuated long chain fatty acid uptake and suppressed de novo synthesis. These findings underscore the broad impact of a single phosphatase on mitochondrial function and provide a rationale for therapeutically targeting PGAM5 to disrupt lipid metabolism in hepatocellular carcinoma.

Aging causes a progressive loss of tissue homeostasis, with stem cell exhaustion as a major hallmark. Age-associated decline in organ function is widely perceived as emanating from progressive accumulation of cellular damage in adult tissues. However, whether aging trajectories are established early on during development remains an open question. Here, we demonstrate that genetic modulation of cellular aging pathways in larval adult midgut progenitors (AMPs), which serve as the precursors of adult intestinal stem cells and differentiated epithelial cells, dictates the long-term trajectory of intestinal aging in Drosophila. Accelerated cellular aging by genetic perturbation employing Toll or Imd pathway overactivation or elevation of reactive oxygen species (ROS) using ND42 (mitochondrial complex I) knockdown in the AMPs results in aberrant progenitor proliferation, skewed lineage allocation, epithelial barrier dysfunction, and genomic instability. These alterations are accompanied by marked destabilization of AMP islet architecture and widespread changes in age-related molecular signatures, as revealed by bulk transcriptomic analysis. In contrast, decelerated cellular aging mediated by Foxo or Atg8a overexpression results in a decrease in enteroendocrine population and the intestinal barrier remained unaffected. Intriguingly, early-life activation of immune and oxidative stress signaling manifested later in the adult gut as elevated enteroendocrine differentiation, highlighting lasting effects on intestinal regenerative capacity and lineage balance. Together, our findings demonstrate that cellular aging is tightly regulated early on in development and its perturbation can cause developmental disruption hampering adult gut homeostasis, establishing AMPs as key developmental determinants that regulate the trajectory of intestinal aging in Drosophila.

PolDIP2 is a multifunctional mitochondrial protein implicated in redox regulation, mitochondrial proteostasis, and diverse mtDNA-associated processes, yet the principles underlying its regulation remain unclear. Crystallographic analysis revealed that PolDIP2 forms a redox-dependent disulfide-linked homodimer via a conserved Cys143 residue within its N-terminal YccV-like domain, and cellular and in vitro assays confirmed that this residue is essential for dimer formation. Oxidative stress enhanced dimerization of endogenous and ectopically expressed PolDIP2, and dimers were detected exclusively within mitochondria, requiring proper mitochondrial import. WT and C143A PolDIP2 overexpression produced similarly modest effects on mtDNA replication in cells, suggesting that dimerization has limited impact on mtDNA-associated processes. Proteomic analysis and biochemical validation identified both previously known and not yet characterized mitochondrial interactors of PolDIP2, and highlighted CHCHD2 as a specific binding partner. A conserved glycine-rich motif in the C-terminal ApaG/DUF525-like domain proved essential for this interaction, and disruption of the motif enhanced Cys143-dependent dimerization while abolishing CHCHD2 association, which preferentially occurs with monomeric PolDIP2. These findings define redox-controlled dimerization and a conserved ApaG-domain motif as key structural features shaping PolDIP2s interaction state within mitochondria and provide a basis for exploring its roles in redox-sensitive mitochondrial pathways.

Epithelial-mesenchymal transition (EMT) and glycolytic metabolism are well-characterized drivers of cancer progression and metastasis. However, most primary breast tumors and metastases express E-cadherin and the epithelial phenotype is associated with mitochondrial oxidative metabolism, yet the causality and relevance of these relationships and their underlying mechanisms remain poorly understood. Using a 3D culture model with mechano-stimulation, we found that E-cadherin promotes mitochondrial oxidative phosphorylation (OXPHOS) while reducing oxidative stress. Through pharmacological and genetic manipulations of inflammatory breast cancer (IBC) and/or triple negative breast cancer (TNBC) cell lines, we identified pyruvate carboxylase (PC) as an E-cadherin effector. Critically, restoring PC in E-cadherin-silenced cells rescued mitochondrial oxygen consumption and protection from oxidative stress. Co-expression of E-cadherin and PC was confirmed in breast cancer tissues and experimental lung metastases. Mechanistically, E-cadherin induced PC expression and OXPHOS via AKT-mediated activation of YAP/ /TEAD transcription factors, which are better known as supporting EMT. Clinically relevant AKT and TEAD inhibitors reduced both PC expression and oxidative respiration. Importantly, PC inhibition as monotherapy attenuated established experimental lung metastases and primary tumor burden in mice. Taken together, these findings reveal that E-cadherin-mediated cell-cell adhesions directly support mitochondrial metabolism through AKT-YAP/TEAD-PC signaling, identifying a therapeutic vulnerability in metastatic epithelial TNBC.

During viral infection, viral replication perturbs endoplasmic reticulum (ER) homeostasis and triggers the unfolded protein response (UPR). XBP1s, a transcription factor generated by one branch of the UPR, is known to potentiate both innate and adaptive immunity, but its role in antiviral responses remains incompletely understood beyond its ability to augment type I interferon (IFN) mRNA induction. Here, we show that XBP1s positively regulates the RIG-I-like receptors (RLRs), ribonuclease L (RNase L), and protein kinase R (PKR) pathways, indicating that it enhances all three major antiviral response pathways. We further show that RNase L activation rapidly decreases XBP1 mRNA levels in an RNase activity-dependent manner, leading to a prompt reduction in XBP1s expression. Consistent with this, RNase L deletion significantly increased both thapsigargin-mediated XBP1s induction and XBP1s expression following Japan encephalitis virus infection. Poly(I:C)-induced IFNB mRNA expression was significantly enhanced in RNase L-knockout cells. This enhancement was completely abolished by RNase L reconstitution. XBP1 knockdown also significantly attenuated IFNB mRNA expression in RNase L-knockout cells. These findings suggest a negative-feedback loop in which RNase L suppresses XBP1s, thereby fine-tuning antiviral responsiveness during viral infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/713401v1_ufig1.gif" ALT="Figure 1000"> View larger version (19K): org.highwire.dtl.DTLVardef@112d312org.highwire.dtl.DTLVardef@df79a9org.highwire.dtl.DTLVardef@1ac571borg.highwire.dtl.DTLVardef@18ac610_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cellular senescence is a heterogeneous cell state induced by diverse stressors, including telomere attrition, genotoxic agents, oxidative damage, and inflammation. Despite ongoing efforts to identify conserved senescence biomarkers, it remains unclear whether senescence-inducing stimuli converge at the level of individual genes or broader molecular processes. Here, we profiled transcriptomic changes in human primary lung fibroblasts (IMR-90) driven toward senescence by replicative exhaustion, bleomycin, H2O2, or ionizing radiation under matched, dose- or time-resolved conditions. Across all four senescent inducers, global transcriptomic variation aligned along a shared axis of senescence progression, consistent with established machine learning-based senescence classifiers. However, overlap at the level of individual genes was limited, with most responses being inducer-specific or only partially conserved. In contrast, pathway-level analysis revealed far more consistent enrichment across all conditions, including downregulation of proliferation-associated pathways and activation of stress-related and pro-inflammatory pathways, accompanied by distinct inducer-specific patterns. These results support a hierarchical organization of the senescent transcriptome, in which diverse senescence inducers converge on shared pathway-level features while maintaining gene-level heterogeneity. These results provide a foundational basis for interpreting senescence signatures and may facilitate the development of more robust transcriptome-based markers of cellular senescence in aging and disease.

3-mercaptopyruvate sulfurtransferase (3-MST) is a mammalian enzyme that contributes to hydrogen sulfide and reactive sulfur species generation. Here we show that 3-MST is markedly upregulated in colorectal cancer stem cells (CSCs) and functions as a critical metabolic support mechanism for this therapy-resistant tumor cell population. CSCs exhibit low proliferation rate, high membrane rigidity and a metabolically restrained phenotype characterized by low oxidative phosphorylation rate, combined with a reduced rate of glycolysis. Genetic or pharmacological inhibition of 3-MST further suppresses cellular bioenergetics in CSCs, and this bioenergetic collapse impairs CSC proliferation, spheroid formation, migration and promotes cell death and attenuates tumor growth. Integrated transcriptomic, proteomic, metabolomic, and lipidomic analyses reveal extensive metabolic remodeling of the CSCs following 3-MST inhibition, including disruption of the glycolysis-TCA axis and marked remodeling of membrane lipid composition, including enrichment of ceramides and sphingolipids and increased incorporation of polyunsaturated phospholipids, resulting in increased membrane fluidity. 3-MST inhibition induced an activation of integrated stress pathways, proteotoxic stress responses and inflammatory signaling, linking the metabolic failure of CSCs to the induction of mixed-mode cell death. These findings identify 3-MST as a metabolic vulnerability in colorectal CSCs. Targeting this enzyme may be a translatable strategy to eliminate therapy-resistant tumor stem cell populations.