Cell Death & Differentiation

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.

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.

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.

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.

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.

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

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.

Immunosuppressive tumor microenvironment (TME) inactivates CD8+ cytotoxic lymphocytes (CTLs). Here, we identify SPTBN2 spectrin as a key immunosuppressive regulator induced in CTLs in response to nutritional deficit. In human pancreatic and colorectal cancers, SPTBN2 expression negatively correlated with CTL infiltration and patients survival. In TME of mouse pancreatic and colorectal adenocarcinomas, SPTBN2 inactivated intratumoral CTLs, stimulated tumor growth and conferred cross-resistance to anti-cancer therapies. SPTBN2 knockout protected CAR T-cells from trogocytosis and increased their memory state. SPTBN2 maintained levels of cell surface proteins such as BTLA that undermine CAR T-cell cytotoxicity and promote exhaustion. Re-expression of BTLA largely reversed phenotypes in SPTBN2-deficient CAR T-cells. In manufactured CAR T cells, SPTBN2 was associated with their clinical failure in pediatric patients with leukemia. Accordingly, ablation of SPTBN2 in CAR T-cells increased their cytotoxicity, in vivo persistence and therapeutic effects indicating that SPTBN2 can be targeted to increase the efficacy of anti-cancer therapies.

TP53 mutations occur in 80-90% of triple-negative breast cancers (TNBCs) and drive genomic instability and metastatic progression. Poly (ADP-ribose) polymerase (PARP) is critical for DNA repair and replication fork stability. How oncogenic signaling influences PARP function to sustain proliferation during replication stress remains unclear. Mutant p53 (mtp53) R273H associates tightly with chromatin, forms complexes with PARP, and enhances PARP recruitment to replication forks [1-3]. The C-terminal region of mtp53 mediates mtp53-PARP and mtp53-Poly (ADP-ribose) (PAR) interactions that facilitate S phase progression [4, 5]. The PARP inhibitor talazoparib (TAL) combined with the alkylating agent temozolomide (TMZ) produces synergistic cytotoxicity selectively in mtp53, but not wild-type p53 (wtp53), breast cancer cells and organoids. Herein we evaluated the mechanism of mtp53-associated cell death and tested if this could translate to a preclinical xenograft model. We found that TMZ+TAL treatment induced elevated cleaved PARP and {gamma}H2AX and reduced the metastasis-promoting oncoprotein MDMX. In orthotopic xenografts expressing mtp53 R273H, but not wtp53, combination therapy significantly decreased circulating tumor cells (CTCs) and lung metastases. Transcriptomic profiling of tumors from combination treated animals demonstrated downregulation of MDMX, VEGF, and NF-{kappa}B, consistent with the observed suppression of CTCs and lung metastasis, and increased {gamma}H2AX, indicative of replication stress in mtp53 xenografts. Inhibition of metastasis was also observed in mtp53 R273H WHIM25 and p53-undetectable WHIM6 TNBC patient-derived xenografts (PDX). The mtp53 C-terminal domain (347-393) demonstrated a critical tumor promoting function, as CRISPR-mediated deletion impaired replication fork progression, tumor growth, and metastatic dissemination. DNA fiber combing showed that expression of full-length mtp53 R273H, but not C-terminal deleted {Delta}347-393, supported sustained single-stranded DNA gaps (ssGAPs) following Poly (ADP-ribose) glycohydrolase (PARG) inhibition. These findings support that mtp53 uses C-terminal amino acids to exploit PARP to enable replication stress adaptation and that mtp53 is a predictive biomarker for combined PARP inhibitor and DNA damaging therapies targeting TNBC. Significance statementTP53 mutations are the most common genetic alterations in TNBC and a major driver of replication stress and metastasis. This study shows that missense mutant p53 uses C-terminal amino acids to reprogram PARP activity to maintain tumor cell survival under replication stress. We demonstrate that p53 status governs the response to combined PARP inhibitor (PARPi) and DNA-damaging chemotherapy, establishing an additional molecular basis beyond BRCA1 mutations for treating TNBC with PARPi therapy. These findings reveal a previously unrecognized mechanism by which the mutant p53-PARP axis enables replication stress tolerance and drives cancer metastasis. We show mutation of p53 in TNBC provides an additional biomarker-guided framework to improve PARPi therapeutic outcomes.

The RAS guanine nucleotide exchange factors Son of Sevenless 1 and 2 (SOS1 and SOS2) are key regulators of RAS signaling pathways controlling cellular proliferation, differentiation, and survival processes that are essential for correct tissue homeostasis. While mice lacking both SOS1 and SOS2 die precipitously, we demonstrate herein that the combined genetic ablation of SOS1 and SOS2 triggers spontaneous, gut-derived, lethal bacteremia. Double-knockout (DKO) SOS1/2 mice exhibit extensive intestinal tissue damage, massive bacterial leakage out of the gut, and rapid progression to multi-organ failure and death. At the cellular level, loss of both SOS1 and SOS2 leads to profound immune cell depletion and a marked reduction in intestinal stem cell abundance and proliferative capacity, which is accompanied by severe disruption of intestinal architecture and increased epithelial permeability, indicating a breakdown of gut barrier integrity. Notably, therapeutic interventions aimed at enhancing cellular stemness significantly improve survival in SOS1/2 DKO mice, restoring intestinal proliferation and tissue organization. Collectively, our findings identify SOS1 and SOS2 as critical regulators of intestinal homeostasis and regenerative capacity during systemic infection and reveal stemness reinforcement as a potential strategy to overcome lethal susceptibility to sepsis.

Rab GTPases orchestrate vesicular trafficking, but their contributions to mitochondrial quality control are not fully defined, despite links to multiple mitochondria-related human diseases. We conducted a family-wide siRNA-based screen using mt-mKeima/YFP-Parkin HeLa cells to identify regulators of depolarization-induced mitophagy. The screen identified several candidate Rabs, and follow-up studies validated Rab12 as a negative regulator of mitophagy. Rab12 knockdown or knockout augments clearance of damaged mitochondria basally and/or after FCCP-induced depolarization, with findings reproduced across distinct cell types. Rab12 depletion increased mitochondrial content, lowered mitochondrial membrane potential, and reduced mitochondrial DNA damage, without detectable changes in overall cellular bioenergetic capacity. Together, these results indicate that Rab12 restrains mitophagic engagement and its loss permits accumulation of lower-functioning mitochondria that are hypersensitive to mitophagy-inducing stress. Rab12 thus emerges as a novel effector linking vesicular trafficking machinery and mitochondrial homeostasis, with potential implications for neurodegenerative disorders and other Rab-associated diseases.

Myocardial ischemia-reperfusion injury significantly exacerbates cardiac damage and worsens clinical outcomes, with oxidative stress in cardiomyocytes representing a central pathological mechanism. In this study, we reveal that APEX1, a key redox regulator, is markedly downregulated in cardiomyocytes under oxidative stress conditions. Functional analyses demonstrate that APEX1 knockdown intensifies oxidative stress-induced cardiomyocyte injury, whereas APEX1 overexpression confers robust protection against hypoxia reoxygenation mediated damage. Mechanistically, APEX1 exerts its cardioprotective effects by stabilizing the p53 protein and modulating its ubiquitination status. These findings establish APEX1 as a critical defender against oxidative injury in cardiomyocytes through direct regulation of p53 protein stability, highlighting its potential as a therapeutic target for ischemia-reperfusion related heart disease.

Endoplasmic reticulum (ER) stress in pancreatic beta cells contributes to impaired function and type 2 diabetes (T2D). In this study we performed genome-wide perturbation screens and genomic profiling in beta cells to identify novel mediators of ER stress responses and diabetes risk. We defined gene regulatory networks in beta cells and identified specific beta cell networks enriched for T2D risk variants with altered expression in ER stress. We performed a loss-of-function CRISPR screen for survival under ER stress in EndoC-{beta}H1 cells, which identified 167 pro-survival and 47 pro-death genes involved in processes related to insulin secretion, mitochondrial transport and protein ubiquitination. Beta cell survival genes collectively had limited genomic change in stress yet showed significant, independent enrichment for T2D risk variants, including novel T2D candidate gene DTNB which we validated protects against beta cell death during stress. Overall, our results revealed mediators of ER stress responses in beta cells and identified new therapeutic targets to preserve beta cells in diabetes pathogenesis.

Signalling via the epidermal growth factor receptor (EGFR) is indispensable for morphogenesis and tissue homeostasis. It is activated by extracellular ligands, typically released from transmembrane precursors by proteolysis. Ligand shedding activity is provided by the conserved rhomboid intramembrane serine proteases in Drosophila, but by the unrelated ADAM family metalloproteases in mammals, leaving the functions of mammalian non-mitochondrial rhomboids underexplored. Using quantitative proteomics, we show that EGFR is the main endogenous substrate of the human rhomboid protease RHBDL2 in keratinocytes. By shedding the EGFR ectodomain, thus producing a decoy receptor, RHBDL2 suppresses EGFR signalling, limiting cell migration and invasion. Conspicuously, RHBDL2 activity is upregulated by elevated intracellular calcium concentration, a condition typical for keratinocyte differentiation. These effects are recapitulated in primary human keratinocytes, and human skin equivalents deficient in RHBDL2 display incomplete differentiation and are morphologically disordered compared to wild type cells. We propose that context-specific fine-tuning of EGFR signalling and sensitivity to cross-talk from other signalling pathways could be important and hitherto overlooked roles of rhomboid proteases in mammals.

Oncolytic adenovirus (ADV) therapy faces heterogeneous responses, implying tumor-intrinsic resistance. We identify interleukin-17 (IL-17) signaling as a novel potential barrier associated with multi-modal cellular reprogramming. Transcriptomic analysis of ADV-treated 4T1 murine mammary carcinoma cells revealed specific upregulation of Il17rb, Il17rd, and Il17f, indicating viral induction of this inflammatory axis. The IL-17 signature correlates strongly with a cancer stemness phenotype. Metabolically, it associates with increased lipid metabolism and suppressed glycolysis, suggesting a state resistant to viral replication. Furthermore, it broadly negatively correlates with programmed cell death pathways (apoptosis, necrosis) while positively associating with pro-survival autophagy. IL-17 component expression effectively stratifies samples into distinct metastatic risk categories, underscoring its prognostic potential. Our findings reveal a previously unrecognized, tumor-intrinsic role for IL-17 signaling in ADV resistance, associated with enhanced stemness, altered metabolism, and impaired cell death. This nominates the IL-17 pathway as both a predictive biomarker and a therapeutic target for combination strategies. HighlightsO_LIOncolytic adenovirus infection selectively upregulates IL-17 receptor subunits (IL17RB, IL17RD) and IL17F ligand in 4T1 tumor cells C_LIO_LIIL-17 receptor expression strongly correlates with cancer stemness gene signatures, particularly through IL17RB and IL17RD C_LIO_LIThe IL-17 axis associates with broad suppression of lytic cell death pathways (apoptosis, necrosis, necroptosis) while positively correlating with autophagy C_LIO_LIIL-17 pathway activity correlates with metabolic reprogramming favoring lipid turnover over glycolysis C_LIO_LIIL-17 expression levels stratify samples into distinct metastatic risk categories, suggesting biomarker potential C_LI

Despite lacking a robust interferon response, pluripotent stem cells remain highly resistant to viral infection, in part through the constitutive expression of immune genes traditionally classified as interferon-stimulated genes. While interferon signaling has been shown to be incompatible with the maintenance of pluripotency, the molecular mechanisms underlying this relationship remain poorly understood. Here, we investigate the transcriptional response of human embryonic stem cells (hESCs) to infection with a potent activator of the interferon response, an influenza A virus mutant lacking the viral NS1 protein. Single-cell RNA sequencing revealed that while most hESCs remain unresponsive to infection, a distinct subpopulation expresses type I and III interferons. Notably, only interferon-expressing cells mounted a robust antiviral response, characterized by strong induction of interferon-stimulated genes. In contrast to the bulk hESC population, interferon responding cells exhibited reduced expression of core pluripotency factors as well as negative regulators of interferon signaling, such as SOCS1 and SPRY4. Depletion of SOCS1 enabled hESCs to respond robustly to interferon stimulation, showing that this negative regulator is a key suppressor of interferon signaling in pluripotent stem cells. We further show that SOCS1 and additional negative regulators of IFN signaling are intrinsically expressed in hESCs and are transcriptionally controlled by pluripotency factors, such as NANOG, SOX2 and OCT4. Together, our findings support a model in which pluripotency factors regulate intrinsic immune gene expression, including negative regulators of interferon signaling, thereby suppressing canonical interferon signaling to preserve pluripotency while maintaining antiviral resistance. IMPORTANCEBy combining single-cell transcriptomics with functional studies, we demonstrate that the pluripotency transcriptional program active in pluripotent stem cells coordinately regulates pluripotency factors, antiviral genes, and negative regulators of interferon signaling. This integrated control enables pluripotent stem cells to achieve effective protection against viral infection while preserving their differentiation potential, providing new insights into how innate immunity is selectively constrained in pluripotent stem cells. These findings have important implications for stem cell-based therapies, where transient modulation of antiviral responses without disrupting pluripotency could improve therapeutic efficacy. More broadly, this work advances our understanding of interferon regulation that could inform the development of antiviral strategies that enhance protective immune responses while limiting harmful or unwanted inflammatory signaling.

Transactive response DNA binding protein 43 kDa (TDP-43) pathology, is a central molecular hallmark of amyotrophic lateral sclerosis (ALS). However, the underlying triggers are incompletely understood. Here, we show that infection with herpes simplex virus (HSV) induces molecular hallmarks of ALS in various in vitro and in vivo models and is associated with an increased risk of ALS in human population data. German healthcare provider data (n = 238,440) and herpesvirus serology of an ALS patient and control cohort (n = 1,100) showed that HSV infection elevated the ALS risk by 210% and odds by [~]65%, respectively. On a molecular level, HSV infection promoted TDP-43 pathology in neuronal cell models, human iPSC-derived motoneurons and cerebral organoids, mice, and human tissue sections. This effect was triggered by HSV-1 or 2, but not by several other related herpesviruses. Mechanistically, the infected cell protein 0 (ICP0) of HSV-1/2 drives TDP-43 pathology by disturbance of promyelocytic leukemia nuclear bodies (PML-NBs), thereby abrogating TDP-43 SUMO2/3ylation. Taken together, we reveal a previously unrecognized association between HSV infection and ALS and clarify the underlying molecular mechanism that drives TDP-43 pathology. Our data may guide future studies into therapeutic and prophylactic interventions against ALS.

Triple negative breast cancer (TNBC) patients harboring residual cancer burden following completion of conventional neoadjuvant chemo-immunotherapy regimens have poor relapse-free and overall survival rates despite recent advances in immunotherapies and antibody drug conjugates. We and others have demonstrated the requirement of mitochondrial function for survival of chemo-refractory TNBC, as well as its pervasive association with chemoresistance in human and patient-derived xenograft (PDX) cohorts. We sought to gain new mechanistic insights into the mitochondrial vulnerability of TNBC. Analyses of human and PDX mass spectrometry proteomics datasets revealed that mitochondrial protein translation-related signatures were among the top significantly associated with chemoresistance. Those signatures encompassed many core mitoribosome components as well as the mitoribosome accessory protein, Oxidase (Cytochrome C) Assembly 1-Like (OXA1L), which was consistently enriched in chemoresistant versus chemosensitive TNBCs across datasets. OXA1L, while not yet characterized in cancer, has been reported to be crucial for the termination of translation of the 13 mtDNA-encoded electron transport chain (ETC) proteins and for the insertion of those proteins, as well as nDNA-encoded ETC proteins, into the inner mitochondrial membrane. Together, those functions are crucial for the proper formation and function of the ETC. Therefore, we hypothesized that mitochondrial translation supported by OXA1L supports mitochondrial dependence and chemoresistance in TNBC. Knockdown (KD) of OXA1L in human TNBC cells reduced ETC protein levels, mitochondrial respirasome supercomplex levels, ATP production, and oxidative phosphorylation (oxphos). Of note, OXA1L was required for the characteristic oxphos elevation induced by carboplatin (CRB), and KD significantly enhanced CRB sensitivity. To explore the translational potential of targeting the mitoribosome in TNBC, we leveraged the bacterial ancestry of mitochondria to repurpose the FDA-approved antibiotic tigecycline (TIG) as a chemo-sensitizing drug based on its mitoribosome inhibitory function. Direct measurement of mitochondrial nascent peptide levels revealed that, while CRB elevated mitochondrial translation, TIG potently diminished mitochondrial translation as monotherapy and when combined with CRB or docetaxel (DTX). Further, TIG abolished CRB-induced oxphos, decreased oxphos in combination with DTX, and significantly improved sensitivity to chemotherapies in human TNBC cell lines, PDX-derived spheroids, and in an in vivo PDX trial. These findings identify OXA1L-dependent mitochondrial translation and ETC formation as critical determinants of mitochondrial function that support TNBC chemoresistance, justifying further exploration of the clinical potential of repurposed antibiotics for TNBC. DISCLOSURESGVE is co-founder, Chief Scientific Officer, and an equity stakeholder of Nemea Therapeutics. G.V.E. formerly received sponsored research funding from Chimerix Inc. G.V.E. receives experimental compounds from the Lead Discovery Center of Germany and from Jazz Pharmaceuticals. MLB is a co-inventor at Nemea Therapeutics. MTL is a founder and limited partner in StemMed Ltd. and a manager in StemMed Holdings, its general partner. He is a founder and equity stakeholder in Tvardi Therapeutics Inc. Some PDX models, including BCM-4272 and BCM-7649, are exclusively licensed to StemMed Ltd., resulting in royalty income to MTL when used for commercial purposes. LED is a compensated employee of StemMed Ltd. Some PDX models, none of which are included in this study, are exclusively licensed to StemMed Ltd., resulting in royalty income to LED. All other authors have nothing to disclose.

To elucidate how cancer cells generate resistance and defined forms of tissue structure in response to therapeutic stress, we tracked the temporal dynamics of life cycle of polyploid giant cancer cells (PGCCs) induced by the mitotic destabilizer vincristine (VCR). Live-cell fluorescence imaging revealed that VCR activated a distinct endoreplication-based life cycle, which replaced canonical mitosis. PGCCs exhibited reduced proliferative activity, enhanced epithelial-mesenchymal transition (EMT), progressive acquisition of blastomere-like features, and broad multilineage differentiation potential. Both PGCC populations and single PGCC-derived progeny displayed time- and dose-dependent acquisition of malignant traits in vitro and tumorigenic capacity in vivo. PGCC-derived spheroids exhibited ability to differentiate into the cells of origin from three germ layers. Importantly, pre-budding PGCCs induced by higher VCR concentrations exhibited enhanced ability for glandular structure formation and tissue differentiation. Morphologically, the nuclei of PGCC-derived spheroids underwent growth in size, formation of luminal structure, and followed by maturation of lumen. Mechanistically, PGCCs entered a senescent state characterized by elevated senescence-associated secretory phenotype (SASP)-manifested by rich proinflammatory cytokines. Notably, silencing IL1{beta}, IL6, or IL8, or pharmacological inhibition of their receptors, suppressed PGCC formation, budding, EMT, and blastomere-like reprogramming into structured tissue. Our studies provide novel mechanistic insights into early embryogenesis and tumorigenesis at the tissue structural developmental level.