Science Signaling

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.

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.

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.

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.

Cell surface receptors are regulated via a variety of mechanisms including proteolytic inactivation by metalloproteases. Cells often co-express both the receptor and its cognate protease simultaneously on the cell surface, often with the proteases in an active state. Despite this, receptor cleavage is generally not constitutive, and rather must be induced. How a receptor and its inactivating protease can both be present in their active form on the cell surface with no cleavage of the target receptor is unclear. Using the efferocytic receptor MERTK and its inactivating protease ADAM17 as a model, along with super-resolution microscopy approaches, we demonstrate that MERTK and ADAM17 are localized to distinct sub-regions of the plasma membrane. These microdomains are distinct subcellular structures, with MERTK contained within actin-based corrals and ADAM17 contained within caveolae. Induction of MERTK cleavage by PMA results in the reorganization of the membrane-proximal actin cytoskeleton that enables MERTK to diffuse into ADAM17-containing microdomains. Only after localizing to ADAM17-containing microdomains is MERTK cleaved. These results demonstrate that the spatial organization of the plasma membrane is dynamically regulated, with reorganization of membrane microdomains playing a role in the regulation of ADAM17 activity.

Introduction: Sporadic colorectal cancers (CRCs) form following initiating APC mutations, inducing constitutive {beta}-catenin/TCF signaling. Similarly, the guanylyl cyclase C (GUCY2C) tumor suppressor axis is universally silenced in CRC. While the GUCY2C receptor is retained, loss of its paracrine hormones guanylin (GUCA2A) and uroguanylin (GUCA2B) is among the earliest events in transformation. Mutant APC/{beta}-catenin/TCF signaling driving CRC transcriptionally silences GUCY2C hormone expression while suppressing that oncogenic pathway reconstitutes hormone production opposing tumorigenesis. p38 opposes tumor initiation, and its loss promotes transformation in chemical and genetic mouse models of CRC. In healthy intestine, p38 supports a gradient of epithelial differentiation along the crypt-surface axis. Here, we reveal that p38 links regulation of oncogenic APC/{beta}-catenin/TCF signaling and GUCY2C hormone expression. Methods: Regulation of p38 by APC/{beta}-catenin/TCF signaling was defined in four genetically engineered human CRC cell lines with conditional oncogenic APC/{beta}-catenin/TCF signaling and healthy human colon organoids. Expression of p38 was eliminated from conditional human CRC cell lines by pharmacological inhibition, RNA knockdown, and CRISPR/Cas9 gene editing. Transcriptomic analysis of these cells by RNA sequencing defined the set of genes regulated by APC/{beta}-catenin/TCF signaling controlled through p38. Results: In CRC cell lines, APC/{beta}-catenin/TCF signaling suppressed p38 phosphorylation and activity, whereas inhibition of APC/{beta}-catenin/TCF restored p38 signaling. In the absence of APC/{beta}-catenin/TCF signaling, inhibiting p38 repressed GUCY2C hormone expression. Inhibiting APC/{beta}-catenin/TCF signaling enriched phosphorylated p38 at the GUCA2A promoter. In that context, analysis of the CPTAC proteomic database revealed that phosphorylation of p38 and its upstream kinases MKK3 and MKK4 was reduced in human CRC. Finally, transcriptomic analysis revealed that p38 is a critical signaling node controlling the expression of a substantial subset of APC/{beta}-catenin/TCF-regulated genes in human CRC cells and patient tumors. Conclusions: APC/{beta}-catenin/TCF signaling inhibits p38 phosphorylation and transcriptional activity, suppressing GUCA2A and GUCA2B expression and functionally silencing the retained GUCY2C receptor. These findings identify p38 as a signaling intermediary integrating oncogenic APC/{beta}-catenin/TCF activity to extinction of the GUCY2C tumor-suppressor axis and reveal a broader p38-dependent component of the APC/{beta}-catenin/TCF-regulated transcriptome.

The mechanism governing singular odorant receptor (OR) gene choice restricts each olfactory sensory neuron (OSN) to expressing a single OR allele from a repertoire of more than 1,100 receptor genes. Consequently, only a small fraction of OSNs express any given OR, limiting studies of OR function, axonal wiring, and odor coding. Here, we show that multimerizing a 21-bp homeodomain enhancer dramatically increases the probability of receptor choice. We engineered a single-copy Olfr151 minigene carrying a 9x21 enhancer and the human odorant receptor OR10G4, integrated at a genomic site distant from all endogenous OR clusters. This transgene drives singular OR10G4 expression in approximately 75% of OSNs and is accompanied by widespread reductions in endogenous Class I, Class II, and TAAR receptor transcripts and proteins, indicating that cis-regulatory elements govern the probability of OR selection. The abundance of OR10G4-expressing cilia enabled highly sensitive ligand screening using CELIA (Cilia-based Evaluation of Ligands and receptor InterActions). Profiling guaiacol derivatives revealed a strong correspondence among receptor activation, cAMP production, and published human psychophysical responses. Although OR10G4 responds to guaiacol-related odorants, multiple canonical vanilla odorants failed to activate the receptor, arguing against a primary role in vanilla perception. Instead, the lowest EC50 ligands were enriched for compounds human subjects described as smoky. Together, these findings link activation of a single human odorant receptor to a defined perceptual odor-quality dimension and establish a general strategy for decoding the molecular basis of human odor perception.

Tertiary lymphoid structures (TLS) predict response to checkpoint inhibition, and conventional dendritic cells (cDC) are required to build and maintain them. What renders a niche permissive for their assembly is unknown: chemokines recruit the cellular constituents but do not specify how they organize. Here we show that the organizing signal is a matrix fragment. Proteolysis of the proteoglycan versican (VCAN) releases the matrikine versikine, constitutively in germinal center light zones and inducibly in tumor stroma. Versikine activates CD11b+ cDC and expands cDC1 to assemble TLS-archetypal triads, apposed DC: CD4+ T: CD8+ T units. Versikine-conditioned DC engage progenitor-exhausted CD8+ T cells (Tpex) and Th1-polarized follicular helper CD4+ T cells. Excess unproteolyzed VCAN inverts this toward DC-Treg crosstalk and Tpex loss. Versikine supplied as mRNA or recombinant protein sensitizes refractory tumors to PD-L1 blockade. VCAN proteolysis prospectively predicted outcomes after PD-1 blockade in metastatic colorectal cancer. Matrikines organize chemokine-recruited cells into functional immune niches.

MAPK-activated protein kinase 2 (MAPKAPK2, MK2) is a downstream substrate of p38 MAPK that regulates inflammatory signaling and cytokine synthesis, making it an attractive therapeutic target. Although several crystal structures of MAPKAPK2 with ATP-competitive inhibitors have supported drug discovery, structural elucidation of selective non-ATP-competitive inhibitors has remained challenging. MK-25 is a non-ATP-competitive furan-2-carboxamide MAPKAPK2 inhibitor developed at Merck to overcome the selectivity and cellular-efficacy limitations of ATP-competitive chemotypes. Here, we show through isothermal titration calorimetry that MK-25 binds the activated MAPKAPK2, consistent with state-dependent allosteric binding. To determine the binding mode of MK-25, we solved a 2.8 A resolution crystal structure of an activated MAPKAPK2 construct in ternary complex with the ATP-site inhibitor PF-3644022 and a bromo-analogue of MK-25. The ligand occupies a pocket in the C-lobe overlapping the protein-substrate recognition region and formed through rearrangements of D and G helices and the activation segment. These interactions are consistent with the structure-activity relationships reported for the furan-2-carboxamide chemotype and identify a ligandable allosteric pocket that may facilitate future structure-guided design of selective, non-ATP-competitive MAPKAPK2 inhibitors.

Mechanical remodeling by cancer-associated fibroblasts (CAFs) stiffens tumors and can restrict molecular transport, yet the time-resolved coupling between cellular force and matrix mechanical state is poorly defined. Here we use a microfabricated force sensor integrated with 3D CAF-collagen tissue to measure CAF-generated force and collagen stiffness continuously during pharmacological perturbation. ROCK or non-muscle myosin II inhibition reduced force and remarkably drove collagen stiffness below its initial value rather than merely arresting stiffening. This softening was prevented by broad-spectrum MMP inhibition, but only modestly altered by lysyl oxidase inhibition, supporting a competition between force-driven collagen compaction and proteolytic remodeling. Second-harmonic generation imaging showed shorter, thinner and less aligned fibers after ROCK inhibition, while fluorescence recovery after photobleaching indicated faster dextran transport. Moreover, CAF contractile adaptation depended strongly on the duration of inhibition, with reversible recovery after brief treatment, a hyper-contractile rebound following prolonged treatment and washout, and partial force recovery during continuous exposure. These findings identify CAF-matrix mechanics as a dynamic, treatment-history-dependent balance and provide a framework for designing stromal interventions that alter stiffness and transport.

Aberrant signaling of receptor tyrosine kinases (RTKs), including the hepatocyte growth factor receptor MET, contributes to colorectal cancer (CRC) progression and is tightly regulated by endocytic trafficking. We previously showed that combined deletion of the endosomal sorting proteins SNX1 and SNX2 disrupts MET trafficking and enhances downstream signaling in HCT116 CRC cells. However, their individual contributions remained unresolved. Using CRISPR/Cas9-engineered SNX1- and SNX2-KO HCT116 cells, we examined their roles in MET trafficking and signaling. Loss of SNX2, but not SNX1, markedly reduced SNX5 and SNX6 protein levels without affecting transcript abundance. SNX2 deficiency also increased total MET under serum-containing conditions without altering MET transcript or cell-surface receptor levels. Following HGF stimulation, loss of SNX2, but not SNX1, transiently delayed MET trafficking to EEA1+ early and RAB7+ late endosomes, while recycling and lysosomal trafficking remained largely unaffected. This was accompanied by enhanced and sustained HGF-induced AKT activation. Together, these findings identify SNX2 as a predominant regulator of early MET endosomal trafficking in HCT116 cells and reveal functional asymmetry between SNX1 and SNX2. They support a role for SNX2 in spatial regulation of MET signaling through receptor trafficking, consistent with the association of low SNX2 expression with poorer survival in CRC patients.

Mitochondrial DNA (mtDNA) instability is an emerging feature of cancer, yet its biological effects on the tumor microenvironment (TME) and tumor progression remain incompletely defined. Using melanocyte-specific conditional silencing of mitochondrial transcription factor A (TFAM) in an autochthonous BrafV600E/Pten-/- melanoma model, we demonstrate that tumor cell-intrinsic mtDNA instability accelerates melanoma growth and metastatic dissemination in a cGAS-dependent manner. Transcriptomic profiling of early and late tumor stages revealed that mtDNA instability drives temporal remodeling of the tumor microenvironment (TME), characterized by an early type I interferon (IFN)-rich inflammatory state that licenses immunoregulatory programs as tumors progress. Tumor-associated macrophages emerged as the principal immune cell type reshaped by tumor cell-intrinsic mtDNA stress signaling, with early enrichment of inflammatory states and a shift toward efferocytosis-associated, tissue-adaptive states. The early mtDNA instability-associated macrophage signatures mapped to human melanoma states associated with favorable survival and therapy response, whereas the late signatures mapped to states associated with poor prognosis and therapy resistance across independent cohorts. Anti-PD-1 treatment delayed progression of mtDNA-instable tumors, consistent with a therapeutic sensitivity window associated with the early IFN-rich immune state. These findings establish tumor cell-intrinsic mtDNA instability as a driver of temporal immune-stromal remodeling that promotes melanoma progression and shapes therapeutic response.

Precise timing of astrogenesis during brain development is essential for proper neural circuit assembly, yet the molecular mechanisms governing this process remain incompletely understood. Here, we identify a novel nuclear signaling pathway initiated by the presenilin-dependent proteolytic release of the ErbB4 receptor intracellular domain (E4ICD) that regulates astrocyte differentiation via direct interaction with Poly(ADP-ribose) Polymerase 1 (PARP1). Upon ligand stimulation, E4ICD forms a complex with PARP1, inducing PARP1 tyrosine phosphorylation and enzymatic activation. This complex translocates to the nucleus, where it binds to the GFAP promoter, repressing its transcription. Loss of ErbB4 or PARP1 abolishes these effects, leading to increased differentiation of neural precursor cells into astrocytes in vitro, and elevated astrocytic gene expression in the neonatal brain in vivo. Notably, the E4ICD binding sites in the GFAP promoter overlap with those of Notch signaling, suggesting that these signaling pathways have antagonistic transcriptional roles in the control of glial fate. These findings establish a previously unappreciated RTK-PARP1 nuclear signaling axis that integrates extracellular cues to regulate neurodevelopmental lineage decisions, expanding the known functional repertoire of PARP1 beyond DNA damage and stress responses.

KRAS signaling is spatially organized within the dynamic proteolipid nanoclusters on the plasma membrane (PM) that selectively concentrate anionic phospholipids, yet the molecular determinants governing this lipid sorting remain incompletely defined. Here we identify phospholipid acyl chain composition as a critical regulator of KRAS spatial organization and signal output. Using genetic and pharmacological perturbation of lysophosphatidylcholine acyltransferase 3 (LPCAT3), a key enzyme that incorporates polyunsaturated fatty acids into phospholipids, we demonstrate that remodeling of membrane acyl chain unsaturation alters the phosphatidylserine (PS) lipidome without changing total PS abundance. Loss of LPCAT3 depletes polyunsaturated PS species, disrupts KRAS PM association and nanoclustering, and attenuates downstream MAPK signaling and oncogenic activity across multiple KRAS mutant contexts. Acute restoration of polyunsaturated, but not saturated, lipids rescue KRAS nanoclustering, establishing a direct requirement for lipid unsaturation in sustaining signaling-competent assemblies. Mechanistically, these findings define membrane acyl chain composition as a determinant of proteolipid organization that encodes KRAS signaling capacity. Consistent with this model, LPCAT3 inhibition selectively impairs KRAS-dependent proliferation and sensitizes tumor cells to direct KRAS inhibition. Together, this work uncovers lipid acyl chain remodeling as a fundamental regulator of membrane-associated signaling and identifies membrane composition as an exploitable vulnerability in oncogenic KRAS systems.

Macrophages are critical regulators of inflammation and tissue homeostasis, yet aberrant macrophage activation contributes to a wide spectrum of inflammatory and malignant diseases. Therapeutic strategies that directly reduce macrophage numbers have shown promise, but macrophage survival pathways remain incompletely defined, limiting the development of targeted therapeutic strategies. Here, we establish a high-throughput screening platform to identify small molecule inhibitors that impair macrophage survival. Screening a library of more than 2,000 targeted compounds in a cell survival assay, combined with in silico and in vitro analysis of macrophage specificity, revealed the identification of three potent inhibitors: BIX-01294, GSK-J4, and Masitinib. All three compounds downregulated leukemia inhibitory factor receptor (LIFR), whose inhibition markedly reduced macrophage viability. In vivo, these inhibitors effectively depleted large peritoneal macrophages, ameliorated key symptoms of macrophage activation syndrome (MAS), and suppressed tumor growth in a syngeneic transplanted melanoma model as well as in an autochthonous lung cancer model. Together, these findings identify small molecule-mediated macrophage depletion as a promising therapeutic strategy and establish an experimental approach to uncover regulators of macrophage survival.