Cell Death & Differentiation

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.

Cytotoxic lymphocytes induce cancer cell death through death receptor-ligand interactions and the perforin-granzyme pathway. These pathways are generally thought to converge on the activation of executioner caspases to drive apoptosis. Here, we employed a reductionist approach to systematically disrupt key cell death mediators in a cytotoxic lymphocyte killing system to define their roles in determining cancer cell fate. We found that loss of executioner caspases conferred only limited resistance to cytotoxic lymphocyte-mediated killing. To identify cancer cell-intrinsic regulators that function beyond executioner caspases, we performed unbiased genome-wide CRISPR screens in executioner caspase-deficient cells. Unexpectedly, disruption of Fas or FADD--core components of the death receptor pathway--conferred substantial resistance to cytotoxic lymphocyte-mediated killing even in the absence of executioner caspases. This resistance persisted following additional disruption of known downstream mediators of Fas-FADD-caspase-8 (CASP8) signaling. Together, these findings identify the Fas-FADD-CASP8 axis as a central cancer cell-intrinsic determinant of susceptibility to cytotoxic lymphocyte-mediated killing whose function is not fully explained by canonical apoptotic or non-apoptotic effector pathways. Our results further suggest that CASP8 engages additional downstream substrates or mechanisms to promote cytotoxic lymphocyte-induced cancer cell death.

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.

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.

Necroptosis is best known as a lytic, proinflammatory cell-death pathway mediated by RIPK3 and MLKL. Effective wound repair requires the rapid resolution of inflammation, and ongoing necroptotic activity would only exacerbate tissue damage, delaying healing. However, damaged skin presents a trigger-rich environment for necroptotic signalling, an apparent paradox that remains unresolved. Using genetic ablation and pharmacological inhibition across multiple wound models, we show that inhibiting necroptosis accelerates wound closure, revealing that necroptotic signalling normally restrains repair. Surprisingly, we found that MLKL activation in wild-type keratinocytes induces differentiation and membrane repair rather than cell lysis. This adaptive, non-lethal mode of necroptotic signalling preserves barrier integrity but slows re-epithelialisation. Our findings redefine epidermal necroptotic signalling as a stress-responsive program that modulates keratinocyte fate in a trigger-rich environment. Temporarily dampening this pathway may enhance regeneration after barrier loss without compromising immune defence, revealing necroptosis as a tunable mechanism balancing tissue repair and inflammation.

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.

The transition from tumor necrosis factor (TNF)-induced plasma membrane-bound complex I to cytosolic death-inducing complex II switches cells from survival to death. However, the precise regulation of this fatal decision is incompletely understood. Here, we show that mTORC1 promotes this transition by destabilizing later-stage complex I without affecting its initial assembly. Inhibition of mTORC1 unleashes ATG9A and FIP200 activity, thereby promoting the accumulation of CHUK (IKK) in complex I. CHUK scaffolds the kinase-active IKK{beta} to stabilize complex I and prevent complex II formation. Activation of this ATG9A/FIP200-CHUK/IKK{beta} axis protects against TNF-induced fulminant hepatitis while compromises antibacterial defense against Staphylococcus aureus. This mTORC1-governed life-or-death transition provides therapeutic insight into TNF-related pathologies--including cancer, metabolic tissue injury, and microbial infections--where mTORC1 activity is frequently suppressed.

The liver is a metabolic hub with a high protein turnover that renders it uniquely susceptible to proteotoxic stress. Perturbation of proteostasis, either by proteasomal inhibitors or in chronic liver diseases, could adversely impact liver physiology. Here, we show that proteasomal inhibition unexpectedly suppresses basal type I interferon (IFN-I) signaling in the murine liver. Proteasomal inhibition by bortezomib selectively downregulates a subset of interferon-stimulated genes (ISGs), among which USP18 and ISG15 emerge as critical determinants of hepatocellular survival. We identify USP18 as a central cytoprotective factor that prevents proteotoxic apoptosis independently of its deubiquitinase activity, but strictly requires its scaffolding function mediated by isoleucine-60 and interaction with STAT2. Mechanistically, proteotoxic stress disrupts IRF9 nuclear translocation, attenuating USP18 transcription, and drives USP18 and other ISGs into insoluble aggregates with kinetics distinct from canonical IFN-I-induced insolubility. Strikingly, IFN-I priming preserves ISG solubility, restores USP18 abundance, and confers resistance to proteotoxic cell death. Together, these findings uncover an unanticipated link between proteostasis and innate immune signaling, and establish the USP18-STAT2 axis to enhance hepatic resilience under proteotoxic stress.

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.

Upon treatment, cancer cells engage non-genetic adaptations, including tolerance and subsequent persistence, to survive therapy. Eliciting programmed cancer cell death in these persister cells (PCs) remains a primary goal in oncology. We found that ferroptosis is the programmed cell death mechanism most deregulated in persisters by some of the most widely used therapeutic regimens, including platinum-based therapies, which combined with ferroptosis inducers ablate persister colorectal cancer cells. Conversely, persisters emerging from topoisomerase inhibitor regimens withstand ferroptosis and ferroptotic inducers, increasing instead intracellular iron concentration. We found that topoisomerase inhibitors trigger the Xc- antiporter axis (via SLC7A11 and CD44) increasing both intracellular cystine, to activate GPX4, and extracellular glutamate. Glutamate then engages the NMDA receptors (NMDARs), which are essential in neurotransmission but recently reported to be deregulated also in cancer cells. In PCs, NMDARs stimulate intracellular Ca2+ uptake and trigger the AKT/NFE2L2 axis, thereby engaging a cytoprotective program to cope with oxidative stress. Furthermore, we found that NFE2L2 increases the distance between the endoplasmic reticulum and mitochondria while reducing mitochondrial ROS in PCs. The synergistic inhibition of both the standard (Xc- antiporter) and this novel NMDAR/NFE2L2 axis resensitizes PCs to ferroptosis. These data provide new opportunities to improve the efficacy of widely used therapeutic regimens. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/738168v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1f32868org.highwire.dtl.DTLVardef@e1c7eborg.highwire.dtl.DTLVardef@10c4d25org.highwire.dtl.DTLVardef@9ce6d6_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIPersister cells induced by FOLFOX are sensitive to ferroptosis while resistant upon FOLFIRI treatment C_LIO_LIIncreased extracellular glutamate activates a NMDAR/NFE2L2 axis C_LIO_LINFE2L2 copes with oxidative stress by inhibiting juxtaposition between ER and mitochondria C_LIO_LIInhibition of Xc- antiporter alongside NMDAR/NFE2L2 is required to trigger ferroptosis in FOLFIRI persister cells C_LI

Mitophagy is the process of selective autophagic clearance of damaged mitochondria and is closely implicated in neurodegenerative disease. PTEN-induced kinase 1 (PINK1) and a RBR E3 ubiquitin-protein ligase (Parkin) constitute a positive feedback loop in mitophagy initiation. It is known that reactive oxygen species (ROS) modulate mitophagy, while the exact regulatory mechanism remains largely elusive. Here, we found that exogenously applied ROS effectively block mitophagy induced by acute mitochondrial damage agents, which could be reversed by antioxidants. Mechanistically, ROS activate poly(ADP-ribose) polymerase 1 (PARP1), and suppression of PARP1 eliminates the inhibitory effect of ROS on mitophagy. Notably, PARP1 directly interacts with PINK1 and mediates its PARylation at residue E417, thereby negatively regulating PINK1 function. Collectively, our study identifies PARylation as a new form of post-translational modification of PINK1 and reveals a novel mechanism underlying the regulatory role of ROS in mitophagy by PARP1 activation and PARylation of PINK1. In briefGao et al. demonstrate that exogenous ROS inhibit mitophagy. Mechanistically, ROS activate PARP1, which mediates PARylation of PINK1, a central regulator of mitophagy, leading to its functional impairment. This study reveals a novel regulatory mechanism of ROS on mitophagy through PARP1 activation and identifies PARylation as a novel form of post-translational modification of PINK1. HighlightsO_LIROS block PINK1-Parkin-mediated mitophagy. C_LIO_LIROS activate PARP1. C_LIO_LIPARP1 suppression eliminates the inhibitory effect of ROS on mitophagy. C_LIO_LIPARylation of PINK1 by PARP1 impairs its activity and mitophagy. C_LI

Although KRAS G12C-specific inhibitors such as sotorasib have been approved by US FDA and currently used in clinic, treating non-G12C mutants and overcoming acquired resistance for these inhibitors remain critical challenges. Here, we introduce a reciprocal feedback blockade therapy combining the MEK inhibitor trametinib and the multi-tyrosine kinase inhibitor imatinib to overcome these limitations. Our study reveals their compensatory roles: trametinib suppresses MEK activity yet promotes tyrosine kinase signaling and angiogenesis, while imatinib, a pan-tyrosine kinase inhibitor unleashes the MEK/ERK pathway via phosphatase suppression. Combining these agents blocks the reciprocal survival signals, inducing robust cell death across diverse KRAS-mutant models. Mechanistically, this combination reprograms cellular metabolism, leading to autophagy-dependent lipid peroxidation accumulation and ferroptosis. This strategy was effective in sotorasib-resistant lung cancer cells and various mouse models, including pancreatic cancer patient-derived xenograft. Furthermore, a pilot clinical trial for KRAS-mutant pancreatic cancer yielded encouraging responses. Consequently, the trametinib-imatinib combination represents a promising, broad-spectrum therapeutic strategy to overcome the constraints of current KRAS-targeted therapies.

Iron is essential for tumor proliferation and metabolic adaptation but becomes cytotoxic when unbuffered, creating a potential metabolic vulnerability. Ferritin, a conserved iron-storage complex, limits labile iron and establishes the upper threshold of iron tolerance in cancer cells. Here, we report the first ferritin heavy chain (FTH) knockout in a brain tumor model system. Although FTH loss was tolerated under basal conditions through adaptive remodeling of iron metabolism, it exposed profound vulnerabilities under iron stress. FTH deficiency lowered the threshold for iron toxicity, sensitizing medulloblastoma (MB) cells to both canonical ferroptosis and a mechanistically distinct iron-dependent cell death pathway. Oxidative iron stress impaired tumor growth and prolonged survival in orthotopic xenografts, whereas vitamin C-induced iron reduction triggered a selective, iron-dependent, but non-ferroptotic elimination of MB-like cells in tumor organoids. Notably, sensitivity to iron toxicity correlated strongly with cellular phenotype, with mesenchymal-like cells displaying greater susceptibility than epithelial-like counterparts. Collectively, these findings identify ferritin as a central regulator of iron tolerance in MB and establish iron toxicity, not via iron deprivation, as a therapeutically exploitable vulnerability. More broadly, this work provides a mechanistic framework for targeting iron metabolism through modulation of ferritin-dependent iron buffering and iron redox homeostasis in cancers. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/739635v1_ufig1.gif" ALT="Figure 1000"> View larger version (53K): org.highwire.dtl.DTLVardef@e8ba94org.highwire.dtl.DTLVardef@1317618org.highwire.dtl.DTLVardef@4060d7org.highwire.dtl.DTLVardef@95fac6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Vacuolar ATPases (V-ATPases) are highly conserved multi-subunit proton pumps that drive the acidification of intracellular vesicles, especially endosomes and lysosomes. By regulating progressive acidification of the endolysosomal pathway, V-ATPase activity impacts signaling transduction pathways both positively (e.g., internalization and activation of receptor-ligand complexes in endosomes) and negatively (e.g., degradation of pathway mediators in lysosomes). While the role of V-ATPases in human neurodegenerative diseases and cancer has been extensively studied, the requirement for these proteins in intestinal restitution remains poorly understood. Here, we use Drosophila to study the role of V-ATPases in regulating intestinal-injury and repair driven by excessive oxidative stress. We find that RNAi driven depletion of multiple subunits of the V-ATPase complex suppressed oxidative stress-induced lethality. By contrast, depletion of the main lysosomal catabolic enzyme in Drosophila (Cathepsin-D) had no effect. On a cellular level, these effects map to absorptive enterocytes (ECs) of the Drosophila intestine. Molecular analysis of intestines following injury by oxidative stress compared to uninjured controls reveals increased cell death, increased JNK-pathway activity, and increased IMD/NF-{kappa}B pathway signaling reporter expression compared to uninjured controls. Depletion of Vha44 (subunit C of the V1 complex) was sufficient to suppress the increased cell death, JNK pathway, and IMD/NF-{kappa}B pathway markers induced by oxidative stress in the intestine. Furthermore, overexpression of the MAP3K TAK1 enhanced death, JNK pathway and IMD/NF-{kappa}B pathway activation in a Vha44 dependent manner. These findings suggest that inhibition of V-ATPase activity can protect against intestinal injury caused by excessive oxidative stress. On a molecular level, we find that attenuation of endolysosomal acidification dampens pro-apoptotic JNK and IMD/NF-{kappa}B pathways, highlighting endosomal acidification as a potential amplifier of excessive oxidative stress.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and more evidence suggests that glucose metabolism plays a significant role in the development and progression with glycosylation at multiple sites potentially being a key characteristic. However, the underlying mechanisms remain insufficiently studied. Here, we first confirmed the presence of O-GlcNAc glycosylation modifications at Thr50 on PKM, which is highly expressed in PDAC and strongly correlated with poor prognosis. Further, we found that PKM expression was significantly positively correlated with CDC27 levels, and mutation of the Thr50 O-GlcNAc site in PKM abolished the upregulation of CDC27. We confirmed that O-GlcNAc-modified PKM enhances nuclear translocation of ARNT, which binds to the CDC27 promoter to upregulate its expression. Finally, we demonstrated that reduced expression of CDC27, as a key component of the APC/C complex, leads to downregulation of ubiquitination at the K11 site of PPP2CA, resulting in upregulation of PPP2CA expression, in turn, reduces AKT phosphorylation, ultimately driving PDAC regression by inhibiting aerobic glycolysis. Thus, we delineate a novel O-GlcNAcylation-dependent pathway where PKM drives PDAC progression through ARNT-mediated CDC27 transcriptional activation and AKT-mediated glycolysis.

Background & AimsProhaptoglobin (proHp), a precursor of haptoglobin (Hp), has recently emerged as a cancer-associated biomarker, but its functional role in pancreatic cancer remains unclear. We investigated whether proHp promotes malignant phenotypes of pancreatic cancer and explored signaling pathways involved. MethodsSerum proHp was examined in patients with pancreatic cancer and healthy controls. HP expression in pancreatic tumors and cell lines was analyzed using transcriptomic datasets. ProHp function was evaluated using PSN1 HP knockout (HPKO) cells, exogenous proHp supplementation, xenograft models, and RNA sequencing of PSN1 wild-type and HPKO cells. YAP activity was assessed by target gene expression, subcellular localization, YAP overexpression, and inhibition of YAP-TEAD interaction. ResultsSerum proHp was significantly elevated in patients with pancreatic cancer, and a subset of tumors and pancreatic cancer cell lines showed HP expression comparable to liver, indicating a tumor-derived source of proHp. In PSN1 cells, HPKO reduced motility, whereas exogenous proHp partially rescued this defect and enhanced motility in additional pancreatic cancer cell lines. Wild-type PSN1 cells continued to proliferate beyond confluence and formed rapidly growing xenograft tumors, which were abolished in HPKO cells. At high density, wild-type cells maintained YAP-related gene expression and nuclear YAP despite Hippo activation, whereas HPKO exhibited reduced nuclear YAP, indicating noncanonical YAP regulation by proHp. YAP restoration in HPKO cells rescued high-density proliferation and cell motility, while a YAP-TEAD inhibitor selectively reduced high-density proliferation of wild-type but not HPKO cells. ConclusionProHp promotes pancreatic cancer progression in a context-dependent manner by sustaining YAP activity and enabling cells to partially overcome contact-dependent growth inhibition. SynopsisProhaptoglobin, a precursor of haptoglobin, is elevated in pancreatic cancer and produced by tumor cells. It promotes cell motility, supports tumor growth under high-density conditions, and maintains YAP-dependent transcription that overrides contact-dependent growth inhibition. What You Need to Know BackgroundProhaptoglobin, a precursor of haptoglobin, is elevated in pancreatic cancer, but its tumor-derived origin and functional role are unknown. We examined whether prohaptoglobin drives progression by sustaining YAP signaling. ImpactWe show that tumor-derived prohaptoglobin sustains nuclear YAP activity, allowing pancreatic cancer cells to bypass contact inhibition and proliferate, revealing prohaptoglobin as a context-dependent driver rather than a passive biomarker. Future DirectionsDefining how prohaptoglobin engages upstream Hippo-YAP regulators and whether prohaptoglobin-YAP signaling is targetable in vivo may uncover new biomarkers and therapeutic vulnerabilities for pancreatic cancer.

Epithelial cells can encode prior damage into lasting epigenetic and functional states, enabling a primed response to future insults. In the pancreas, acute injury induces reversible acinar cell reprogramming toward a progenitor-like identity that persists beyond repair, supporting resilience to recurrent injury but creating a permissive state for malignant transformation. Given the central role of the tissue niche in stem cell regulation, we investigated microenvironmental adaptations that sustain this primed epithelial state. Using genetic mouse models and ex vivo organoid co-cultures, we identify a sex-specific sensory neural memory after pancreatitis that sustains long-term epithelial plasticity through a CGRP-dependent neuron-epithelial axis. We show that sex differences in acute inflammation drive neutrophil-dependent suppression of neural activation in females, decoupling neural memory from epithelial plasticity after repair. In males, neural memory promotes post-injury plasticity, revealing tissue memory as coordinated adaptation between epithelial progenitors and their niche.

A-type lamins are critical for nuclear integrity and mechanotransduction in cardiomyocytes, and their disruption is a major cause of inherited cardiomyopathy. To compare the consequences of lamin A/C loss versus defective lamin A maturation, we generated CRISPR/Cas9-edited hiPSC lines lacking LMNA or ZMPSTE24 and differentiated them into iPSC-derived cardiomyocytes. LMNA knockout caused progressive nuclear deformation, loss of culture stability, and contractile vulnerability in iCM. ZMPSTE24 knockout led to subtler nuclear abnormalities and reduced calcium transient activity, temporally correlating with prelamin A accrual. Transcriptomics profiling revealed aberrant mechanical responses in both LMNA and ZMPSTE24 bi-allelic knockouts as well as unique perturbations in inflammatory signaling and epigenetic pathways. Interestingly, both knockout models shared a marked defect in proteostasis, as confirmed by reduced proteasome activity. Together, these results show that loss of lamin A/C and accumulation of prelamin A trigger both converging and distinct cardiomyocyte stress responses. In addition, the newly generated models offer an attractive platform to study lamin-associated cardiomyopathy and its therapeutic targeting.

Human pluripotent stem cells (hPSCs) rely predominantly on glycolysis and exhibit relatively low mitochondrial respiration. Under these conditions, the mitochondrial F1Fo ATP synthase tends to operate in reverse mode, hydrolyzing ATP. ATP synthase inhibitory factor subunit 1 (IF1) inhibits this F1Fo ATP hydrolysis, but its role in hPSCs remains unclear. Here, we generated human induced pluripotent stem cells (hiPSCs) with stable IF1 knockdown (IF1-KD). IF1-KD enhanced F1Fo ATP hydrolysis and elevated the mitochondrial membrane potential (MMP). Although core pluripotency transcription factors were maintained, IF1-KD cells exhibited a partial epithelial-mesenchymal transition (EMT)-like state and biased trilineage differentiation. Mechanistically, the elevated MMP was accompanied by enhanced store-operated Ca{superscript 2} entry (SOCE) and nuclear translocation of NFATc3. Moreover, lowering the MMP attenuated SOCE, and NFATc3 overexpression reproduced the EMT-like gene expression. These results support a model in which IF1, by inhibiting F1Fo ATP hydrolysis, prevents excessive elevation of the MMP and thereby suppresses the transition to a partial EMT-like state via the MMP-SOCE-NFAT axis, contributing to the maintenance of the epithelial state associated with hiPSC pluripotency.

Hypoxia and NF-{kappa}B signaling are well-established drivers of cancer progression and treatment failure, yet the oxygen-dependent regulation of non-canonical NF-{kappa}B signaling remains poorly defined. Here, we identify hypoxia-inducible factor prolyl-4-hydroxylase-1 (HIF-P4H-1/EGLN2) as a key modulator of the non-canonical NF-{kappa}B pathway. Using integrated biochemical, genetic, proteomic, and transcriptomic analyses across human cell lines, mouse models, and clinical tumor samples, we demonstrate that HIF-P4H-1 directly interacts with and hydroxylates IKK at proline 367, thereby promoting its ubiquitination and proteasomal degradation. Loss or inhibition of HIF-P4H-1 results in accumulation of IKK, impaired NF-{kappa}B2/p100 processing to p52, destabilization of NF-{kappa}B-inducing kinase (NIK), and suppression of non-canonical NF-{kappa}B-dependent survival gene expression. Structural modeling and mutagenesis identify proline 367 hydroxylation as a critical determinant of IKK turnover. Analysis of TCGA cohorts reveals an inverse correlation between HIF-P4H-1 and IKK expression, with elevated HIF-P4H-1 associating with advanced tumor stage and reduced overall survival in clear cell renal cell carcinoma. Functionally, targeting HIF-P4H-1 sensitizes cancer cells to cell death and impairs proliferation, clonogenic growth, and migration. Together, our findings define a previously unrecognized oxygen-dependent mechanism regulating non-canonical NF-{kappa}B signaling through direct control of IKK stability and nominate the HIF-P4H-1-IKK axis as a potential therapeutic vulnerability in hypoxia-adapted malignancies.