Science Signaling

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that eliminate virus-infected and transformed cells through the formation of a specialized immune synapse. Effective target cell killing requires coordinated plasma membrane remodeling and dynamic reorganization of the actin and microtubule cytoskeletons, enabling centrosome polarization and directed secretion of lytic granules. The scaffold protein CIP4 has emerged as an important regulator of cytoskeletal coordination in NK cells, yet how its subcellular localization is controlled during NK cell activation is unknown. CIP4 contains a unique protein kinase A (PKA) phosphorylation site (threonine 225, T225) within its F-BAR domain, a domain that mediates interactions with microtubules and the plasma membrane. We hypothesized that localized PKA signaling controls CIP4 redistribution during immune synapse assembly. To test this hypothesis, we analyzed CIP4 localization and phosphorylation in NK cells engaged with sensitive target cells using biochemical and imaging approaches. We show that NK-target cell interaction enhances PKA activity and promotes phosphorylation of CIP4, coinciding with its delocalization from microtubules and accumulation at the immune synapse. Importantly, this relocalization process requires the PKA-anchoring protein AKAP350, which positions PKA and CIP4 within the same protein complex, thereby facilitating CIP4 phosphorylation. Consistently, pharmacological inhibition of PKA prevented CIP4 delocalization from microtubules and reduced its accumulation at the immune synapse. The non-phosphorylatable CIP4 mutant T225A displayed increased association with microtubules compared with a phosphomimetic mutant, identifying phosphorylation at T225 as a key determinant of CIP4 spatial regulation. Together, these findings identify a signaling mechanism that links compartmentalized PKA activity to the spatial control of CIP4 during immune synapse formation, providing new insight into the molecular mechanisms governing immune synapse maturation.

Migration of leukocytes in the context of immune homeostasis or inflammatory diseases is regulated by activation of chemokine receptors by chemokine ligands. To elucidate how these interactions give rise to cell migration, we mapped the chemokine-stimulated signal transduction network in monocytic THP-1 cells. Global phosphoproteomics revealed 630 time-resolved changes in phosphorylated proteins downstream of the chemokine receptor CCR2. We used the "PHONEMeS" network modeling algorithm to generate the most parsimonious signal transduction network consistent with the observed protein phosphorylation data. The CCR2 signaling network is highly divergent, acting via multiple branches to regulate proteins required for cell migration. We validated this model using kinase inhibitors targeting different branches of the network and successfully blocked chemokine-stimulated cell migration. Thus, chemotaxis is an emergent property resulting from an integrated cellular response to divergent signaling pathways. This paradigm suggests that physiological regulation or pharmacological blockade of chemokine-driven inflammation could potentially be achieved by inhibiting any of the divergent pathways within the network.

RAF kinases interpret signals from the three major RAS isoforms to initiate MAPK pathway activation, yet the molecular logic that governs isoform-specific RAS recruitment and the early events that relieve RAF autoinhibition are not yet fully understood. In particular, how the modular N-terminal regulatory architecture of CRAF and ARAF, anchored by the multifunctional cysteine-rich domain (CRD), discriminates among HRAS, KRAS, and NRAS has remained a central unresolved question. Here, we combine quantitative biophysical measurements with structural and dynamic analyses to define how RAS isoform identity and CRD engagement shape the earliest steps of RAF activation. These studies reveal unexpectedly divergent modes of RAS recognition between CRAF and ARAF and expose previously unappreciated functions of the CRD in modulating RAS affinity and intramolecular regulatory contacts. We further identify a direct link between RAS binding and destabilization of RAF autoinhibition, providing mechanistic insight into how RAS initiates the transition from an inactive monomer to an activation-competent assembly. Finally, we show that emerging KRAS inhibitors variably perturb KRAS-CRAF interactions, offering insight into how these therapeutics influence early RAS-RAF signaling events. Together, this work uncovers distinct biophysical principles that govern RAS-RAF selectivity and reveals a regulatory role for the CRD that reframes our understanding of RAF activation and its dysregulation in RAS-driven cancers. SignificanceProteins in the RAS-RAF signaling pathway control cell growth and are frequently mutated in cancer. Despite their importance, how different RAS proteins selectively recruit RAF kinases has remained incompletely understood. This study reveals that the cysteine-rich regulatory region of RAF plays a central role in distinguishing RAS isoforms and controlling RAF activation. These insights clarify early steps in MAPK signaling and may guide the development of improved therapies targeting RAS-driven cancers.

Identification of signaling networks is an essential goal in systems biology. Here, we use CRISPR/Cas9 knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gs-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R) pathway. Screening was done using a specially-designed fluorescence-based reporter cell line with green-fluorescent protein (GFP) co-transcribed with Aqp2, a gene whose transcription is dependent on vasopressin-mediated activation of protein kinase A (PKA). Positive regulators (n=14) included PKA-catalytic subunit (Prkaca) and Dyrk1a (minibrain homolog). Negative regulators (n=12) included PKA-regulatory subunit type I, Stk11 (catalytic subunit of liver kinase B1 [LKB1] complex), and three TGF-{beta} receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). Dyrk1a knockout cell lines failed to express AQP2 protein and exhibited a profound decrease in AQP2 mRNA. RNA-sequencing demonstrated widespread increases in cell-cycle transcripts, with a general defect in cell differentiation, accounting for AQP2 loss. TGF-{beta} exposure to un-transformed cells results in a profound decrease in V2R and AQP2 mRNA abundance along with multiple additional transcriptional targets of V2R-PKA signaling, consistent with prior findings in TGF-{beta}-mediated vasopressin escape. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA, even in the absence of vasopressin. RNA-sequencing showed a marked similarity between the responses to Stk11/LKB1 deletion and vasopressin exposure in untransformed cells. Phospho-proteomic data point to opposing roles of Stk11/LKB1 and PKA in the regulation of cAMP-responsive transcriptional coactivator (CRTC) proteins in the transcriptional response to V2R-PKA signaling. Significance StatementCells throughout the body are regulated by extracellular signals like the hormone, vasopressin. Hormonal effects on cellular function are mediated by membrane receptors that trigger biochemical changes, often by inducing chemical modification of the amino acids making up individual proteins, such as addition of function-altering phosphate groups (phosphorylation). Protein phosphorylation is mediated by enzymes known as "protein kinases". Here, we have screened all known protein kinases using modern CRISPR/Cas9 technology to identify those involved in vasopressin action in the kidney. As expected from prior knowledge, the screen identified protein kinase A and one of its regulatory subunits, but also identified several protein kinases not previously implicated in vasopressin action in the kidney.

Systemic acquired resistance (SAR) is a long-lasting, broad-spectrum immune response induced in distal tissues by signals generated at primary infection sites. Although numerous mobile immune signals have been implicated in SAR, how these signals are perceived and mechanistically coupled to transcriptional reprogramming in systemic tissues remains poorly understood. Extracellular NAD(P) [eNAD(P)] functions as a key integrative SAR signal that activates immunity through the plasma membrane-localized lectin receptor kinase LecRK-VI.2 and the master immune coactivator NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1). However, the mechanism linking eNAD(P) perception to activation of NPR1 has remained unknown. Here, we show that LecRK-VI.2 constitutively associates with NPR1 and directly phosphorylates NPR1 at T359 and likely S356 upon eNAD(P) perception. NADP+-induced phosphorylation of NPR1 occurs rapidly in vivo and requires LecRK-VI.2. Nonphosphorylatable NPR1 variants abolish eNADP+-induced local and systemic immunity as well as biologically induced SAR, whereas phosphomimetic variants retain NPR1 function. Mechanistically, LecRK-VI.2-mediated phosphorylation promotes NPR1 interaction with TGACG-binding transcription factors (TGAs) and the Mediator subunit MED15, thereby enhancing assembly of a transcriptional activation complex required for defense gene expression. We further demonstrate that NPR1 facilitates TGA-MED15 association in a phosphorylation-dependent manner. Together, these findings establish a receptor-to-coactivator signaling mechanism that directly links extracellular immune signal perception to transcriptional activation. This work closes a major mechanistic gap in the SAR signaling pathway and reveals receptor-mediated coactivator activation as a mechanism for rapid conversion of extracellular immune cues into coordinated transcriptional outputs during systemic immunity.

Atypical chemokine receptors (ACKRs) are non-signaling GPCRs that regulate ligand availability, with ACKR3 functioning as a dual scavenger of chemokines and opioid peptides. Here, we demonstrate that following ligand stimulation, besides the canonical internalization, ACKR3 is released on extracellular vesicles (EVs). ACKR3 was also found on EVs released under basal conditions, although to a lesser extent. These observations were confirmed across multiple cellular contexts, including endogenous systems. Mechanistically, basal and ligand-induced EV release are independent of GRKs and {beta}-arrestin but each relies on distinct trafficking routes and C-terminal determinants. Ligand-induced EV release is associated with plasma membrane localization and receptor recycling pathways. In contrast, basal EV release is governed by intracellular sorting processes and influenced by receptor ubiquitination and RAMP3. Functionally, EV-associated ACKR3 retains high-affinity ligand binding, enabling sequestration of CXCL12 and opioid peptides and thereby attenuating their signaling through CXCR4 and MOR. We also show that the release on EVs, in particular under basal conditions, is observed for other receptors such as KOR, CXCR4 and several ACKRs. Collectively, these findings establish EVs as regulators in chemokine and opioid systems and as a previously underappreciated dimension of ACKR3 and more broadly GPCR biology.

G protein-coupled receptors (GPCRs) direct neutrophil chemotaxis through heterotrimeric G proteins, yet the downstream effectors of the predominant Gai isoform, Gai2, remain incompletely defined. Here we identify the Ras GTPase-activating protein CAPRI (RASA4) as a functional effector of Gai2 that links GPCR signaling to Ras adaptation. Using AlphaFold3-based structural modeling and binding free-energy calculations and experimental verification, we reveal that constitutively active and structurally altered Gai2 mutants (Q205L and T182A) exhibit enhanced binding to CAPRI. Neutrophils expressing these mutants display elevated basal Ras activity, heightened sensitivity to chemoattractant, and improved chemotaxis in low- or subsensitive-concentration gradients. However, these cells exhibit excessive Ras activation and impaired chemotaxis at high, saturating chemoattractant concentrations, while maintaining near-normal responses at intermediate concentrations. These results reveal an upward shift in the concentration range for efficient chemotaxis. Our findings not only establish a previously unrecognized Gai2-CAPRI signaling axis that tunes Ras adaptation but also define a mechanism by which heterotrimeric G proteins calibrate leukocyte navigation across diverse chemoattractant gradients.

Src family kinases (SFKs) share highly conserved catalytic domains yet display distinct biological functions, raising the question of how substrate specificity is achieved. Here, we investigate the molecular basis of differential ITAM recognition by Lck and Src, combining cellular assays with structural analysis and docking simulations. In-cell assays demonstrated that, contrary to Lck, Src was completely incapable of phosphorylating the TCR ITAMs when ectopically expressed in a T cell environment. Domain-swapping experiments further revealed that substitution of the Src kinase domain with that of Lck was sufficient to confer ITAM phosphorylation and trigger downstream TCR signaling responses, whereas exchange of adaptor domains had minimal effect. Comparative structural analysis revealed that, despite their overall conserved fold, Lck exhibits a more open and solvent accessible pocket located between the N- and C-lobes of the kinase domain, adjacent to the activation loop, compared to Src. Consistent with this, docking simulations showed that Lck accommodates ITAM peptides in multiple favourable conformations, whereas Src displays a markedly reduced number of non-productive binding poses. Residue-level contact analysis identified a defined interaction surface in Lck, spanning the inter-lobal regions and activation loop. Our results highlight the importance of kinase domain conformational landscape in shaping substrate selectivity and have implications for the rational design of selective SFK inhibitors.

The small GTPase RAB7a is a key regulator of melanoma progression by enhancing the activity of the endolysosomal two-pore cation channel TPC2. In this study, we demonstrate that CCZ1--a core component of the RAB7a guanine nucleotide exchange factor (GEF) complex--is essential for mediating this RAB7a-dependent enhancement of TPC2. Unexpectedly, we find that constitutively active (GTP-locked) RAB7a fails to bind and regulate TPC2 in the absence of CCZ1, indicating that CCZ1 contributes to the RAB7a-TPC2 interaction through mechanisms beyond its GEF activity. Furthermore, the CCZ1 facilitated GTPase-activating function on RAB5 is dispensable for modulating TPC2. Notably, in the absence of CCZ1, TPC2 exhibits increased affinity for its agonist, PI(3,5)P2, along with markedly upregulated channel activity. In melanoma cell lines, this upregulation enhances migratory capacity. Our findings identify CCZ1 as a functional inhibitor of TPC2 and highlight its critical role in regulating cancer cell migration.

IntroductionThe RAS(ON) multi-selective daraxonrasib (RMC-6236) inhibitor is effective in patients with NSCLC KRAS mutant cancers. Tolerance to daraxonrasib invariably develops by mechanisms that remain unclear. There is no known involvement of the M1C oncogenic protein in daraxonrasib resistance. MethodsNSCLC H358 KRAS(G12C), H2122 KRAS(G12C) and patient derived MGH1112 KRAS(G12C) cells with acquired daraxonrasib resistance were investigated for M1C dependence in studies of SHP2, STAT1/3 and NF-KB activation, clonogenicity, and self-renewal capacity. ResultsWe demonstrate that M1C is induced as a protective response in NSCLC KRAS(G12C) mutant cells treated with daraxonrasib. We report that M1C forms novel cell membrane-associated biomolecular condensates with the SHP2 protein tyrosine phosphatase in driving daraxonrasib resistance. M1C integrates SHP2 activation with induction of (i) oncostatin-m/gp130/STAT3 signaling, and (ii) the NF-{kappa}B-mediated epithelial-mesenchymal transition (EMT) pathway. The functional significance of this M1C-driven pathway is supported by the demonstration that targeting STAT3 and NF-{kappa}B reverses daraxonrasib resistance. Consistent with M1C dependence, we also show that targeting M1C is effective against daraxonrasib-resistant NSCLC KRAS mutant cell line and tumor models. In contrast, M1C drives sotorasib resistance by STAT1-mediated inflammatory signaling, demonstrating that M1C confers resistance to KRAS(G12C)-selective and RAS(ON) tri-complex inhibitors by noncongruent mechanisms. ConclusionsThese findings demonstrate that M1C is required for daraxonrasib tolerance and is a potential target for the treatment of patients with NSCLC KRAS(G12C) mutant tumors refractory to this agent.

Tumor-associated macrophages (TAMs) comprise functionally diverse states that can suppress anti-tumor immunity and promote tumor progression, yet the tumor microenvironmental cues and signaling programs that generate these states remain incompletely defined. Here, we systematically stimulate primary human monocyte-derived macrophages with a panel of cytokines and metabolites abundant in the tumor microenvironment (TME), and profile their transcriptomic and phosphoproteomic responses to resolve stimulus-specific molecular programs. We observe that potassium (K+) and adenosine (Ado) stimulation, which accumulate in necrotic tumor cores, downregulate antigen-presentation genes and their master regulator CIITA. K+ stimulation results in the upregulated fibronectin 1 expression, associated with immunosuppressive, metastasis-promoting TAM subsets. Ado induces upregulated expression of tryptophan (Trp) catabolism genes, myeloid checkpoints and metallothioneins (MTs). Although MT-high TAM states have been recurrently observed across tumor single cell RNA sequencing studies, their function remains poorly defined. We show that elevated MT expression in tumor tissue is associated with shorter overall survival. By aligning in vitro transcriptomes with single-cell RNA sequencing (scRNA-seq) signatures from a pan-cancer TAM atlas, we identify significant similarities between several in vitro states and clinically observed TAM populations, with Ado-stimulated macrophages closely resembling a MT-expressing TAM cluster. Overall, this work provides a systematic molecular context linking tumor microenvironmental cues to clinically relevant TAM states and offers a framework for recapitulating their functions in vitro. STATEMENT OF SIGNIFICANCEThis study explores how cytokines and metabolites from the tumor microenvironment shape macrophage molecular phenotypes and lead to the upregulation of clinically relevant marker genes and recapitulation of functional states of interest.

The Arabidopsis thaliana S-domain receptor kinase LORE senses bacterial medium-chain 3-hydroxy fatty acids (mc-3-OH-FAs) as microbe-associated molecular patterns to activate pattern-triggered immunity. How LORE recognises these fatty acid ligands at the molecular level remains unknown. Here, we combined protein structure prediction, protein-ligand interaction modelling and molecular dynamics (MD) simulations with ligand-binding assays using chimeric and mutant receptor ectodomains, and functional analysis of receptor activation to characterise the mc-3-OH-FA binding mechanism. Domain-swap experiments between LORE and its non-binding paralog AtSD1-23 identify the lectin 2 (L2) domain as the ligand-binding domain. Mutational analysis and reverse engineering confirm a hydrophobic pocket in the L2 core as the primary ligand-binding site. Multiple walker Supervised MD (mwSuMD) simulations reveal that the acyl tail enters the pocket first, whilst polar interactions between the headgroup and a flexible L2 loop guide and stabilise the bound state. In support of this model, 3-OH-C10:0 analogues with bulky headgroup modifications dock into the pocket but act as antagonists, presumably by preventing the loop from adopting the conformation required for signalling. Together, these data suggest that the flexible L2 loop has multiple functions: it acts as a dynamic gate regulating pocket access, provides essential anchoring points once the ligand is bound, and contributes to receptor activation. These findings provide a mechanistic framework for immunogenic mc-3-OH-FA sensing by LORE.

The cellular response to DNA replication stress (DRS) provoked by anticancer drugs involves activation of the G2/M checkpoint (which promotes transient cell cycle arrest at G2 phase) and DNA repair, followed by induction of apoptosis or senescence. Here, we activated the p53-p21 pathway and ATR using DRS-inducing drugs, and found that that the transition to senescence depends on the duration of the G2 phase. Shortening of G2 duration by G2/M checkpoint inhibitors led not only to a switch in cell fate from senescence to mitotic entry, but also to effective cell death through carry-over of chromosomal aberrations (generated by DRS-inducing drugs) into mitosis and subsequent mitotic progression. Such enhanced cell death was also observed in p53 deficient cells, which do not normally undergo senescence. Thus, we propose that temporal regulation of G2 phase is an approach to enhancing the effects of DRS-inducing drugs in a manner that is independent of p53 status.

Macrophages are abundant in the colorectal tumour microenvironment and can alter drug response. Using mouse Apc/Kras/Trp53 (AKP) colorectal cancer organoids, we found that macrophages and/or macrophage-conditioned medium reduced sensitivity to the MEK inhibitor trametinib and the pan-RAS inhibitor RMC-6236. In contrast, macrophage-conditioned medium had little effect on regorafenib and increased sensitivity to dabrafenib, suggesting that resistance depends on the inhibitory profile of each drug. Secretome profiling identified PDGF-BB and GDF-15 as candidate mediators. Adding both ligands to organoid medium reproduced much of the conditioned-medium effect, whereas either ligand alone was insufficient. Inhibition of PDGFR or RET partially reduced drug resistance, suggesting that PDGF-BB and GDF-15 likely act through canonical signalling by that additional macrophage-derived signals also contribute. Kinome profiling pointed to increased tyrosine kinase signalling during trametinib treatment, with SRC family kinases emerging as a key downstream node. Consistent with this, SRC inhibition reduced the difference between control and conditioned-medium responses. The multi-kinase inhibitor masitinib--which targets several kinases along this resistance network--strongly restored sensitivity to trametinib and RMC-6236. Together, these data define a macrophage-driven resistance network in KRAS-mutant colorectal cancer organoids and support combined inhibition of RAS-pathway and tyrosine kinase signalling.

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.

The neurotransmitter dopamine (DA) is central to synaptic regulation that support diverse behavioral functions, including both learning and forgetting. This multi-functional role of DA is due to receptor specific signaling in specific subcellular environments that remain uncharacterized. Here we utilized proximity labelling proteomics in human cells to characterize the proximal environments of two Drosophila D1-like DA receptors (Dop1R1 and Dop1R2) in basal and DA activation environments. While DA drives both receptors to recruit Beta-Arrestin 2, Dop1R1 alone showed ligand driven recruitment of G-protein Receptor Kinase 2/3, proximity to clathrin mediated endocytosis, and WASH complex mediated endosomal trafficking. Additionally, we show evidence that Dop1R1 and Dop1R2 reside in distinct domains at the cell surface. In vivo disruption of Drosophila orthologs of Dop1R proximal proteins revealed three trafficking proteins, Sec24AB, Krz, and CG13887, that regulate R1-mediated learning, starvation induced attraction to odors, and DA-mediated cAMP responses in memory circuits. In addition to revealing DA receptor trafficking proteins that support learning, our comparative characterization of the cellular environments D1-like receptors offers insights into how DA differentially regulates diverse behavioral and synaptic functions. For TOC only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/728438v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@656e56org.highwire.dtl.DTLVardef@12f0084org.highwire.dtl.DTLVardef@cb05cdorg.highwire.dtl.DTLVardef@e9d623_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tumor reliance on antioxidant defenses creates a vulnerability to ferroptosis, yet strategies to therapeutically disable these systems remain limited. Here, we identify targeted degradation of the selenium uptake receptor LRP8 as an effective approach to decrease the abundance of the ferroptosis-protective enzyme glutathione peroxidase 4 (GPX4). Using bispecific cytokine receptor-targeting chimeras (KineTACs) that couple LRP8 to cytokine receptor internalization pathways, we selectively direct LRP8 to the lysosome for degradation. LRP8 degradation reduces the abundance of several selenoproteins, including GPX4, lowering the cellular threshold for lipid peroxidation and sensitizing cancer cells to ferroptosis. These findings establish receptor-mediated selenium uptake as a critical, targetable node in ferroptosis resistance and demonstrate that extracellular protein degradation can be leveraged to reprogram intracellular translational dependencies in cancer cells. More broadly, this work provides a framework for exploiting nutrient acquisition pathways to overcome therapy resistance.

BRCA-deficient high-grade serous ovarian cancer is characterized by profound genomic instability and elevated replication-associated DNA damage, rendering these tumors initially sensitive to platinum-based chemotherapy and PARP inhibition. However, despite this vulnerability, most patients ultimately develop resistance, underscoring the need for therapeutic strategies that extend beyond DNA repair-targeted mechanisms. Here, we introduce the MTDH-SND1 complex as a complementary therapeutic target that may expose additional stress vulnerabilities in ovarian cancer cells. We show that pharmacological disruption of the MTDH-SND1 interaction using C26-A6 increases susceptibility to ferroptosis-associated stress, an iron-dependent form of regulated cell death and that BRCA-deficient models are particularly more sensitive to this perturbation. Notably, when combined with PARP inhibition, MTDH-SND1 disruption is associated with increased MHC class I expression in tumor cells, suggesting enhanced tumor visibility to the immune system. Together, these findings support a combination strategy that couples DNA repair disruption with metabolic and immunogenic remodeling in BRCA-deficient ovarian cancer.

The unfolded protein response (UPR) safeguards endoplasmic reticulum (ER) homeostasis by integrating signals arising from both protein-folding defects and membrane stress. While activation of Ire1 by unfolded proteins has been extensively characterized, the contribution of membrane lipid composition to this process remains incompletely understood. It is unclear whether ergosterol acts as a primary activating signal for Ire1 or instead modulates the activation threshold and amplitude of the UPR response. Using {beta}-mercaptoethanol (BME) as a proteotoxic perturbation, we found that this reducing agent exerts opposing effects on the two major inputs that converge on Ire1 signaling. Although BME induces proteotoxic stress, it triggers only a moderate UPR response while simultaneously causing a pronounced reduction in ergosterol biosynthesis, a behavior distinct from that generated by classical ER stressors. Pharmacological, proteomic, genetic, and lipidomic analyses revealed a causal relationship between reduced ergosterol levels and attenuated Ire1 activation. Importantly, elevated ergosterol levels alone were insufficient to activate the UPR, indicating that sterols do not directly trigger the pathway. Instead, our findings support a model in which ergosterol functions as a permissive determinant of Ire1 responsiveness, tuning the amplitude and gain of UPR signaling in response to ER proteotoxic stress. Moreover, the differential effects of endogenous and exogenous sterol accumulation on Ire1 activation raise the possibility that not only total ergosterol abundance, but also its intracellular distribution and accessibility contribute to amplify Ire1 response. Together, these results identify sterol homeostasis as a key regulator of ER stress signaling and reveal how membrane composition influences the efficiency with which luminal stress information is translated into productive Ire1 activation.

Two prominent mechanisms by which tumors fend off immune control are by constraining the ability of T cells and CAR T cells to survive and expand in the tumor, and by restraining their ability to sustain full cytotoxic capacity. We identified I{kappa}B{delta}, encoded by Nfkbid, a poorly characterized I{kappa}B family member, as a molecular lever that overcomes both of these constraints on anti-tumor CD8+ tumor-infiltrating lymphocytes (TILs). Nfkbid is an NFAT target gene that is expressed in CD8+ effector T cells and, at modest levels, in CD8+ TILs. We found that Nfkbid depletion impaired TIL accumulation, exacerbating the growth of solid tumors. On the other hand, ectopic I{kappa}B{delta} overexpression enhanced TIL expansion, reduced the expression of exhaustion-associated transcription factors and inhibitory receptors, and elevated cytotoxic molecule production, leading to enhanced tumor control. I{kappa}B{delta} has a shorter protein isoform that is identical in a core region spanning the ankyrin-repeat domain known to interact with NF{kappa}B proteins, but that lacks the [~]150-residue N-terminal region. We showed that the shared core region is sufficient to drive T cell accumulation, whereas the N-terminal peptide region is required for robust effector function and to counter exhaustion, underscoring that tumor-infiltrating CD8+ T cell accumulation and effector differentiation are separable programs. Our current study provides evidence that I{kappa}B{delta}, an atypical member of the NF{kappa}B family, is a lever to overcome two cardinal deficits that limit CD8+ TIL anti-tumor efficacy: impaired accumulation in the tumor and diminished effector function.