Science Signaling

NUDT2 is an emerging candidate for therapeutic intervention in cancer, and its inhibition or loss is known to elevate adenosine-containing dinucleoside polyphosphates (ApnNs), including diadenosine tetraphosphate (Ap4A). Ap4A is a stress- and immune-associated nucleotide metabolite proposed to act as a second messenger, raising the possibility that NUDT2 targeting may unintentionally affect important nucleotide-sensitive signaling pathways. One such pathway is cGAS-STING signaling, a central innate immune axis that detects cytosolic DNA, produces the nucleotide second messenger 2'3'-cGAMP, and drives type I interferon responses. Because cGAS-STING also contributes to antitumor immunity and is being pharmacologically targeted in cancer, we asked whether sustained Ap4A accumulation perturbs this pathway. We systematically evaluated Ap4A and related dinucleoside polyphosphates across the cGAS-STING-TBK1 axis using biophysical, enzymatic, structural, and cellular approaches. Contrary to a previous model, STING did not bind Ap4A, Ap3A, or Ap4G, despite robust binding of canonical cyclic dinucleotides. Although cGAS bound these nucleotides with micromolar affinities, DNA-activated cGAMP synthesis was inhibited only at high, supra-substrate ratios. Similarly, TBK1 inhibition required extreme Ap4A ratios beyond physiologically relevant levels. In a THP-1 cell model, NUDT2 knockout caused strong Ap4A accumulation, but the resulting intracellular dinucleoside polyphosphate levels remained below the ratios required to inhibit cGAS or TBK1 in vitro. This study thus distinguishes biochemical possibility from physiological relevance and argues that NUDT2-linked Ap4A accumulation is unlikely to directly compromise cGAS-STING pathway activity.

Because ErbB receptors play distinct roles in regulating diverse cellular functions, the mechanisms governing ErbB receptor activation are likely to be more diverse than previously recognized. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] positively regulates ErbB1 kinase activity, but the role of PI(4,5)P2 in regulating other ErbB family members in living cells remains poorly understood. We show that disruption of PI(4,5)P2 binding enhances ErbB4 oligomerization and kinase activity while reducing both processes in ErbB1. Analysis of chimeric receptors identified the juxtamembrane (JM) regions of ErbB1 and ErbB4 as key determinants of their distinct responses to PI(4,5)P2 during receptor oligomerization and kinase activation. Furthermore, the JM-kinase module of ErbB1 is more active in the presence of PI(4,5)P2, whereas that of ErbB4 is activated by the disruption of PI(4,5)P2 binding. In contrast, the JM-kinase module of ErbB2 exhibits weak dependence on PI(4,5)P2. ErbB2 preferentially promotes ErbB4 oligomerization over ErbB1 oligomerization, thereby enhancing ErbB4 activation. Collectively, these findings identify plasma membrane PI(4,5)P2 availability and ErbB2 abundance as two factors that jointly govern ErbB receptor oligomerization and activation.

Toll-like receptor (TLR) signaling must be activated rapidly and then terminated to support host defense without sustained inflammation. We developed a rule-based model of mouse macrophage TLR4 signaling at the molecular-interaction level using measured protein copy numbers, RNA-seq-based abundance estimates, literature- and structure-informed reaction rates, and 979 dynamic experimental constraints. The trained model reproduced much of the TLR4-induced NF-{kappa}B and MAP kinase response but consistently failed to capture deactivation of MyD88, TRAF6-associated species, and IKK/{beta}. The recurrent model failure conveyed important biological information, localizing missing regulation to the proximal MyD88-IRAK-TRAF6 module and guiding experimental evaluation of IKK{varepsilon} and its scaffold TANK. Loss of IKK{varepsilon} enhanced transcriptional, cytokine, MAP kinase, and NF-{kappa}B responses to MyD88-specific TLR ligands. TANK deficiency produced a similar cellular phenotype and abolished stimulus-induced IKK{varepsilon} phosphorylation. Deficiency of either protein increased IRAK1 and TRAF6 ubiquitination without increasing MyD88 ubiquitination, placing the inhibitory checkpoint at or immediately downstream of the IRAK1-TRAF6 ubiquitin-signaling node. Overlapping but non-identical in vivo phenotypes further supported a shared regulatory axis with additional protein-specific functions. Our study presents a model-experiment discovery cycle where quantitative pathway discordance identifies missing biology and reveals a TANK-dependent IKK{varepsilon} checkpoint that restrains MyD88-driven inflammation.

Abstract: Endocannabinoids (eCBs) like anandamide and 2-arachidonoylglycerol are endogenous lipid ligands for CB1 and CB2 G protein-coupled receptors (GPCRs). Here, we show that phosphorylation of biologically important anandamide generates naturally occurring anandamide phosphate (AEAp), which switches GPCR ligand specificity to lysophosphatidic acid (LPA) receptors (LPARs) and the primate-specific bile acid sensory receptor MRGPRX4. The kinase responsible for anandamide phosphorylation was identified asdiacylglycerol kinase (DGK) theta (DGKq or DGKQ) using activity-guided brain fractionation, inhibitor profiling, recombinant reconstitution, and DGK isozyme screening. DGKQ showed noncanonical biphasic lipid kinetics and was inhibited by phytocannabinoids, most notably tetrahydrocannabinolic acid. Thus, eCBs are not only cannabinoid receptor ligands but also enzymatically adaptable lipid signals activating LPARs and MRGPRX4, thus linking metabolism of the distinct lipid LPA with Cannabis pharmacology.

Type I and type III interferons (IFNs) activate the shared Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway but generate distinct inflammatory transcriptional responses in addition to their shared antiviral transcriptional induction. While some mechanisms including cytokine-receptor affinity and receptor abundance have been proposed for regulating type I vs type III IFN specificity, they account for differences in signal amplitude but do not fully explain IFN-specific changes in JAK-STAT activity over time (i.e. the signaling temporal dynamics) which has been shown to be predictive of inflammatory gene activation. Here, we investigated mechanisms that could explain how type I and III IFNs produce distinct temporal dynamics of the IFN-activated transcription factor IFN-stimulated gene factor 3 (ISGF3), leading to IFN-type specific activation of inflammatory genes in lung epithelial cells. We show that IFN-{beta}, a type I IFN, and IFN-{lambda}3, a type III IFN, induced comparable initial ISGF3 activation but subsequently generated distinct signaling trajectories, with IFN-{beta} undergoing potentiation and IFN-{lambda}3 limiting the duration of high ISGF3 activity, preventing robust inflammatory gene induction despite prolonged lower-level activity. Inflammatory gene induction was associated with the magnitude and duration of ISGF3 activity above an activation threshold during the intermediate phase of the signal response. The characteristic IFN-{lambda}3-induced ISGF3 signaling dynamics were maintained at saturating doses of IFN-{lambda}3 indicating the lack of signal activation above the threshold was independent of the dose. Additionally, differences in the abundance of the ISGF3 complex subunits STAT1, STAT2, and interferon regulatory factor 9 (IRF9), or in the induction of known negative regulators did not readily explain the response. Blocking protein synthesis caused rapid loss of IFN-{lambda}3-induced STAT phosphorylation and ISGF3 activity, supporting the presence of a pre-existing negative regulator limiting IFN-{lambda}3-mediated JAK-STAT pathway activation above the threshold required for activating inflammatory genes, while IFN-{beta} and IFN-{lambda}3 co-stimulation localized this regulation upstream of the shared ISGF3 complex. Increasing pathway competence through IFN-{beta} priming enhanced the IFN-{lambda}3-induced ISGF3 response above the inflammatory gene activation threshold leading to induction of inflammatory genes, albeit transiently. Together, these findings support a model in which the magnitude and duration of ISGF3 activity above an inflammatory gene activation threshold, rather than pathway activation alone, contribute to stimulus-specific transcriptional responses and suggest that a negative regulatory mechanism upstream of the ISGF3 complex constrains the temporal dynamics of IFN-{lambda}3 signaling.

Dysregulation of cGAS-STING pathway contributes to various disorders, including autoimmunity, infectious diseases and cancer. Transcription factors, particularly IRF3 which presumably recruits CBP/p300 as transcription coactivators, drive innate immune responses to STING activation. Here we show that CBP/p300 inhibitors (p300i) boost, not repress as expected, the cGAMP-induced transcription of innate immune genes. Mechanistically, p300i and a CBP/p300 degrader markedly enhance the cGAMP-induced IRF3 phosphorylation and slightly increase NF-{kappa}B activation. Furthermore, cGAMP stimulation increases the presence of p300, not CBP, in the cytoplasm to interrupt interactions between TBK1 and IRF3. Conversely, p300i enhances TBK1-IRF3 interactions, resulting in higher IRF3 phosphorylation. After STING activation, CBP, not p300, is recruited to chromatin along with IRF3 and is coupled with H3K27ac upregulation. At later stages of STING activation, nearly all CBP and p300 are detached from chromatin, which is associated with global histone deacetylation, IRF3 chromatin eviction, and the termination of IRF3-driven transcription programs. p300i robustly enhances IRF3 chromatin engagement which consequently amplifies type 1 interferon responses to STING activation. Together, these findings suggest that CBP, not p300, acts as a transcription coactivator in the nucleus. In contrast, p300, not CBP, is a suppressor of STING signaling in the cytoplasm.

ZNF217 is a potent oncogene that drives ovarian cancer progression and therapeutic resistance. We show that ZNF217 overexpression markedly increases ERBB2 levels in ovarian cancer cells, suggesting its potential as a biomarker to identify ovarian tumors that will respond to ERBB2-targeted therapeutics. Unexpectedly, ZNF217-high ovarian cancer cells exhibit resistance to multiple ERBB2 inhibitors, revealing a disconnect between receptor abundance and drug sensitivity. Mechanistically, ZNF217 drives chemoresistance by elevating the expression of several key receptor tyrosine kinases, most notably AXL, that activates MAPK signaling. Further, ZNF217 also upregulates ERK1/2 levels in ovarian cancer cells. This induces rewiring of downstream signaling, promoting an ERK-dominant state that sustains survival despite ERBB2 inhibition. Therapeutic targeting of the AXL-ERK axis reduced cell viability and metastatic potential in vitro, while suppressing tumor burden and prolonging survival in xenograft models. Thus, by elevating the expression of several key signaling receptors and their downstream effectors in ovarian cancer cells, ZNF217 establishes signaling plasticity that defines a novel mechanism of chemoresistance. These findings identify ZNF217 as a key determinant of signaling state and drug response and provides rationale to evaluate targeting AXL and ERK signaling in ZNF217-high ovarian tumors.

T cell-based immunotherapies have remained ineffective against high-grade serous ovarian carcinoma (HGSOC). The metabolic environment of HGSOC suppresses the activity of cellular therapies, however, the metabolites that enhance or suppress T cell antitumor activity are not fully understood. Here, a pooled CRISPR-Cas9 knockout screen in primary human T cells cultured with patient-derived ascites was used to identify metabolic enzymes that inhibit effector cytokine production and cytolytic function. The screen identified PRDX1 as a negative regulator of T cell effector function. Targeted deletion of PRDX1 increased the frequency of IFN-{gamma}-producing T cells, enhanced glucose uptake, increased mitochondrial mass, and improved T cell viability under suppressive ascites conditions. Mechanistically, PRDX1 deficiency increased intracellular reactive oxygen species (ROS) and impaired autophagic flux. The effects of PRDX1 deletion enhanced aspects of T cell function, while its effects on chimeric antigen receptor (CAR)-T cell cytotoxicity were donor dependent. Collectively, this study identifies PRDX1 as a regulator of T cell activation, metabolism, and effector function in the inhibitory physiological suppressive environment of HGSOC ascites.

Macrophages are central regulators of innate immunity, yet the post-translational mechanisms that enable rapid inflammatory responses remain incompletely understood. Here, we integrated transcriptomics, unenriched proteomics, phosphoproteomics, metabolomics, and cysteine reactivity profiling to comprehensively characterize primary human macrophages following Toll-like receptor and Stimulator of Interferon Genes (STING) activation. This approach revealed substantial discordance between transcript- and protein-level changes, as well as extensive post-translational remodeling. Differentially reactive cysteines were enriched in proteins involved in small-GTPase signaling, vesicular trafficking, and the endo-lysosomal system. Reactivity changes in two subunits of the endosomal mRNA-transport FERRY complex nominated it as a candidate target of inflammation-associated post-translational regulation. Biochemical and imaging analyses established FERRY complex assembly and early endosomal localization in primary human macrophages and identified DISC1 as a previously unrecognized FERRY-associated protein. This work provides a comprehensive resource of biochemical and post-translational remodeling during macrophage activation and implicates endosome-associated mRNA transport in innate immune regulation.

Colorectal cancer (CRC) subtypes differ fundamentally in their reliance on WNT signaling, yet the sources and regulation of WNT ligands as well as the subtype-specific requirements for ligand-driven activation remain poorly defined. Here, we generated genetically-engineered organoid models of Apc- and Braf-mutant CRC to systematically dissect ligand dependency across distinct genetic backgrounds and CRC subtypes. Through genetic perturbation and pharmacological inhibition of WNT secretion, we find that BRAF-driven organoids critically depend on autocrine WNT ligand production for survival and proliferation. In contrast, APC-mutant organoids remain viable but undergo discernible transcriptional changes upon ligand withdrawal. We identify Wnt7b as a non-redundant epithelial WNT ligand required to sustain beta-catenin-dependent signaling specifically in BRAF-mutant CRC. Chromatin accessibility and transcriptional profiling reveal that Wnt7b activation occurs early during transformation and coincides with cancer-specific opening of regulatory elements at the Wnt7b locus. Integrative regulatory analyses nominate the transcription factor RFX7 as a candidate upstream regulator of epithelial Wnt7b expression. Consistent with these findings, analysis of human CRC specimens demonstrates WNT7B expression in a subset of BRAF-mutant tumors and reveals an association with poor patient survival. Together, these results redefine ligand dependency in colorectal cancer and uncover autocrine WNT7B signaling as a mechanistically defined vulnerability in aggressive BRAF-mutant disease.

Functional ovarian plasticity depends on the granulosa cell (GC) fate determination, differentiation, oocyte support and steroid production. After birth, GC identity must be maintained in differentiating follicles, while from puberty, ovarian plasticity is cyclically challenged by gonadotropic hormones. TRIM28-FOXL2 interactions form a key chromatin component maintaining ovarian identity programs and repressing testis-specific genes. This TRIM28 role depends on its E3-SUMO-ligase activity, suggesting SUMOylation in GC can be regulated by transient hormonal cues, although hormonal involvement in modulating GC identity remains to be established. Here, we demonstrated the in vivo interplay between gonadotropic-PKA-signaling and hypo-SUMOylation waves underlying ovarian GC plasticity. In vitro TRIM28 mutagenesis and phospho-proteomics demonstrated direct PKA inhibitory control on TRIM28. This prompted us to highlight SUMOylation alterations in Carney Complex ovarian lesions resulting from chronic PKA activity from rare genetic mutations. Using mutant models, transcriptomics and TRIM28/FOXL2-ChIP-Seq analyses, we specified the in vivo PKA inhibitory mechanisms on TRIM28/FOXL2 chromatin binding and its consequences on adult ovarian somatic cell identity. Together, our data support an integrative model where gonadotrophin pulses create transient windows of PKA-mediated TRIM28 phosphorylation, hypo-SUMOylation, and FOXL2-TRIM28 destabilization. This positions SUMOylation dynamics as central integrator of hormonal signaling and epigenetic identity maintenance in the ovary.

Repetitive cytosolic Ca2+ spikes in pancreatic acinar cells, elicited by low (physiological) concentrations of acetylcholine (ACh), cholecystokinin (CCK) and gastrin releasing peptide (GRP), control secretion of digestive enzymes, whereas high-intensity stimulation induces sustained Ca2+ elevation initiating acute pancreatitis. Since inositol trisphosphate (IP3) was discovered as an intracellular Ca2+ releasing messenger, it has been assumed that a major class of G-protein coupled receptors relies on this pathway. We have now compared the mechanisms of action of the three physiological stimulants, all acting on different receptors, but each coupled to the IP3 pathway. Low concentrations of CCK and GRP cannot elicit Ca2+ signals without co-operation of an additional intracellular mechanism. CCK-elicited Ca2+ signalling requires activation of intracellular receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), whereas this is not the case for the action of GRP that nevertheless relies on the operation of CD38, the enzyme involved in the synthesis of both cyclic ADP ribose and NAADP. Even Ca2+ signals elicited by ACh are partially dependent on CD38. It is engagement of these additional non-IP3 pathways that allows low concentrations of secretagogues to elicit safe Ca2+ spiking and therefore secretion, obviating the need for potentially toxic high levels of secretagogues.

The mild hypothermia response (MHR) is a conserved mammalian cytoprotective program activated upon exposure to mild hypothermia (32 degrees C) that contributes to the neuroprotective effects of therapeutic hypothermia following hypoxic injury. Although rapid changes in intracellular calcium occur upon cooling, the mechanisms linking calcium dynamics to the activation of core MHR factors such as SP1 and RBM3 remain incompletely defined. In this study, we used siRNA-mediated knockdown (KD) of candidate regulators in conjunction with novel mild hypothermia indicator (MHI) reporters to identify upstream modulators of MHR-associated transcription. We identify SARAF, a negative regulator of store-operated calcium entry (SOCE), as a repressor of both SP1 and RBM3 under normothermic conditions. SARAF depletion is associated with increased intracellular calcium release and enhanced SP1- and RBM3-linked transcriptional outputs. We identify JUN as an important downstream factor mediating SARAF depletion-dependent de-repression of the MHR and demonstrate that it undergoes activation rapidly upon cooling. Finally, SARAF depletion conferred significant cytoprotection against hypoxia-induced early apoptosis. Collectively, these findings establish SARAF as an upstream regulator of MHR-associated transcription and provide a functional link between cold-induced intracellular calcium dynamics and the induction of core MHR effectors.

CD8+ T cells in solid cancers progressively lose anti-tumor activity, yet the cell-intrinsic mechanisms driving this loss of function remain incompletely defined. Here, we performed matched proteomic and transcriptomic profiling of dysfunctional and bystander CD8+ tumor-infiltrating T cells isolated from primary tumors of treatment-naive non-small cell lung cancer patients. Proteomic analysis revealed widespread discordance with mRNA expression, with 8% of all quantified proteins displaying differential expression exclusively at the protein level. Genetic perturbation of such differentially expressed proteins identified the chromatin remodeler CHD4 and fatty acid synthase (FASN) as cell-intrinsic regulators of T cell function. CHD4 deletion resulted in altered gene-regulatory networks that promoted effector differentiation and enhanced cytokine production. In contrast, FASN deletion preserved mitochondrial fitness and sustained T cell functionality under chronic T cell receptor stimulation. Together, these findings demonstrate that proteomic profiling uncovers regulators of T cell functionality that are not apparent from transcriptomic analyses alone, highlighting an additional layer of regulatory control.

H2O2 released to the extracellular space (ECS) regulates diverse physiological processes, yet its concentrations and spatial distribution in tissues remain poorly defined. This uncertainty hampers mechanistic understanding of redox signaling. Here, we used reaction-diffusion modeling to estimate extracellular H2O2 concentrations and transport ranges in various scenarios. Idealized analytical models were combined with numerical models incorporating localized NADPH oxidase (NOX) clusters, ECS microstructure, membrane permeability, and the thioredoxin- and GSH-dependent clearance systems. Using maximal neutrophil and NOX superoxide/H2O2 release rates, we obtained upper bounds for extracellular H2O2. Adjacent to isolated average-sized, fully active NOX2 clusters H2O2 peaked at ~540 nM at adhesion cell-cell separations, and decreased radially over ~50-100 nm. At the receptor cell surface, peak concentration decreased inversely with intercellular separation, to <5 nM at 1 m separation. Radial decrease here, for this wide separation, was over ~2.5 m. Even the former maximal extracellular concentrations induce just a minimal, highly localized oxidation of the intracellular Prdx, Trx and GSH pools. In turn, maximally activated neutrophils carry ~2000 such NOX2 clusters, inducing 10s of M peak ECS H2O2 concentrations. These cause extensive Prdx and Trx oxidation near the exposed membranes. However, the GSH-dependent system still sustains a strong transmembrane gradient if the permeation barrier remains intact, and ECS H2O2 concentrations decay to sub-M within a few m of the source cell. Extracellular H2O2 concentrations scaled linearly with source flux in all the examined conditions. These results establish stringent constraints on autocrine, juxtacrine and next-cell paracrine H2O2 signaling.

Caspase-11 (Casp11) is a murine zymogen that becomes active upon sensing cytosolic lipopolysaccharide (LPS), enabling it to form the non-canonical inflammasome and execute an inflammatory cell death known as pyroptosis. The lipid A acylation state of LPS is thought to be the primary regulator of Casp11 activation. However, our data demonstrate that the polysaccharide moiety of LPS, and particularly the polysaccharide phosphates, are critical for eliciting maximal Casp11 activation. Accordingly, the polysaccharide was required for Rhodobacter sphaeroides LPS to act as an inflammasome antagonist. Furthermore, isolated polysaccharide was sufficient to antagonize Casp11 activation. Mechanistically, our findings support a two-step model of non-canonical inflammasome activation where the polysaccharide recruits Casp11 to LPS and the lipid A acylation state governs oligomerization efficiency. These findings provide new insight into the molecular basis of LPS-driven Casp11 activation, with implications for the design of future therapies that target the non-canonical inflammasome.

Target-derived neurotrophins regulate neuronal survival and pruning via retrograde signaling. Unlike Trk receptors, which are transported as transmembrane proteins, the p75 neurotrophin receptor (p75NTR) undergoes regulated intramembrane proteolysis to release a soluble intracellular domain (ICD) that must somehow associate with axonal endosomes for retrograde transport. Here, we identify palmitoylation of a conserved juxta membrane cysteine (C281) as a critical requirement for this process. In sympathetic neurons, we demonstrate that p75NTR palmitoylation is dispensable for surface trafficking and proteolytic cleavage but is strictly required for ligand-induced internalization and the biogenesis of p75ICD-positive signaling endosomes. Consequently, blocking palmitoylation, either pharmacologically or via a palmitoylation-deficient C281A mutation, prevents the accumulation of p75ICD in axonal endosomes and blocks axon-initiated apoptosis. In vivo, C281A knock-in mice exhibit reduced developmental apoptosis of sympathetic neurons, similar to p75NTR deletion. These findings define palmitoylation as an essential checkpoint that couples p75NTR cleavage to endosomal segregation, thereby enabling the long-distance transmission of regressive signals.

BRAF mutant colorectal cancer (CRC) remains difficult to treat despite the clinical use of combined BRAF and EGFR inhibition, highlighting a need to define tumour-intrinsic mechanisms that limit therapeutic response. Here, using genome-wide CRISPR-Cas9 screening in BRAF-mutant CRC cells, we identify MEN1, encoding the chromatin-associated protein Menin, as a selective determinant of sensitivity to combined encorafenib and cetuximab (EC). MEN1 loss markedly enhanced EC-mediated inhibition of cell proliferation and ERK activity while having comparatively little effect in untreated cells, and re-expression of Menin restored resistance. Transcriptomic and chromatin profiling revealed that Menin supports the transcriptional response associated with MAPK signalling. Menin occupied promoters of MAPK/BRAF-responsive genes and EC treatment caused widespread displacement of Menin from chromatin. Phosphoproteomic analysis demonstrated extensive remodelling of MAPK signalling following EC treatment, whereas proximity proteomics showed that the Menin-associated protein complexes remained largely intact despite loss of Menin chromatin occupancy. Importantly, MLL1 loss did not reproduce the sensitising effect of MEN1 deletion, and pharmacological Menin inhibition with revumenib failed to phenocopy either genetic MEN1 loss or acute Menin degradation, indicating that this phenotype is independent of Menin-MLL activity. Together, these findings identify a previously unrecognised, MLL-independent role for Menin in buffering the response of BRAF-mutant CRC cells to MAPK pathway inhibition and suggest targeting Menin, rather than disruption of its interaction with MLL, may provide a strategy for enhancing the response to BRAF-targeted therapy for CRC.

Anti-angiogenic cancer therapies attempt to withhold necessary nutrients and oxygen from growing tumors by targeting the major promoters of endothelial cell (EC) angiogenesis: vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2). Unfortunately, these treatments are often insufficient, even when coupled with chemotherapies, and fail to significantly increase survival rates. The tumor microenvironment (TME) is mechanically distinct compared to normal tissue, including increased matrix deformations or strains caused by cancer-associated fibroblasts (CAFs). In this report, we detail the specific and independent roles of two tyrosine residues, Y1054 and Y1214, on mechanical activation of VEGFR-2. Furthermore, we characterize CAF biochemical and mechanical signaling and demonstrate how ECs exhibit decreased vessel growth when co-cultured with CAFs and treated with a contractility inhibitor. Using non-phosphorylatable VEGFR-2 mutants, we reveal Y1054 and Y1214 are each necessary for EC angiogenesis, particularly in response to strain. Overall, this research highlights the need to study how mechanics in the TME promote vessel growth and thus tumor progression, which is important to consider when developing future anti-angiogenic therapies.

Plasma membrane ceramide (PMCer) is a recognized cellular signal that regulates stress responses, such as chemotherapy and membrane damage, triggering apoptosis and plasma membrane repair. Acid sphingomyelinase (ASM) has been proposed to generate PMCer by directly hydrolyzing plasma membrane sphingomyelin into ceramide. However, the enzymatic requirements of ASM are difficult to reconcile with the extracellular environment on the outer leaflet of the plasma membrane. Here, we identify several cellular stress inducers, with acidic stress yielding a stronger response in PMCer generation. Using this inducer, we defined a lysosome-to-plasma membrane pathway for stress-induced PMCer generation. Acidic stress increased PMCer in an ASM-dependent manner, whereas ASM loss or catalytic inactivation abolished this response. Although secreted ASM was enzymatically active when tested in vitro, neither secreted nor exogenous recombinant ASM generated PMCer. Instead, acidic stress induced lysosomal exocytosis, while disruption of lysosomal fusion reduced PMCer accumulation. Modulating lysosomal ceramide translated into PMCer, showing that lysosomal ceramide is directly transferred into the plasma membrane. These findings redefine stress-related PMCer signaling through lysosomal ceramide exocytosis.