Abstract Introduction Post-transcriptional regulation (PTR) plays a crucial role in shaping T-cell gene expression programs that adapt to their microenvironment. Among RNA-binding proteins (RBPs) involved in PTR, Human antigen R (HuR; ELAVL1) binds to AU-rich elements within mRNAs encoding regulators of proliferation, inflammation, and immune responses. While HuR controls cytokine expression and T-cell development, its role in effector function and persistence remains unclear. Methods We utilized a T cell—specific HuR knockout (HuRfl/fl CD4-Cre) mouse model and employed flow cytometry, RIP-seq, and transcriptomic profiling to identify HuR-bound targets and their downstream effects. Functional analyses included chronic stimulation followed by characterization, ELISA, HuR and SerpinB9 overexpression, adoptive transfer into Rag ko mice (colitis), and the B16-F10 tumor model to assess T-cell function and persistence. Results Using this approach, we found that HuR-deficient T cells exhibit increased effector cytokine secretion, decreased CD27 expression, elevated KLRG1 levels, and enhanced SA-β-Gal activity, characteristics of replicative senescence. Adoptive transfer of HuR-deficient T cells caused less severe colitis in Rag KO mice despite their effector phenotype, suggesting impaired persistence. Corroborating these findings, HuR expression was significantly reduced in tumor-infiltrating and aged T cells. Conversely, HuR overexpression decreased senescence and restored memory marker expression in both mouse and human aged T cells and TILs. Mechanistically, HuR bound transcripts involved in immune regulation, including SerpinB9, a granzyme B inhibitor vital for cytotoxic T-cell survival. Overexpressing SerpinB9 in wild-type and HuR-deficient T cells enhanced persistence under chronic stimulation and improved tumor control. Conclusion These findings highlight a HuR-SerpinB9 axis that regulates T-cell senescence and persistence, offering a potential therapeutic target in cancer and autoimmune diseases. Funding Source This work was supported by NIH grants R01CA300284, R01CA290201, R01CA282408, R42CA239952, R01DE030013, R01CA250458. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Background: Spinster homolog 2 (SPNS2) exports sphingosine-1-phosphate (S1P) to promote oncogenic signaling. Although SPNS2 is associated with metastatic progression, its tumor-intrinsic role in regulating immunogenic cell death (ICD) and anti-tumor immunity remains unclear. We examined how genetic or pharmacologic inhibition of SPNS2 influences metastasis, ICD induction, and systemic immune activation. Methods: Patient datasets were analyzed in conjunction with breast (4T1, EMT6) and melanoma (B16) models. SPNS2 was ablated using CRISPR or inhibited using a first-in-class small-molecule SPNS2 inhibitor. S1P export, migration, and S1PR1-AKT signaling were assessed in vitro. Orthotopic, tail-vein, co-injection, and vaccination models were used to evaluate tumor growth, metastatic spread, ICD signatures, and systemic immunity. Individual DAMP pathways were disrupted to test mechanistic requirements. Results: High SPNS2 expression correlated with poor survival across multiple cancer types. SPNS2 promoted S1P export, S1PR1-AKT activation, epithelial-mesenchymal transition, stemness, and lung colonization, whereas SPNS2 loss impaired migration and markedly reduced spontaneous and experimental metastases. SPNS2 inhibition elicited hallmark ICD features—including eIF2α phosphorylation, calreticulin exposure, and ATP/HMGB1 release—enhancing antigen presentation, expanding CD4+ and CD8+ T cells, and limiting primary tumor growth, metastasis, and postsurgical relapse. Vaccination with SPNS2-deficient or inhibitor-treated tumor cells protected against rechallenge with 4T1 or antigenically distinct EMT6 tumors. Disruption of individual DAMP pathways attenuated these responses, demonstrating ICD dependence. Conclusions: SPNS2-mediated S1P transport drives metastasis and immune evasion, whereas SPNS2 inhibition induces ICD and potent systemic T-cell immunity. Targeting SPNS2 represents a therapeutic strategy to suppress metastatic progression and generate durable anti-tumor immunity. Disclosure: Generative AI was used to assist in editing this abstract. Citation Format: Han Gyul Lee, Wyatt Wofford, Alhaji H. Janneh, Paramita Chakarborty, Natalia Oleinik, Mohamed Faisal Kassir, Odai Darawshi, Neil Parikh, Stefano Berto, Kevin R. Lynch, Webster L. Santos, Özgür Şahin, Shikhar Mehrotra, Besim Ogretmen. Targeting Spns2 induces immunogenic cell death and systemic anti-tumor immunity to suppress metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3509.

Abstract Immunotherapy has revolutionized cancer treatment, yet only a fraction of patients develop durable responses to immune checkpoint blockade (ICB). Identifying tumor-intrinsic mechanisms driving αPD-1/αPD-L1 resistance remains critical to improving patient outcomes. We recently discovered that reduced ceramide synthase 4 (CerS4) activity and the subsequent loss of C18/20 ceramide impairs response to ICB through intracellular PD-L1/Caprin-1 signaling. To investigate this further, we generated an orthotopic, transplantable TNBC (E0771) model of ICB resistance through serial in vivo αPD-L1 exposure, yielding the 2RA cell line. 2RA tumors are refractory to both αPD-L1 and αPD-1 therapy, and transcriptomic signatures derived from this model strongly predict clinical ICB outcomes, supporting its relevance to human disease. Functionally, 2RA tumors display reduced CerS4 expression, diminished C18/20 ceramide, and increased intracellular PD-L1/Caprin-1 interaction. Bulk RNA-seq revealed marked enrichment in prostaglandin E2 (PGE2) signaling, a potent immunosuppressive pathway, in 2RA tumors. Mechanistically, we identified that CerS4 inversely regulates prostaglandin-endoperoxide synthase 2 (Ptgs2, COX-2) expression and PGE2 production through the PD-L1/Caprin-1 complex, whereby ceramide directly interacts with PD-L1 to restrict Caprin-1 binding. Analysis of TCGA and ICB-treated patient datasets substantiated the CerS4/PD-L1/COX-2 axis across multiple solid tumor subtypes. Immune profiling via flow cytometry and snRNA-seq identified dysfunctional progenitor and effector CD8+ T cells as central to impaired ICB response in 2RA tumors. Genetic or pharmacological disruption of the CerS4/PD-L1/PGE2 axis, achieved through CerS4 restoration or TGF-β inhibition (LY2157299), restored ICB sensitivity, prolonged survival, and induced tumor rejection in vivo. Targeting PGE2 production with celecoxib, but not aspirin, further enhanced responses when combined with LY2157299 and αPD-1 therapy. Targeted lipidomics and multiplex immunofluorescence (mIF) confirmed that this triple combination potently blocks the ceramide/PGE2 axis and stimulated CD8+ T cell responses to control tumor growth. Finally, mIF analysis of nivolumab-treated pre-surgical human HNSCC specimens (responders vs. non-responders) corroborated these findings by demonstrating reduced tumor ceramide abundance, elevated PanCK+COX-2+ceramidelo populations, and decreased intratumoral CD8+ T cell density amongst non-responders. Collectively, these studies (1) establish a relevant model of ICB resistance, (2) define a mechanistic framework linking ceramide metabolism to prostaglandin-mediated immune suppression, and (3) highlight therapeutic strategies to target ICB resistance and improve patient outcomes. Citation Format: Wyatt O. Wofford, Elif Percin, Han G. Lee, Odai Darawshi, Bryan Granger, Lucy Mulligan, Natalia V. Oleinik, Mohamed F. Kassir, Chase Walton, Paramita Chakraborty, Stefano Berto, Raymond N. DuBois, Shikhar Mehrotra, Besim Ogretmen. Crosstalk between ceramide and prostaglandin signaling mediates resistance to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5564.

Background: Sphingolipids are essential structural and signaling lipids that support membrane integrity and govern cell fate decisions. While the consequences of chronic sphingolipid inhibition have been extensively explored, the immediate cellular responses to acute suppression of sphingolipid synthesis remain poorly defined. Methods: We analyzed subcellular proteomic changes following an acute reduction in sphingolipid levels induced by myriocin, an inhibitor of de novo sphingolipid synthesis. We then evaluated the cytotoxicity of co-treatment with myriocin and inhibitors of the altered pathways in cancer cells. Results: We found that de novo sphingolipid synthesis is sensitive to myriocin, an inhibitor of serine palmitoyltransferase (SPT), and can be efficiently inhibited within 4 h of treatment. Cells respond to reduced sphingolipid levels by rapidly remodeling their proteome. Mass spectrometry analysis revealed changes in the abundance of hundreds of proteins across the membrane, cytosolic, and nuclear fractions. Gene set enrichment analysis revealed alterations in the proteome across several pathways involved in protein and lipid homeostasis and stress responses, including upregulation of cholesterol homeostasis and lysosome. Co-treatment with myriocin and cholesterol synthesis or lysosomal function inhibitors synergistically reduced cancer cell viability by promoting apoptosis rather than other forms of programmed cell death. Conclusions: Together, our work provides insights into how cells rapidly rewire the abundance of certain protein classes in response to reduced sphingolipid levels and identifies signaling and metabolic pathways that can be exploited for therapeutic intervention.

Defects in elastin trigger hyperproliferation of smooth muscle cells, which leads to arterial and congenital heart diseases. Research now shows that elastin deficiency induces SPHK1 and S1P signaling by EGR1 in SMCs, and inhibitors of SPHK1 or S1PR1 attenuate smooth muscle cell proliferation and mitigate aortic disease.

BACKGROUND/OBJECTIVES:T cells within solid tumors often switch from a recirculating to a tissue-resident state, which may blunt antitumor activity, but the signal driving this switch in vivo remains unclear. We asked whether alterations in sphingosine-1-phosphate (S1P) signaling in tumor or the surrounding stromal cells are associated with T cell residency in glioblastoma (GBM). METHODS:We analyzed five single-cell RNA-sequencing cohorts: three human glioma datasets, an in-house mouse CT2A glioblastoma cohort, and a human melanoma tumor-infiltrating lymphocyte cohort. T cell egress and tissue-residency programs, together with stromal S1P production and degradation, were scored per cell using curated gene modules. Cell-state contrasts were quantified as Cohen's d, and sample-level coupling as Spearman ρ. RESULTS:In human GBM, T cell residency programs were elevated in CD4+ helper and regulatory T cells in tumors compared with low-grade glioma controls. In mouse CT2A-derived GBM tumors, stromal S1P production correlated negatively with T cell residency across four independent stromal cell types. In human GBM microglia, S1P production was reduced compared with control microglia. The same CD8+ residency phenotype was replicated in CD3-sorted GBM tumor-infiltrating lymphocytes (TILs) and in melanoma TILs. CONCLUSIONS:A loss of stromal S1P production accompanies T cell tissue residency in GBM. Thus, stromal S1P metabolism is a candidate axis for modulating T cell recirculation and TIL biology in GBM. These findings are transcriptomic associations from single-cell RNA sequencing that do not directly measure S1P metabolite levels or signaling activity and will require functional and lipidomic validation.

Abstract Sphingolipid metabolism, specifically sphingosine 1-phosphate (S1P), has been demonstrated to regulate cancer progression and metastasis. Our previous research showed that oncogenic S1P and S1P receptor 1 (S1PR1) signaling activated intracellular C3 complement processing to enhance migration/metastasis through inflammasome activation by the C3-PPIL1 complex. This study addressed how the S1PR1/C3 axis mediates inflammasome/NLRP3 activation in various solid tumors, including melanoma and triple-negative breast cancer (TNBC). To better understand the roles of S1PR1 and C3 in mouse mammary tumorigenesis and metastasis, we crossed the MMTV-Cre S1pr1fl/fl; MMTV-Cre or C3Tdt; MMTV-Cre mice with MMTV-PyMt expressing mice to generate S1pr1fl/fl; MMTV-Cre; MMTV-PyMt or C3Tdt; and MMTV-Cre; MMTV-PyMt (controls) animals. Our preliminary data show that silencing C3 and S1PR1 selectively in mammary tumors significantly reduces tumor burden in MMTV-PyMt mice, consistent with decreased inflammasome signaling. Mechanistically, our data also showed that PPIL1-C3 complex requires SNU13 to induce cell migration in response to S1P signaling by mediating the alternative splicing of various factors involved in activating NLRP3/inflammasome in metastatic tumors. These findings suggest that attenuation of the S1PR1/C3 and PPIL1-SNU13-inflammasome signaling inhibits cancer cell migration and metastasis. Citation Format: Salih Gencer, Alhaji H. Janneh, Natalia Oleinik, Charles Chalfant, Besim Ogretmen. Intracellular complement signaling plays a critical role in s1p/s1pr1 mediated inflammasome activation and tumor metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2247.

Background: Ceramides are bioactive sphingolipids involved in cellular stress responses, inflammation, and apoptosis. Dysregulated ceramide metabolism has been associated with chronic kidney disease (CKD) in both clinical and experimental studies. Elevated ceramide levels have been reported in patients with reduced kidney function, supporting clinical interest in their role in disease progression. Among different ceramide species, long-chain ceramides have been implicated in cellular injury pathways and have been suggested to play a role in renal damage. However, the distribution of long-chain ceramides in human kidney tissue across different kidney diseases and renal compartments is not well defined. Clarifying these patterns may help identify markers of active injury and support the development of new therapeutic strategies. Methods: Formalin-fixed, paraffin-embedded human kidney biopsy samples with confirmed diagnoses were stained with a long-chain ceramide-selective antibody (Enzo Life Sciences, Cat# ALX-804-196). We further analyzed diabetic nephropathy (DN) and histologically normal controls from both sexes (male DN: 47±8 years, eGFR 53±11 mL/min/1.73m 2 ; female DN: 39±4 years, eGFR 73±10; male controls: 53±9 years, eGFR 74±7; female controls: 61±8 years, eGFR 77±15; n=4 per group). Female samples from lupus nephritis (LN) and focal segmental glomerulosclerosis (FSGS) were evaluated in two age groups (LN: 45±2 years, eGFR 66±3, and 26±2 years, eGFR 69±20; FSGS: 62±5 years, eGFR 33±5, and 26±2 years, eGFR 67±12; n=4 per group). Ceramide signal was evaluated in glomerular and tubulointerstitial compartments. Further, immunofluorescence staining was used to assess long-chain ceramide localization in podocytes and tubular epithelial cells. Staining intensity was quantified in regions of interest and analyzed using mean gray value measurements. Quantification was performed in a blinded manner and stratified by diagnosis, sex, and age. Results: Long-chain ceramide abundance showed distinct disease- and compartment-specific patterns. In control kidneys, males had higher baseline ceramide levels than females in glomeruli (23.5±2.1 vs 7.6±1.0; p< 0.01) and tubules (47.2±2.8 vs 27.3±2.7; p< 0.01), indicating overall sex differences in abundance. DN exhibited the highest accumulation, particularly in the tubulointerstitial region (64.2±5.7 in males and 43.0±3.8 in females; p< 0.01 vs control), and elevated glomerular levels (46.4±4.7 and 30.9±2.2; p< 0.01 vs control). LN demonstrated robust increases in glomerular ceramide abundance (35.5±3.2 and 34.2±3.1; p< 0.001 vs female control in both older and younger groups). FSGS showed moderate increases in glomerular ceramides (22.4±2.1 and 17.1±2.1; p< 0.01 vs female control in older and younger female groups) while tubular ceramide levels in FSGS did not differ significantly from controls. Conclusion: Across CKD groups, DN showed the highest levels of long-chain ceramide accumulation, followed by LN and FSGS. Sex-related differences were observed in controls and DN, and age-associated differences were present within LN and FSGS samples. These findings indicate that long-chain ceramide abundance differs across CKD types and renal compartments. The results support the potential value of long-chain ceramides as compartment-specific tissue markers of kidney injury and highlight the need for further studies to evaluate their diagnostic and therapeutic relevance in CKD. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) limit the efficacy of adoptive T cell therapies, highlighting the need to overcome tumor-associated immunosuppression. Sphingosine-1-phosphate (S1P), is an abundant signaling lipid in the TME. Here, we show that inhibition of sphingosine kinase-2 (SphK2), the enzyme generating S1P in MDSCs, reduces the suppressive activity of monocytic MDSCs (M-MDSCs) while promoting their differentiation toward a mature, immunogenic phenotype characterized by enhanced antigen presentation. Pharmacological SphK2 inhibition enhances the response to anti-PD-1 therapy in preclinical models of checkpoint-resistant breast, bladder, and melanoma cancers by mitigating MDSC-mediated suppression and limiting tumor progression. Mechanistically, S1P directly binds acetyl-CoA carboxylase-1 (ACC1) to inhibit its activity, thereby rewiring fatty-acid metabolism. Lowering intracellular S1P restores ACC activity, promotes phosphatidylcholine synthesis, and reduces MDSC immunosuppression. These findings identify the SphK2-ACC-phospholipid axis as a metabolic checkpoint controlling the immunogenicity of MDSCs and a potential therapeutic target for enhancing cancer immunotherapy.

ABSTRACT Introduction Opaganib is a first‐in‐class oral sphingolipid metabolism inhibitor that inhibits sphingosinekinase 2 (SphK2) and dihydroceramide desaturase (DES) and that has a demonstrated safety and preliminary anti‐cancer activity signal in a Phase I study. Methods In this phase II trial, patients with metastatic castration‐resistant prostate cancer who had disease progression on novel hormonal agents (NHAs) abiraterone or enzalutamide were enrolled and treated with opaganib while continuing their NHA. After safety lead‐in cohorts, the trial enrolled cohort 2 (abiraterone + opaganib 500 mg Q 12 h) and cohort 3 (enzalutamide + opaganib 500 mg Q 12 h). The primary efficacy endpoint was the proportion of patients with disease control at Day 113. The postulated disease control rate was 10%. Secondary efficacy endpoints include prostate‐specific antigen (PSA) progression‐free survival (PSA‐PFS) and PSA response rates. The primary safety endpoint was the incidence of adverse events (AEs). Results The disease control rates were 15% (95% CI = 4%–35%, 4 of 26 patients) in cohort 2 and 9% (95% CI = 2%–24%, 3 of 34 patients) in cohort 3. The median PSA‐PFS was 56 days (95% CI = 35–112 days) in cohort 2 and 55 days (95% CI = 35–56 days) in cohort 3. The most common AEs of grade 3 or higher were hypertension (8%) and musculoskeletal AEs (8%) in cohort 2 and grade 3 anemia (18%) in cohort 3. Conclusion The trial did not meet its primary objective of demonstrating 30% disease control at 113 days. However, subjects who experienced a PSA response or stabilization warrant further exploration for biomarkers of response. Trial Registration ClinicalTrials.gov number: NCT04207255

High metabolic heterogeneity and plasticity of triple-negative breast cancer (TNBC) contribute to therapy resistance, necessitating identification of therapeutic vulnerabilities. Here, we identify non-canonical functions of the extracellular matrix (ECM) remodeler, lysyl oxidase (LOX), in regulating glucose metabolism and mitochondrial homeostasis and show that inhibiting LOX generates targetable vulnerability to ferroptosis. Mechanistically, LOX interacts with PARKIN and its upstream kinase PINK1, which we identified as a substrate of LOX. LOX-mediated PINK1 oxidation suppresses PARKIN phosphorylation, stabilizing hypoxia-inducible factor 1-alpha (HIF-1α) and increasing glycolysis. Concomitantly, LOX inhibits PARKIN-mediated mitophagy and maintains mitochondria-ER contacts through VDAC1 stabilization, while the LOX-HSP90 complex promotes mitochondrial Ca2+ transport and ATP production. Inhibiting LOX suppresses glycolysis, disrupts mitochondrial dynamics, reduces OXPHOS and GPX4/FSP1, and induces compensatory DHODH activity. Our “one-two punch” approach combining LOX inhibition with clinical DHODH inhibitor suppresses tumor growth in vivo in chemo-free setting. Notably, LOX protein correlates with HIF-1α/GLUT1/GPX4 in TNBC patient tumors, supporting its clinical relevance.

Lipid accumulation in the tumor microenvironment is a hallmark of solid tumors, with increased palmitate (PA) availability fostering tumor progression. Although PA's direct effects on cancer cells are well described, its impact on CD8 T cells [cytotoxic T lymphocytes (CTLs)] remains unclear. Here, we show that PA irreversibly impairs CTL mitochondrial metabolism, leading to the loss of effector functions and compromised antitumor immunity. PA-induced mitochondrial dysfunction reduced histone acetylation and chromatin accessibility, suppressing transcription of genes involved in T cell replication and effector programs. We identified sphingosine kinase 2 (SPHK2) as a key mediator of PA-induced dysfunction, with pharmacological inhibition of SPHK2 restoring mitochondrial fitness, rescuing CTL effector function, and promoting antitumor activity. These findings uncover a distinct mechanism by which PA drives immune evasion in tumors and highlight SPHK2 as a potential therapeutic target to enhance T cell-based immunotherapies.

List of diagnostic codes for cancer ascertainment in the Medical Expenditure Panel Survey.

This file contains a key resources table that includes details of key experimental materials and supplies used in this study.

Demographic characteristics of human APP-high vs APP-low samples.

The misclassification of functional genomic loci as pseudogenes has long obscured critical regulators of cellular homeostasis, particularly in aging-related pathways. One such locus, originally annotated as RPL29P31, encodes a 17-kDa protein now redefined as PERMIT (Protein that Mediates ER-Mitochondria Trafficking). Through rigorous experimental validation-including antibody development, gene editing, lipidomics, and translational models-p17/PERMIT has emerged as a previously unrecognized mitochondrial trafficking chaperone. Under aging or injury-induced stress, p17 mediates the ER-to-mitochondria translocation of Ceramide Synthase 1 (CerS1), facilitating localized C18-ceramide synthesis and autophagosome recruitment to initiate mitophagy. Loss of p17 impairs mitochondrial quality control, accelerating neurodegeneration, and sensorimotor decline in both injury and aging models. This Perspective highlights p17 as a paradigm-shifting discovery at the intersection of lipid signaling, mitochondrial biology, and genome reannotation, and calls for a broader reassessment of the "noncoding" genome in aging research. We summarize a rigorous multi-platform validation pipeline-including gene editing, antibody generation, lipidomics, proteomics, and functional rescue assays-that reclassified p17 as a bona fide mitochondrial trafficking protein. Positioned at the intersection of lipid metabolism, organelle dynamics, and genome reannotation, p17 exemplifies a growing class of overlooked proteins emerging from loci historically labeled as pseudogenes, urging a systematic reevaluation of the "noncoding" genome in aging research.

T-cells of 3xTg mice, but not APP-PS1 mice, have a functional advantage and protect the mice from tumor growth and development.

LCL768 reduces TCA cycle metabolism in HNSCC cells.