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.

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.

Activation-induced cytidine deaminase (AID), which is essential for antibody diversification, exhibits elevated expression in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here, we demonstrate that AID modulates transcriptional programs linked to cell cycle progression, proliferation, and DLBCL subtype identity. AID loss in ABC-type DLBCL cell lines negatively impacts MYC and E2F pathway activity, while AID re-expression restores activity, establishing a causal link. Consequently, loss of AID delays G1/S cell cycle transition and reduces proliferation. In addition, AID expression skews transcriptional programs towards ABC-type DLBCL in cell lines. In agreement, AID expression correlates with ABC-type gene expression in primary DLBCL patient samples. Moreover, AID overexpression resulted in increased IRF4 protein levels, and enhanced NF-{kappa}B activity, supporting AIDs role in reinforcing the ABC-type identity. Shared enrichment of the IRF4 co-factor BATF in AID-high tumors of both ABC- and GCB-subtypes points towards a common mechanism driving subtype skewing. These findings underscore a broader role for AID in DLBCL pathogenesis, establishing AID as a key regulator of transcriptional programs linked to cell cycle progression and DLBCL subtype.

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.

Y box-binding protein 1 (YB-1; Ybx1/ybx1) is essential for zebrafish development. Maternal ybx1-/- mutants exhibited embryonic lethality, whereas zygotic mutants (Zybx1-/-) showed high postnatal mortality between 10 and 20 dpf, although a small fraction survived to adulthood. Western blot and immunohistochemical analysis revealed strong, transient expression of Ybx1 in intestinal enterocytes from 3 to 5 days post-fertilization (dpf), followed by rapid ubiquitin-mediated degradation at 6 dpf, coinciding with defective intestinal development and compromised gut homeostasis. RNA-seq analysis identified elevated reactive oxygen species (ROS) and upregulation of matrix metalloproteinases mmp9 and mmp13a in Zybx1-/- larvae. Antioxidant treatment with ascorbic acid rescued postnatal lethality and alleviated intestinal defects, whereas prooxidant exposure exacerbated them. Pharmacological inhibition of Mmp9 or Mmp13a similarly prevented lethality, highlighting a ROS-MMP axis driving tissue damage. By 30 dpf, surviving mutants exhibited progressive intestinal impairment and severe pathology. These findings demonstrate that Ybx1 deficiency triggers ROS-dependent intestinal inflammation, MMP-mediated gut damage, and postnatal lethality, establishing Ybx1-deficient zebrafish as a robust model for studying inflammatory bowel disease (IBD)-like intestinal disorders.

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.

Despite progress in recent decades, breast cancers remain the most diagnosed malignancies, and second leading cause of cancer death, in women. Although improved screening and systemic and endocrine adjuvant approaches have contributed to major declines in breast cancer mortality, current standard of care drugs are extremely toxic, and many women continue to be overtreated. Although nearly two-thirds of breast cancers are hormone-responsive, aggressive subtypes, particularly Triple Negative Breast Cancer (TNBC), still lack safe oral medications. Recently the roles that mitochondria play in TNBC carcinogenesis, metastasis, and resistance to treatment have garnered a great deal of attention. Contrary to the popular dogma that cancer cells are powered by glycolysis, metastatic breast cancer cells have enhanced mitochondrial function. Our work identified that Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1; also, CHCHD2, PARK22), a key coordinator of mitochondrial-nuclear crosstalk that is physically present in both compartments, is overexpressed in TNBC cells and is an important regulator of metastasis signaling. We have identified Heat Shock Factor 1 (HSF1) as the main transcription factor that activates the MNRR1 promoter in TNBC cell lines. In the mitochondria, MNRR1 protein facilitates ATP production and inhibits apoptosis, whereas in the nucleus it regulates the transcription of stress-responsive genes including several required for epithelial to mesenchymal transition (EMT), metabolic flexibility, and cell growth. Thus, each of the bi-organellar functions of MNRR1 constitutes processes regarded as hallmarks of cancer. For reasons that are not yet fully understood, MNRR1 levels display a significant and robust ancestry bias, showing increased expression in tumor samples from Non-Hispanic Black (NHB) women when compared to disease-matched tumors from Non-Hispanic White (NHW) patients. It is possible that increased levels of MNRR1 may underlie the aggressive metastatic phenotype observed in many NHB patients. In further support of this observation, loss of MNRR1 function, either genetically or by use of inhibitors, reduces TNBC growth and metastasis. MNRR1 therefore is an attractive therapeutic target that could be exploited for design of novel therapies or as adjuncts to existing ones.

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.

The biogenesis of integral membrane proteins is complex, as revealed by an ever-growing number of cellular components shown to be dedicated to the insertion, folding, surveillance, rectification, or quality control of specific client membrane proteins. The zinc metalloprotease ZMPSTE24 and its yeast homolog Ste24 have well-established roles in the proteolytic maturation of the nuclear scaffold protein lamin A and yeast a-factor, respectively. Additionally, Ste24 has been implicated through yeast genetic screens in a variety of membrane processes, including ER- associated degradation (ERAD), Sec61 translocon "unclogging," the unfolded protein response (UPR), and potentially as a membrane protein topology determinant. Recently, an interaction was demonstrated between ZMPSTE24 and the antiviral interferon induced transmembrane protein IFITM3, although the functional significance of this interaction is not well-understood. IFITM3 is a tail-anchored protein with a cytoplasmic N-terminus, a single transmembrane span, and a lumenal/exocellular C-terminus. Here, we show that a catalytic-dead version of ZMPSTE24, ZMPSTE24E336A, exhibits enhanced binding to IFITM3, and this bound species of IFITM3 is hypo-palmitoylated. Using a split fluorescence topology reporter, we demonstrate that ZMPSTE24E336A "traps" and stabilizes a subpopulation of IFITM3 molecules with an atypical membrane topology, whose C-terminus is cytosolic instead of lumenal. Such inverted forms of IFITM3 are also detected in the presence of ERAD inhibitors when ZMPSTE24E336A is absent. We hypothesize the ZMPSTE24E336A trap mutant reveals a normally transient isoform of IFITM3 whose transmembrane span is inverted and that ZMPSTE24 is involved in the quality control of IFITM3 topology, either inverting, correcting or assisting in removal of aberrant IFITM3 molecules.

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.

Centrosome amplification (CA) is a hallmark of aggressive cancers, including pancreatic ductal adenocarcinoma (PDAC), and is linked to genomic instability and poor prognosis. While CA promotes tumor evolution, it also imposes substantial intracellular stress that cells must overcome to survive. However, the specific metabolic adaptations that enable cancer cells to tolerate stress induced by supernumerary centrosomes remain poorly understood. Here, we show that PDAC cells with CA acquire distinct metabolic dependencies that sustain survival. A metabolism-focused CRISPR-Cas9 screen, coupled with functional validations, identified critical vulnerabilities in three inter-connected axes: redox homeostasis, nucleotide sugar metabolism, and the unfolded protein response (UPR). Specifically, CA elevates intracellular reactive oxygen species (ROS), creating a reliance on glutamine metabolism and NRF2-driven antioxidant signaling. CRISPR screen hits in the hexosamine and uronic acid pathways revealed dependencies that converge on hyaluronic acid (HA) metabolism, and functional assays demonstrated that the HA-CD44 axis is required for centrosome clustering and mitotic fidelity, with its disruption increasing lethal multipolar divisions. In parallel, CA activated all branches of the UPR, and both hyper-activation and suppression of ER stress proved detrimental, indicating a finely tuned proteostatic equilibrium is essential for adaptation. Together, these findings show that, in a PLK4-driven model, centrosome-amplified cells rely on coordinated redox control, proteostatic buffering, and extracellular matrix signaling to tolerate CA-induced stress, revealing selective vulnerabilities that could be therapeutically exploited to target aggressive, therapy-resistant tumor subpopulations.

S100A4, a metastasis promoting calcium binding protein, drives tumor progression through pleiotropic mechanisms, yet its context dependent functions in gestational malignancies remain elusive. To dynamically decode its role in choriocarcinoma pathogenesis, we leveraged label free real time cell analysis (RTCA) to profile malignant phenotypes in JAR cells following siRNA mediated S100A4 silencing, complemented by apoptosis assessment and targeted signaling profiling. Efficient knockdown (verified by qPCR/Western blotting) significantly attenuated cellular proliferation (96 hr cell index slope decreased vs. scramble control; p<0.01) and suppressed migration capacity (p<0.01). Critically, S100A4 depletion did not induce apoptosis (flow cytometry and cleaved caspase 3/9 blotting confirmed no significant change), and invasion through Matrigel coated membranes remained statistically unaltered despite comparable experimental rigor. Mechanistically, S100A4 silencing triggered adaptive signaling rewiring: IRS1 and PI3K expression were elevated, Akt1 was suppressed, while MEK1/2 remained unchanged suggesting compensatory pathway activation.

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.

Mitophagy is generally considered to promote cell survival by removing damaged mitochondria in response to mitochondrial stress, whereas apoptosis occurs during prolonged stress. However, the mechanisms that determine cell survival and cell death under these stress conditions remain poorly understood. Here, we showed that cytoplasmic mRNA granules, designated as mitophagy-induced mRNA granules (mitoRGs), were formed transiently and played an important role in cell fate decisions during PINK1/Parkin-dependent mitophagy. Although some components, such as G3BP1, were shared with stress granules (SGs), mitoRGs were distinct from SGs because mitoRG assembly required the mitochondrial protein phosphatase PGAM5. In response to mitochondrial stress, PGAM5 was released into the cytosol from mitochondria and incorporated into mitoRGs, but was then released back into the cytosol during mitoRG disassembly following prolonged mitochondrial stress, corresponding with the induction of apoptosis. Impairment of mitoRG assembly through G3BP1 depletion sensitized cells to apoptosis during mitophagy in a PGAM5-dependent manner. These results suggest that mitoRGs regulate cell fate decisions by spatiotemporally controlling PGAM5 and its pro-apoptotic activity during PINK1/Parkin mitophagy.

Genetic inhibition of cyclophilin D (CypD) delays the opening of the mitochondrial permeability transition pore (MPTP) and therefore reduces necrotic cell death. Elucidation of factors that impact CypD activity is therefore key to understanding the regulation of MPTP opening. Glycogen synthase kinase-3{beta} (GSK3{beta}) is a serine/threonine kinase that has been shown to modulate MPTP and cell death, potentially through phosphorylation of CypD. Therefore, we hypothesized that the mitochondrial fraction of GSK3{beta} directly phosphorylates CypD and promotes opening of MPTP. Overexpression of full length GSK3{beta} in mouse embryonic fibroblasts sensitized the MPTP and exacerbated oxidative stress-induced necrosis. In contrast, genetic inhibition of GSK3{beta} protected against oxidant-induced cytotoxicity but did not affect the MPTP. Recombinant GSK3{beta} could directly bind to and phosphorylate recombinant CypD. Mass spectrometry revealed several putative GSK3{beta} phosphorylation sites on CypD. However, mutation of these sites did not affect the peptidyl prolyl isomerase activity of CypD and reconstitution of these phosphomutants in CypD-deficient cells increased MPTP sensitivity and oxidative-induced cell death to the same extent as wild-type CypD. Further, targeted overexpression of either wild-type or kinase-inactive GSK3{beta} in the mitochondrial matrix did not impact MPTP or cell death. Moreover, while proteinase-K digestion of cardiac mitochondria showed a significant amount of GSK3{beta} in the mitochondria, it was not localized to the matrix. Finally, overexpression of GSK3{beta} was still able to increase MPTP sensitivity and oxidative stress-induced death in CypD-null cells. Taken together, these data indicate that, while GSK3{beta} can modulate MPTP, this appears to be independent of GSK3{beta}s interaction with, or phosphorylation of CypD.

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.

Chemoresistance is a major contributor to poor clinical outcomes in AML patients and can arise from interactions between AML cells and the bone marrow microenvironment (BME). How immune cells, particularly macrophages (M{varphi}s), facilitate this process requires better clarification. This study shows that M2-like M{varphi}s protect AML cells from apoptosis induced by daunorubicin (DNR) and cytarabine (Ara-C). This protection occurs via co-culture and is linked to enhanced mitochondrial transfer from M{varphi}s to AML cells. M{varphi}s interacted with AML cells via tunneling nanotube (TNT)-like structures. Furthermore, inhibition of mitochondrial transfer using cytochalasin B reduced the protective effect, indicating that mitochondria mediate this process. M{varphi}s transferred functional mitochondria to AML cells as evidenced by enhanced metabolic capacity and reduced reactive oxygen species levels in AML cells under chemotherapy stress. TH-257 (LIMK inhibitor) and metformin blocked mitochondrial transfer and M{varphi}-driven chemoprotection. Moreover, increased transcript expression levels of RhoC and cofilin correlate with inferior overall survival in AML patients. These findings suggest that M2-like M{varphi}s contribute to chemoresistance through TNT-mediated mitochondrial transfer and the LIMK-Cofilin pathway, identifying potential therapeutic targets to circumvent chemoresistance in AML.

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.