Cell Death & Disease

Cellular material in apoptotic cells must be efficiently cleared by phagocytes to maintain tissue homeostasis. Defects in this process can lead to the onset of secondary necrosis and the release of intracellular contents such as damage associated molecular patterns (DAMPs) and autoantigens that are often derived from the nucleus. Therefore, appropriate handling and clearance of apoptotic material is vital to prevent unwanted inflammatory response and the onset of autoimmune disorders. However, how nuclear material is packaged by apoptotic cells for effective clearance by phagocytes is not well understood. By utilising murine models of apoptosis, we observed that a distinct subset of large extracellular vesicles generated from apoptotic thymocytes, known as apoptotic bodies (ApoBDs), can harbour the majority of nuclear contents. Mechanistically, we discovered that apoptotic cells can asymmetrically partition the nucleus into a single large membrane bleb located at one side of the cell, with other cellular contents such as mitochondria and acid organelles distributed to the opposite side. Whilst this newly observed apoptotic morphology, coined as asymmetric cell death morphology (AsyCDM), is morphologically similar to the process of erythroblast enucleation, pharmacological compounds that could interfere with erythroblast enucleation did not block the establishment of AsyCDM during apoptosis. Notably, AsyCDM was reliant on the contractile forces generated by ROCK1-dependent plasma membrane blebbing. Taken together, this study suggests that intracellular contents are partitioned into different ApoBD subsets during apoptosis through a regulated process driven by ROCK1-dependent actomyosin contraction.

Ferroptosis, an intracellular iron-catalyzed form of programmed cell death (PCD) driven by lipid reactive oxygen species induced membrane damage, is mechanistically uncharacterized in its execution process. Here, we investigated ferroptosis execution in mesenchymal-like ovarian cancer cells treated with ferroptosis inducers ML162 and erastin. We showed that YVAD (a pyroptosis-associated inflammatory caspase inhibitor) and disulfiram (preventing gasdermin pore formation on plasma membrane) deterred ferroptotic cell death. Moreover, we also observed LDH release and IL-1{beta} secretion from ferroptotic cells, suggesting that ferroptosis involves a pore-forming process. Intriguingly, ferroptosis is independent of the canonical inflammasome pathway because caspase-1 is dispensable and not activated upon ferroptosis induction. In contrast, we found that caspase-5 was activated while caspase-4 was not during ferroptosis. In addition, depletion of caspase-5 rendered cells not responding to ferroptosis inducers. Also intriguingly, GSDMD, the well-established caspase-5 substrate, was not involved in ferroptosis. We instead detected GSDME cleavage upon ferroptosis induction and knockdown of GSDME reduced cell death induced by ferroptosis inducers. As caspase-5 activity was necessary for ferroptosis and caspase-5 directly cleaved GSDME, we conclude that the axis of caspase-5/GSDME executes ferroptosis in ovarian cancer cells.

TP53 (also called TRP53 or p53) is a critical tumour suppressor that prevents cancer development by inducing a transcriptional program which can lead to diverse cellular responses, most prominently, cell proliferation arrest/senescence with survival of cells or cell death by apoptosis. Why distinct cell types undergo different outcomes after p53 activation remains unclear. Using integrated RNA-sequencing, proteomic and functional analyses across a diverse range of murine primary cell types, we demonstrate that cell fate is governed by the balance between pro-survival BCL-2 and pro-apoptotic BH3-only proteins. Cells resistant to apoptosis displays a higher starting ratio of pro-survival BCL-2 to pro-apoptotic BH3-only proteins, along with transcriptional upregulation of the pro-survival gene Bcl2l1, encoding BCL-XL. This control of cell fate is also seen in human wild-type p53 cancer cell lines. These findings reveal the mechanism for understanding p53-driven cell fate decisions, suggest therapeutic strategies to shift p53-induced cell proliferation arrest/senescence toward apoptotic cell death and allowed generation of an RNAseq data-based predictor of outcome for cancer cells after p53 activation.

Cell replacement represents a potential treatment modality for retinal disorders characterized by photoreceptor loss. However, photoreceptor replacement approaches have not been clinically established. To take this forward, the main goal of this study was to systematically compare human photoreceptors of different ages and identify those that enable functional integration into the degenerative retina. Donor cells were isolated from iPSC-derived retinal organoids generated by a GMP-compliant protocol at differentiation days 120, 150, or 200 and transplanted subretinally into cone photoreceptor function loss 1 (Cpfl1) recipients, an inherited mouse model of cone degeneration. While younger photoreceptors showed slightly improved transplantation outcomes, donor photoreceptors of all culture stages displayed long-term survival, cone identity, structural integration into the host retina, and tight interactions with host Mueller glia, including formation of a continuous outer limiting membrane. Transplanted photoreceptors showed signs of advanced maturation, including correct polarization with generation of apical inner- and outer segments, while basal synapses were formed with host bipolar cells. Electrophysiological assessment of host retinal ganglion cells revealed light-evoked responses in transplant-containing regions, providing evidence for functional incorporation of human photoreceptors into the mouse neuro-retinal circuitry. Thus, GMP-compliant human iPSC-derived photoreceptors are stable over a wide range of differentiation stages and constitute a robust cell source for retinal transplantation and functional repair. The findings provide important prerequisites for the development of standardized procedures towards clinical translation of photoreceptor replacement in the retina.

Mouse models have historically been central to studies of TNF-induced cell death and guided pharmaceutical translation into clinic, based on the assumption that TNF signaling is conserved between human and mouse. Here, our work uncovers critical species-specific differences between the two. By systematically dissecting the roles of RIPK1, TRADD, and sensitivity to RIPK1 inhibitors in TNF signaling--including RIPK1 kinase-dependent and-independent apoptosis--we found that both apoptosis modalities diverge between human and mouse cells. In mouse cells, RIPK1 suppresses TRADD-mediated kinase-independent apoptosis, whereas in human cells, RIPK1 and TRADD act redundantly. Moreover, RIPK1 inhibitors block kinase-dependent apoptosis in mouse but not human cells, despite effectively inhibiting RIPK1 S166 phosphorylation. Cross-species complementation revealed that these discrepancies stem not from RIPK1 itself but from cell-context differences. These findings echo the clinical failures of RIPK1 inhibitors despite efficacy in mouse models and underscore the need for humanized models and therapeutics that more faithfully predict clinical outcomes.

Photoreceptor transplantation is considered a disease-agnostic therapeutic strategy for retinal degenerative diseases with highly heterogenous genetic, molecular, and cellular pathologies. While integration of human photoreceptors enriched from stem cell-derived retinal organoids was noted in previous preclinical studies, the potential influence of retinal degeneration severity on transplantation efficiency has not been systematically assessed. Here, we employed mice presenting mild or severe retinal degeneration as recipients for human induced pluripotent stem cell-derived photoreceptors. Donor cells formed multi-cellular clusters that structurally integrated from 3 weeks post-transplantation (wpt) in mildly degenerated retinas, closely interacting with host Muller glia, resulting in proper maturation characterized by inner/outer segment and synapse formation by 26 wpt. In contrast, in severely degenerated hosts, donor photoreceptors remained mainly singularized and scattered in the subretinal space, showing limited structural integration or signs of maturation. Differential maturation of donor cells in mild vs. severe hosts was confirmed by single-cell RNA-sequencing analysis. However, transplantation at the beginning of the degeneration process of the severe model allowed structural integration and maturation of donor photoreceptors, despite complete loss of endogenous photoreceptors over time. The study thus shows that survival, integration, and maturation of donor photoreceptors depend on the degenerative retinal microenvironment shaping significantly transplantation efficiency.

Retinopathy of prematurity (ROP) is caused by hyperoxic exposure of prematurely born infants. The mouse model of oxygen-induced retinopathy (OIR) recapitulates pathological features of both phase I and phase II ROP. We here looked at the retinal proteins that change in response to hyperoxia in phase I of the mouse model of OIR. Using tandem mass tag labeled proteomics, we found several differentially expressed proteins (DEPs) in phase I of OIR. Of all the DEPs, we investigated the role of previously unknown protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2). NDUFA4L2 protein and its paralog NDUFA4 are both mitochondrial complex I proteins; however, here we demonstrate that NDUFA4L2 changes in both phases of OIR, with no changes in its paralog NDUFA4, implying its unique function in pathophysiology of the disease. We demonstrate that NDUFA4L2 is an oxygen-sensitive protein and regulates retinal endothelial cell migration by rescuing isocitrate dehydrogenase flux impaired by hyperoxia in phase I of OIR.

Leydig cells (LCs) are essential for male reproductive function due to their role in testosterone synthesis, a process critically dependent on mitochondrial cholesterol transport mediated by the Steroidogenic Acute Regulatory protein (StAR). Despite their importance, LCs are highly sensitive to metabolic and exogenous stressors. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a key link between cellular metabolism and cell fate; however, its role in LCs and steroidogenesis remains poorly understood. In this study, we investigated the induction of ferroptosis in LCs and its impact on their steroidogenic capacity. We evaluated cellular responses to canonical ferroptosis inducers (Erastin and RSL3) alongside the transcriptional regulation of key genes. Our results demonstrate that LCs are vulnerable to ferroptotic stress, which significantly downregulates Star expression. Notably, we uncovered a novel endocrine-metabolic crosstalk: hormonal stimulation via hCG effectively rescues LCs from Erastin-induced toxicity and fully sustains maximal steroidogenesis. However, this hormone-driven cytoprotection fails against direct GPX4 inhibition by RSL3, indicating an absolute reliance on functional GPX4. These mechanistic findings highlight the paradoxical dual role of ACSL4 in Leydig cell biology and are further supported by bioinformatic analysis of public transcriptomic profiles from infertile patients, which reveal a detrimental imbalance in the ACSL4/GPX4 axis. Together, our data position ferroptosis as a critical disruptor of male endocrine function and reveal a hormone-mediated metabolic adaptation that could inform novel therapeutic strategies against oxidative stress in the testis. Highlights-Leydig cells exhibit a strong vulnerability to ferroptotic cell death. -Ferroptosis disrupts StAR expression and halts Leydig cell steroidogenesis. -hCG signaling promotes metabolic adaptation against Erastin-induced ferroptosis.

Traumatic brain injury (TBI) induces rapid neuroinflammatory responses not only in the brain but also in anatomically and immunologically connected central nervous system (CNS) compartments, including the retina. In our study, we investigated retinal microglial activation, retinal ganglion cell (RGC) calcium dynamics, and caspase-3 activation in adult mice subjected to severe traumatic brain injury using the Marmarou impact-acceleration model at 24 and 48 h post-injury. Carrying out Ca{superscript 2}-imaging, immunohistochemistry, and ex vivo time-lapse microscopy, we found robust microglial activation in both the superficial and deep retinal layers following TBI, accompanied by increased microglial motility. RGCs exhibited a transient surge in degeneration-induced spontaneous activity at 24 h, followed by a marked reduction below control levels at 48 h, consistent with early degenerative changes. Activated caspase-3 levels were significantly elevated in both microglia and other retinal cell types at both time points, indicating ongoing apoptotic effects. Together, these findings demonstrate that TBI rapidly triggers inflammatory and apoptotic mechanisms in the retina, which are detectable within the first 48 hours. Our results highlight the retina as a sensitive indicator of early CNS pathology after traumatic injury and underscore the potential of retinal analysis for monitoring TBI-induced neurodegeneration for future clinical implementation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/734783v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@5bc694org.highwire.dtl.DTLVardef@14a4ce4org.highwire.dtl.DTLVardef@fe2d32org.highwire.dtl.DTLVardef@149419d_HPS_FORMAT_FIGEXP M_FIG C_FIG

The multiligand endocytic receptor, megalin (LRP2), is expressed in the retinal pigment epithelium (RPE) and patients lacking the receptor develop high myopia. Despite its established role in retinal development, the contribution of megalin to retinal homeostasis in the normally developed/mature eye remains poorly understood. Here, we investigated megalin function using an inducible knockout mouse (KO) model and human iPSC-derived RPE with megalin knockdown (KD) to distinguish post-developmental homeostatic functions from developmental effects. In vivo, megalin ablation caused progressive retinal degeneration and visual impairment, with morphological abnormalities in the RPE but no changes in myopia-associated ocular phenotypes including axial length and intraocular pressure. Proteomic profiling of megalin-KO RPE revealed reduction of autophagy-related proteins. In line with this, megalin deficiency was associated with accumulation of pro-cathepsin D, and perturbed rhodopsin turnover. This was supported in vitro, where trafficking of photoreceptor outer segment (POS) containing phagosomes to lysosomes was reduced, suggesting disturbed phagosome maturation. Megalin KD did not measurably impair initial uptake of POS discs, but delayed rhodopsin degradation, indicating defective post-ingestion processing. Together, these findings establish megalin as a key regulator of retinal homeostasis in the mature eye by controlling phagosome-lysosome fusion in the RPE and suggest that megalin dysfunction contributes to slowly progressive retinal degeneration. This positions megalin as a potential therapeutic target in lysosomal degenerative diseases in the retina.

PurposeCisplatin (CDDP) is the most active chemotherapy for ovarian cancer; primary or acquired resistance signals a poor prognosis. Nelfinavir (NFV), an HIV protease inhibitor, has demonstrated anti-tumor activity in multiple cancer models, but its interaction with CDDP in ovarian cancer has yet to be demonstrated. In this work, we addressed whether the combination of CDDP and NFV provides treatment advantage in platinum (Pt)-resistant ovarian cancer cells. MethodsDrug synergy between NFV and CDDP was assessed using cell vitality assays and Loewe additivity modelling. Apoptotic and pyroptotic signalling were evaluated by immunoblotting, mitochondrial membrane potential analysis, and lactate dehydrogenase (LDH) release, and caspase inhibition. Transcriptomic changes were assessed by bulk mRNA sequencing followed by differential gene expression analysis and gene set enrichment analysis. ResultsNFV synergized with CDDP to reduce the viability of Pt-resistant ovarian cancer cells, promoting a regulated lytic cell death phenotype involving apoptotic and pyroptotic features. Combination treatment induced caspase-8 and caspase-3 activation, and downstream gasdermin E (GSDME) processing. Inhibition of caspase-3 significantly attenuated cell death, and caspase-8 inhibition rescued viability and prevented Bid cleavage, caspase-3 activation, and GSDME cleavage. These effects occurred in the context of enhanced endoplasmic reticulum stress, increased DNA damage with reduced DNA repair, and impaired Akt-driven survival signalling. ConclusionsOur findings establish that NFV synergizes with CDDP in killing Pt-resistant ovarian cancer cells by promoting a caspase-8-dependent apoptotic-to-secondary pyroptotic response, supporting further investigation of NFV as a potential drug to be repurposed to increase the efficacy of Pt-based therapy.

Stem cell dysfunction and loss of renewal capacity are primary characteristics of tissue aging and decremental regeneration in response to injury. Alveolar type 2 cells (AT2) are key progenitor cells responsible for lung repair and are thought to be dysfunctional in diseases such as chronic obstructive pulmonary disease (COPD). AT2 cells are highly metabolic and rely on mitochondria, but how mitochondrial mechanisms influence their maintenance and cell fate is unclear. This gap is critical as no current therapies target lung repair or mitochondrial function in COPD. Here, we report that adenine nucleotide translocase 2 (ANT2), a key ATP/ADP transporter, is reduced in AT2 cells from COPD lungs, and that ANT2 loss impairs bioenergetics (ATP). We also identify, for the first time, ferroptotic susceptibility as a consequence of ANT2 loss in AT2 cells, leading to impaired self-renewal and progenitor capacity in alveolar organoids. Together, loss of ANT2 and the associated cellular dysfunction resulted in worsened lung damage or emphysema due to cigarette smoke in mice. Therapeutic restoration of ANT2 expression resulted in renewed AT2 stem cell function and prevention of emphysema by reducing oxidative stress and ferroptosis. These findings highlight the importance of ANT2 in metabolic regulation, plasticity, and cell resiliency of AT2 cells in the lung and that ANT2 is a potential target for lung repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/737954v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@96d0caorg.highwire.dtl.DTLVardef@165b15dorg.highwire.dtl.DTLVardef@15f86baorg.highwire.dtl.DTLVardef@862745_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hepatocellular carcinomas (HCC) are genetically heterogeneous cancers frequently characterized by MYC gene amplification or hyperactivating {beta}-catenin (CTNNB1) mutations. Analysis of TCGA transcriptomics revealed that MYC-driven HCC tumors have decreased expression of mtDNA-encoded genes, but increased expression of nuclear-encoded mitochondrial genes. To investigate this apparent discrepancy, we generated MYC- and CTNNB1-driven murine HCCs, all of which displayed aberrant mitochondrial metabolism. Notably, MYC-driven tumors exhibited significant reductions in OXPHOS and TCA cycle activity that correlated with increased ROS levels, as well as elevated mitochondrial turnover through mitochondrial fission and mitophagy. MYC induces the expression of nuclear respiratory factor 1 (NRF1), which regulates DRP1 and other genes to promote receptor-mediated mitophagy. Knocking out DRP1 reduced mitophagy and ROS levels and promoted survival of HCC-bearing mice. These results identify elevated mitochondrial turnover as a potential therapeutic target in MYC-driven HCC. SignificanceHepatocellular carcinoma can arise from multiple oncogenes, making targeted therapy more difficult. Here we show that tumors with MYC amplification lose mitochondrial function via fission and mitophagy upregulation. Targeting mitochondrial quality control results in increased survival suggesting a therapeutic window in MYC-driven HCC.

Glaucoma is a group of eye diseases that affects 4 million people in the US and is one of the leading causes of vision loss due to damage to the eyes optic nerve (ON) which is composed of axons from retinal ganglion cells (RGCs) that transmit visual information to the brain. Injury to the ON often triggers RGC death and subsequent loss of visual function. Despite its increasing prevalence worldwide, effective therapies for glaucoma remain elusive. Notably, the thirteen-lined ground squirrel (TLGS) exhibits intrinsic neuroprotection during hibernation; however, reproducing this protective state pharmacologically has proven challenging. To elucidate the metabolic mechanisms underlying this resilience, we conducted untargeted metabolomic analyses on TLGS retinas at 6 hours, 3 days, and 7 days following ON crush. Retinas from awake and hibernating animals were compared to identify temporal and state-dependent metabolic signatures. Distinct metabolomic profiles were observed in hibernating animals relative to their awake counterparts. Pathway analyses revealed coordinated regulation of amino acid, lipid, and purine metabolism that likely contributes to hibernation-induced resilience. Furthermore, our findings indicate that hibernating TLGS retinas increase exosome biogenesis, prompting in vitro validation using TLGS-derived exosomes, which demonstrated robust neuroprotective and anti-inflammatory effects. Proteomic and transcriptomic characterization of exosomal cargo identified conserved miRNAs, mRNAs, and proteins implicated in redox balance, cytoskeletal stabilization, and stress-response regulation. Collectively, these data support the hypothesis that metabolic reprogramming and exosome-mediated intercellular signaling underlie hibernation-associated neuroprotection. Modulating these pathways may provide a blueprint for novel therapeutic strategies to mitigate neurodegeneration and promote recovery following optic nerve injury. Graphical AbstractIllustration depicting state-dependent metabolic responses to optic nerve crush (ONC) injury in Thirteen-lined Ground Squirrels (TLGS). In Awake animals, injury triggers enhanced ATP production through the TCA cycle, leading to excessive reactive oxygen species (ROS) generation and subsequent retinal ganglion cell (RGC) death. In contrast, Hibernating animals shift toward lipid metabolism and utilize ATP for the biosynthesis of ceramides and sphingolipids, promoting membrane integrity and exosomal signaling. Additionally, a range of metabolites associated with hibernation-linked neuroprotection are elevated, contributing to enhanced RGC survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/733742v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@1ef3a7eorg.highwire.dtl.DTLVardef@e9293dorg.highwire.dtl.DTLVardef@192729forg.highwire.dtl.DTLVardef@1a32cb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Internal-tandem-duplication of the receptor tyrosine kinase FLT3 (FLT3-ITD) generates ligand-independent signaling and is highly recurrent in acute myeloid leukemias (AMLs). One way signaling pathways can quickly influence cell fates is by phosphorylating key fate-determining proteins to trigger their proteolysis. We investigated the master transcription factor (MTF) driver of granulo-monocytic lineage-fates, CEBPA, for regulation by this mechanism because we found high CEBPA mRNA but little CEBPA protein in FLT3-ITD versus FLT3-wildtype AML cells, and inhibiting FLT3-ITD signaling with tyrosine kinase inhibitors (TKI) rapidly rescued CEBPA protein. Mass spectrometry analyses of CEBPA and its interactome demonstrated prominent interactions with major ubiquitin-proteosome pathway (UPP) components UHRF1 and USP7. TKI treatments decreased CEBPA and USP7 phosphorylations at serine 21 and serine 18 respectively alongside shifts in CEBPA interactions from degradative ubiquitin-ligase UHRF1 toward protective deubiquitinase USP7. The rescued CEBPA activated granulocytic-differentiation. Supporting that the serine-phosphorylations were phospho-degrons, UPP-inhibitors (bortezomib, MG132) increased phosphorylated and total CEBPA and USP7. The MTF regulator of apoptosis p53 is a known USP7 client, therefore, we also evaluated p53 status: TKIs and UPP-inhibitors stabilized USP7 and p53, triggering apoptosis in addition to granulocytic-differentiation specifically in FLT3-ITD but not FLT3-wildtype AML cells. UPP-inhibitors produced these consequences in TKI-resistant FLT3-ITD AML cells also. These data predicted genetic loss-of-function to CEBPA or TP53 is redundant in the FLT3-ITD context, borne out by mutual exclusivity of the mutations in clinical series. In summary, FLT3-ITD signals for CEBPA and p53 proteolysis to block lineage-maturation and apoptosis, positioning UPP-inhibitors as therapeutic candidates acting downstream of TKIs. KEY POINTSO_LIThe oncoprotein kinase FLT3-ITD signals for CEBPA and p53 proteolysis and hence suppresses lineage-differentiation and apoptosis C_LIO_LIProteosome-inhibitors are candidate remedies to restore CEBPA and p53, acting downstream of presently used FLT3-ITD kinase inhibitors C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/738455v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@6ae211org.highwire.dtl.DTLVardef@12003bforg.highwire.dtl.DTLVardef@d62eb9org.highwire.dtl.DTLVardef@1958693_HPS_FORMAT_FIGEXP M_FIG C_FIG

Despite continuous progress in diagnosis and therapy, breast carcinoma (BC) remains a major health problem. Triple-negative (Estrogen Receptor-/Progesterone Receptor-/HER2-) breast cancer (TNBC) is the most aggressive subtype due to its high metastatic potential and resistance to chemotherapy. The Y-box binding protein 1 (YB-1) transcription factor, a protein present in both cytoplasm and nucleus, is a driver of TNBC malignancy as it stimulates its cancer stem cell phenotype and disrupts cell cycle progression. Here, we hypothesized that YB-1-containing sEVs deliver YB-1 to the nuclear compartment of recipient cancer cells and play a major role in the activation of the metastatic process. We found a selective enrichment of YB-1 in sEVs from MDA and 4T1 cells, with [~]65% and 50% of all sEVs positive for YB-1 by d-STORM. Administration of sEVs from wild-type MDA and 4T1 to their YB-1 knockout counterparts resulted in nuclear translocation of sEV-associated YB-1 and increased tumorsphere formation. Pharmacological blockade of the nuclear transport machinery based on the inhibition of the formation of the "VOR" complex (VAP-A-ORP3-Rab7) by PRR851 impaired both nuclear translocation and the YB-1-induced increase in tumorsphere formation. YB-1 phosphorylation at S102 was required for nuclear localization. In fact, loss of YB-1 phosphorylation inhibited tumorsphere growth and stemness of cancer cells and YB-1-positive sEVs restored the oncogenic behavior of cancer cells expressing phospho-mutant YB-1. Moreover, PRR851 inhibited the nuclear translocation of the phosphorylated form of YB-1 and the oncogenic behavior of the TNBC cells. These data support the conclusion that the nuclear translocation of sEV-associated phosphorylated YB-1 is an important factor in the malignant behavior of TNBC and a potential therapeutic target.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by a dense desmoplastic tumor microenvironment (TME) that limits drug penetration and promotes immune evasion. Effective treatment, therefore, requires simultaneous modulation of multiple signaling pathways. Here, we describe a directed polypharmacological strategy to identify dual modulators of c-MET and Smoothened (SMO), aiming to disrupt the protective stroma through SMO inhibition while directly suppressing tumor cell survival via c-MET targeting. An AI-guided virtual screening workflow combining the machine-learning platform PyRMD, trained on known c-MET and SMO ligands, with structure-based molecular docking was applied to a library of over 9 million compounds. This approach led to the identification of compound 21, an aminopyrimidine-benzamide-phenoxyquinoline derivative, as a dual c-MET/SMO inhibitor. Biochemical and cellular studies demonstrated that compound 21 selectively binds the SMO orthosteric site (pKi = 5.60), inhibits agonist-induced GLI (Glioma-associated oncogene) signaling (pIC50 = 5.50), and potently suppresses c-MET kinase activity (pIC50 = 6.94). Western blot analyses further revealed that compound 21 promotes ubiquitin-proteasome-mediated degradation of c-MET, eliminating receptor availability and limiting compensatory resistance signaling. In 3D heterotypic models comprising MIAPaCa2 pancreatic cancer cells and CAF154-hTERT fibroblasts, dual inhibition of SMO-mediated stromal support and c-MET-driven tumor progression resulted in greater cytotoxicity than the combination of the selective inhibitors Sonidegib and PHA-665752. Overall, compound 21 overcomes stromal-mediated resistance, enhances tumor cell death, and validates dual SMO/c-MET targeting as a promising single-agent therapeutic strategy for PDAC. One Sentence SummaryAn AI-identified dual SMO/c-MET inhibitor overcomes stromal resistance and degrades c-MET to suppress pancreatic cancer.

Photoreceptors are among the most metabolically active cells in the retina and are therefore highly sensitive to fluctuations in oxygen availability. Age-related tissue changes in the eye affect oxygen delivery to the outer retina, which may result in hypoxic stress within photoreceptors and can contribute to disease development and retinal degeneration. To investigate how chronic hypoxic signalling affects photoreceptor metabolism, we examined a rod-pecific Vhl knockout mouse (RodVhl), in which constitutive HIF activation mimics the molecular response to hypoxia. Combining a cell-type-enriched multi-omics approach with metabolic flux analysis, we identified an early metabolic response in the retina of Rod{Delta}Vhl mice prior to degeneration. This response was characterized by a shift towards an oxidative redox environment indicated by a decrease in nucleotide precursors and an increased antioxidant response. While steady-state glycolytic flux remained unchanged, the dynamic 13C-glucose tracing revealed accelerated carbon flow through the three-carbon glycolytic intermediates, indicating a carbon rerouting. Outer segment lipidomics revealed selective remodelling of phosphatidylcholine and phosphatidylethanolamine species toward more oxidation-resistant and elongated acyl chains, supported by early gene upregulation of essential enzymes involved in fatty acid elongation, desaturation and oxidation. Together, these findings indicate a coordinated shift in metabolic and lipid pathways in photoreceptors under chronic hypoxic stress, consistent with an adaptive response that may help preserve outer segment integrity and improve stress resilience.

Dendritic cells (DCs) are an important type of antigen presentation cell that regulate immunity by initiating antigen-specific immunity and tolerance through T cell activation. The interaction between DCs and T cells can be mediated through direct cell-cell contact or via the release of extracellular vesicles (EVs) from DCs that harbour antigen presentation machineries. Although small EVs (<200 nm in diameter) such as exosomes released by DCs have been shown to regulate immunity, whether other EV subtypes, in particular those that are released by dying DCs due to homeostatic turnover or following infection, can modulate immune responses is not defined. In this study, we demonstrated that DCs undergoing apoptosis can generate a subclass of large EVs ([~]1,000-5,000 nm in diameter) known as apoptotic bodies (ApoBDs) via distinct morphological steps. Mechanistically, ApoBD formation by apoptotic DCs is regulated by Rho-associated kinase 1 and T-type calcium channels. Functionally, DC-derived ApoBDs were found to mediate direct antigen presentation. These data demonstrate a novel function of ApoBDs and highlight the ability of apoptotic materials derived from dying DCs to continue mediating intercellular communication and regulating immune responses.

We show that chronic impairment of mitochondrial respiration is associated with marked accumulation of cytochrome c (Cytc) protein. Using SCO2-deficient HCT116 cells lacking functional cytochrome c oxidase and wild-type cells exposed to sustained hypoxia, we found that substantial mitochondrial Cytc accumulation parallels reduced electron flux through Cytc. SCO2-deficient cells exhibited equally elevated Cytc levels under normoxia (19% O2) and hypoxia (0.1-3% O2). Wild-type cells under sustained hypoxia accumulated Cytc, reaching levels comparable to those in SCO2-deficient cells. This effect was reversible upon reoxygenation. Increased Cytc protein levels were also observed in other cell models, including primary cortical neurons cultured under chronic hypoxia and in cerebral cortex tissue from hypoxia-exposed mice. Cytc accumulation occurred independently of CYCS transcription, mRNA translation, HIF activation, ROS production and changes in mitochondrial network. Pharmacological inhibition of complex III was likewise accompanied by increased Cytc levels, whereas mitochondrial uncoupling had no effect, suggesting that impaired electron transfer rather than membrane depolarisation per se underlies this association. Raman spectroscopy revealed enrichment of reduced Cytc and an increased Cytc-to-cytochrome b ratio in respiration-deficient cells. Further supporting a stabilisation-based mechanism, the fraction of membrane-unbound ferro-Cytc was decreased in SCO2-deficient cells, consistent with moderate cardiolipin enrichment, which is known to enhance retention of Cytc at the inner mitochondrial membrane. Despite elevated mitochondrial Cytc content, SCO2-deficient cells were less susceptible to apoptosis induced by intermittent hypoxia or dichloroacetate. Together, these findings indicate that reduced electron flux through complex IV is associated with Cytc accumulation through increased protein stability and membrane retention without enhancing apoptotic sensitivity.