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Key Points Alanyl-transfer RNA synthetase 1 (AARS1) was upregulated and promoted cyst growth in autosomal dominant polycystic kidney disease. AARS1 promoted lysine lactylation of signal transducer and activator of transcription 3 (K140) and NF-κB p65 (K310), enhancing their phosphorylation, interaction, and transcriptional activity. AARS1 transcriptionally repressed its novel targets, Atg5 and Dusp4, to impair autophagy but activate polycystic kidney disease signaling pathways. Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder, characterized by metabolic reprogramming with enhanced glycolysis and lactate accumulation. However, the enzyme that senses lactate and uses it as a substrate for protein lactylation in ADPKD remains unknown. Alanyl-transfer RNA synthetase 1 (AARS1) is a lactate-responsive enzyme with lactyltransferase activity, while its role and mechanisms in ADPKD have not been defined. Methods To investigate the role of AARS1, we generated Pkd1 and Aars1 double conditional knockout Pkd1 fl/fl :Aars1 fl/fl :Ksp-Cre mice and evaluated the effect of AARS1 inhibitor β -alanine in two ADPKD mouse models. Coimmunoprecipitation and Cleavage Under Targets and Tagmentation analyses were performed to identify novel AARS1 substrates and downstream target genes involved in cystogenesis. Results AARS1 was elevated in Pkd1 mutant renal epithelial cells and kidneys, and genetic deletion of Aars1 significantly delayed cyst growth, preserved kidney function, and reduced renal lactylation in Pkd1 mutant mice. Mechanistically, AARS1 promoted lactylation-dependent activation of signal transducer and activator of transcription 3 (STAT3) and NF-κB (p65), specifically at STAT3 K140 and p65 K310, thereby amplifying pro-proliferative and proinflammatory transcriptional programs. AARS1 also associated with promoter regions and mediated histone H3 lysine 14 lactylation, thereby repressing the transcription of Atg5 to impair autophagy and suppressing the transcription of Dusp4 to sustain phosphorylation of cAMP response element–binding protein and retinoblastoma protein. Cytokine signaling through IL-6 and TNF- α further reinforced AARS1 expression through STAT3- and NF-κB–dependent feed-forward loops. Importantly, inhibition of AARS1 with β -alanine markedly slowed cyst progression in Pkd1 mutant mouse models. Conclusions Our study identified AARS1 as a central metabolic sensor linking lactate-driven lysine lactylation to transcriptional, epigenetic, and signaling pathways that drive ADPKD progression.

Introduction Large-scale production of mammalian cells is pivotal for applications in biotechnology, regenerative medicine, and therapeutic manufacturing. However, current bioreactor technologies face significant technical and economic challenges, including excessive cell aggregation, shear stress-induced cell death, batch-to-batch inconsistencies, and limited scalability. We propose that engineering a cell-friendly microenvironment can enhance culture efficiency. Previously, we developed alginate hydrogel microtubes (AlgTubes) that significantly improved cell density and growth rates; however, AlgTubes lack adhesion sites essential for anchorage-dependent cells and frequently break, causing cell leakage and production inconsistencies. Methods To address these limitations, we reinforced AlgTubes with collagen nanofibers, creating collagen-alginate hybrid hydrogel microtubes (ColAlgTubes). Collagen was integrated to form a dense nanofiber network interwoven with the alginate mesh, with the dual aim of enhancing mechanical properties and providing cell adhesion sites. ColAlgTubes were fabricated to maintain cell mass within a 400 μm diameter to ensure efficient nutrient exchange and waste removal. Results ColAlgTubes successfully formed a reinforced hybrid hydrogel structure in which the collagen nanofiber network improved the mechanical integrity of the alginate matrix while supplying adhesion sites for anchorage-dependent cells. The optimized 400 μm diameter microenvironment supported high cell viability, rapid proliferation, and exceptional yields of 5 × 10 8 cells/mL. Discussion These results demonstrate that ColAlgTubes overcome the key limitations of AlgTubes by combining structural reinforcement with biological functionality. The improved mechanical properties reduce tube breakage and cell leakage, while collagen-derived adhesion sites broaden compatibility to anchorage-dependent cell types. With their scalability, cost-effectiveness, and high efficiency, ColAlgTubes represent a transformative solution for large-scale mammalian cell production across biotechnology, regenerative medicine, and therapeutic manufacturing.

ABSTRACT Background Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cyst growth, inflammation, fibrosis, and renal failure. Klotho is a renoprotective protein with antiproliferative, anti-inflammatory, and antioxidant properties, but its mechanistic role in ADPKD remains poorly defined. Methods Klotho expression was analyzed in human ADPKD kidneys, public bulk and single-cell RNA sequencing datasets, Pkd1 mutant renal epithelial cells, and Pkd1 mouse models. The therapeutic effects of recombinant Klotho were evaluated in late-onset Pkd1fl/fl:tamoxifen-Cre mice. Molecular mechanisms were investigated using cell viability assays, western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry, ferroptosis assays, DNA methylation analysis, and chromatin immunoprecipitation assay. Results Klotho expression was markedly reduced in human ADPKD kidneys and Pkd1-deficient cells and mice, particularly in distal nephron segments. Recombinant Klotho treatment significantly reduced cyst burden, kidney enlargement, and blood urea nitrogen levels, while decreasing epithelial proliferation and increasing apoptosis in cyst-lining cells. Mechanistically, Klotho suppressed PKD-associated signaling pathways, including protein kinase B (AKT), signal transducer and activator of transcription 3 (STAT3), and retinoblastoma protein (Rb), reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated inflammation via downregulation of Smyd2, and attenuated transforming growth factor-beta (TGF-β)-Smad-dependent renal fibrosis. Klotho also inhibited ferroptosis by upregulating GPX-4 and reducing lipid peroxidation. Epigenetic analyses revealed DNA methyltransferase 1 (DNMT1)-mediated hypermethylation of the Klotho promoter and gene body in ADPKD kidneys, contributing to Klotho silencing. Inhibition of DNMT1 restored Klotho expression, and recombinant Klotho reduced DNMT1 levels, suggesting a negative feedback loop. Conclusions Klotho is epigenetically repressed in ADPKD and functions as a central regulator of cyst growth, inflammation, fibrosis, and ferroptosis. Restoration of Klotho signaling markedly attenuates disease progression in Pkd1 mutant mice, identifying the Klotho–DNMT1 axis as a promising therapeutic target for ADPKD.

Abstract GNE myopathy (GNEM) is a neuromuscular disorder caused by biallelic mutations in the gene GNE , which encodes a bifunctional enzyme essential for the biosynthesis of sialic acid. Despite extensive characterization, no effective therapies or validated biomarkers exist, and the molecular mechanisms driving disease pathology remain poorly understood. This study identifies the proteoglycan serglycin as a preliminary candidate biomarker in GNEM and investigates the mechanisms underlying its dysregulation. Proteomic analysis of serum samples from GNEM patients revealed reduced circulating serglycin levels. In contrast, increased intracellular serglycin was observed in GNE knockout and GNEM patient iPSC-derived myoblasts, suggesting impaired secretion and/or degradation. To investigate the basis of this accumulation, intracellular trafficking and autophagic pathways were examined. Proteomic profiling from HEK GNE knockout cells revealed alterations in pathways involved in membrane trafficking, Golgi function, and glycoprotein processing, consistent with broader disruptions in the secretory system that may impair serglycin export and contribute to its intracellular retention. In GNEM iPSC-derived myoblasts, serglycin overlapped with LAMP1-positive lysosomal compartments and the ER-phagy receptor FAM134B, suggesting association with lysosomal and ER-phagy related pathways. Functional assays further indicated impaired autophagic flux in mutant cells. Together, these findings support a model in which combined defects in secretion and autophagic clearance contribute to intracellular serglycin accumulation and reduced circulating levels. This study identifies serglycin as a potential circulating biomarker for GNEM and highlights dysregulation of secretory and degradative pathways as converging features of disease pathology.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, arising from both B and T cell lineages (B-ALL and T-ALL). Current therapy exploits ALL cells’ low expression of asparagine synthetase (ASNS) by using L-asparaginase, a bacterial enzyme that depletes circulating asparagine. However, resistance can emerge through induction of ASNS, mediated in part by the amino acid stress sensor GCN2. In this study, we addressed the efficacy of L-asparaginase in combination with genetic or pharmacological inhibition of GCN2 and the ASNS inhibitor ASX-173. Using a Kras G12D -driven mouse model of T-ALL, we found that GCN2 is dispensable for leukemogenesis. However, genetic inactivation or pharmacologic inhibition of GCN2 sensitized ALL cells to asparagine depletion, correlating with impaired ASNS induction. While GCN2 targeting enhanced sensitivity to asparagine depletion, a subset of Gcn2 –/– T-ALL cells retained high ASNS expression and remained resistant to L-asparaginase. Likewise, some human T-ALL cells with elevated ASNS levels were refractory to GCN2 inhibition even under asparagine-depleted conditions. When combined with L-asparaginase, ASX-173 effectively eliminated ASNS hi leukemic cells in vitro and in vivo. These findings suggest that direct targeting of ASNS provides therapeutic benefit in leukemias that express high levels of ASNS and are resistant to GCN2 inhibition under asparagine-depleted conditions.

e16449 Background: Pancreatic cancer (PC) carries a dismal 5-year survival of < 12%, and no FDA-approved agents exist for primary prevention. PC risk is strongly linked to obesity, diabetes, and chronic inflammation. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) directly target obesity-driven inflammatory pathways, reduce metabolic stress, and modulate signaling pathways (PI3K/AKT/mTOR) that drive cancer apoptosis. Based on this biological rationale, we conducted the first large-scale real-world study to evaluate the safety and efficacy of GLP-1RA for primary prevention of PC in high-risk patients. Methods: Leveraging a large real-world database (TriNetX), we identified patients aged 18-90 years with ≥1 PC risk factor (tobacco use, obesity, type 2 diabetes, alcohol use disorder, chronic pancreatitis, first-degree relative with PC, genetic susceptibility to PC) based on ICD-10 codes. Patients were stratified by GLP-1RA exposure into two cohorts: Cohort A (GLP-1RA users with ≥ 2 months of prescriptions) and Cohort B (controls). The primary endpoint was incident PC, and the secondary endpoints were adverse events related to GLP-1RAs. Patients with outcomes before the study window were excluded. Propensity score matching adjusted for confounders. Multivariate logistic regression was used to estimate odds ratios for categorical outcomes, and the Cox proportional hazards model was used to estimate hazard ratios for time-to-event outcomes. Subgroup and sensitivity analyses were employed to validate the strength of our results. Results: Median follow-up was 3,397 days for GLP-1RA users and 2,531 days for controls. Demographics were balanced between cohorts (mean age 42 years; 74% White, 15% Black; 72% female). Among 572,330 high-risk patients (286,165 per cohort), PC incidence was significantly lower among GLP-1RA users (0.05%; 134/286,165) than among non-users (0.13%; 362/286,097), with a 2.6-fold reduction in the absolute incidence of PC among high-risk patients (NNT of 1,255). GLP-1RA use was associated with a 71% reduction in PC incidence (HR 0.289; 95% CI 0.237–0.352). GLP-1RA use was associated with an increased risk of gastrointestinal adverse events, including nausea/vomiting (HR 1.108; 95% CI 1.096–1.121), abdominal pain (HR 1.081; 95% CI 1.066–1.097), diarrhea (HR 1.199; 95% CI 1.182–1.217), and constipation (HR 1.159; 95% CI 1.143–1.176). Conclusions: GLP-1RA use significantly reduced PC incidence by 71% in high-risk patients, the strongest real-world signal yet for a pharmacologic prevention of this lethal malignancy. Although the absolute incidence remained low, the magnitude of the risk reduction underscores the transformative potential of GLP-1RA in preventive oncology. These findings address a critical unmet need in pancreatic oncology. Prospective mechanistic and randomized studies are warranted to validate these associations.

4562 Background: Approximately 50-80% of patients (pts) with la/mUC have tumors with HER2 protein expression. As HER2-directed therapies enter development, a robust understanding of HER2 prevalence and real-world outcomes with standard-of-care (SOC) therapies across HER2 expression subgroups is needed. SGNDV-003 (NCT05902494) is a global retrospective chart review study in which HER2 status was assessed via central lab testing and outcomes were analyzed by HER2 status in pts with la/mUC. Methods: SGNDV-003, conducted 6/2023-7/2025, included pts with la/mUC who received ≥1 prior treatment (tx) (incl. 1 line of platinum-based chemotherapy [chemo]). HER2 status was centrally tested using a UC-specific investigational immunohistochemistry [IHC] and in situ hybridization (ISH) assays. HER2 status was defined as: HER2-positive (IHC3+ or IHC2+/ISH+), HER2-low (IHC2+/ISH- or IHC1+), HER2-expressing (IHC3+, 2+, 1+), and HER2-zero (IHC0). Index line of therapy (LOT) was defined as the first LOT received after platinum-based chemo. Outcomes included real-world objective response rate (rwORR) (per RECIST 1.1 or physician-assessed response) to index LOT, real-world progression-free survival (rwPFS), and overall survival (OS) from start of index LOT. Data was analyzed via descriptive statistics. Results: A total of 353 pts with la/mUC and available HER2 status were analyzed (male, 76%; median age, 70 years). 70% of pts were HER2 expressing (19% HER2-positive, 51% HER2-low); 30% were HER2-zero. Disease characteristics were well balanced by HER2 status. Index LOT tx and real-world outcomes are shown in the Table. Immuno-oncology therapies (IO) and enfortumab vedotin (EV) were the most common index LOTs. For IO, rwORRs were 21% for HER2-expressing and 17% for HER2-zero. For EV mono, rwORRs were 36% (HER2-expressing) and 38% (HER2-zero), respectively. Conclusions: This global study provides a robust estimate of HER2 distribution in la/mUC based on standardized central lab testing, showing high prevalence of HER2 expression. Real-world outcomes with current SOC therapies were broadly consistent across HER2 subgroups, establishing an important benchmark for the clinical development of HER2-directed therapies in this setting. HER2-positive (n=67) HER2-low (n=181) HER2-expressing (n=248) HER2-zero (n=105) Total (n=353) Real-world Outcomes rwORR, n (%)[95% CI] 17 (25.4)[15.5, 37.5] 50 (27.6)[21.3, 34.7] 67 (27.0)[21.6, 33.0] 22 (21.0)[13.6, 30.0] 89 (25.2)[20.8, 30.1] Median rwPFS (95% CI), mo 2.8(2.0, 4.1) 3.7(2.7, 5.1) 3.0(2.7, 4.5) 2.6(2.1, 3.4) 2.9 (2.6, 3.5) Median OS(95% CI), mo 12.2(9.3, 16.5) 11.5(9.5, 13.5) 11.9(10.3, 13.4) 7.7(5.9, 9.9) 10.8(9.1, 12.3) Index LOT, n% IO EV monoChemoOther tx 37 (55)17 (25)10 (15)3 (5) 94 (52)51 (28)24 (13)14 (8) 131 (53)68 (27)34 (14)17 (7) 65 (62)21 (20)12 (11)7 (7) 196 (56)89 (25)46 (13)24 (7)

3146 Background: The MTA-cooperative PRMT5 inhibitor BMS-986504 selectively binds to the PRMT5-MTA complex, a synthetic lethal target in MTAP -del cancer cells, while sparing MTAP –wild-type normal cells. In the phase 1 CA240-0007 study, BMS-986504 was well tolerated and had antitumor activity in pts with MTAP -del advanced solid tumors (ORR, 23%; median DOR, 10.5 mo). BMS-986504 also demonstrated dose-dependent reductions in plasma symmetric dimethylarginine (SDMA) levels, a PD biomarker of PRMT5 inhibition. Here, we report expanded PD and genomic analyses and initial results of exploratory proteomic analyses from CA240-0007. Methods: Pts with advanced, unresectable, or metastatic solid tumors with homozygous MTAP -del received BMS-986504 (50–800 mg) in 3-wk cycles. Analyses of SDMA levels and proteomic profiles from paired plasma samples (baseline and C2D1) were performed using mass spectrometry and SomaScan 11K, respectively. Tumor genomic information was extracted from local pathology reports. ORR was assessed per RECIST v1.1. Results: Among 78 pts with paired samples at data cutoff (22 Sept 2025; median f/u, 15.9 mo), dose-dependent reductions were seen in plasma SDMA levels, with the greatest reductions at 400 mg QD (median, 60.3%; n = 31) and 600 mg QD (median, 60.3%; n = 23). Proteomic pathway enrichment analysis demonstrated downregulation of PI3K/Akt/mTOR, Myc target, cell cycle, and DNA damage repair (DDR) pathways with BMS-986504 on C2D1 (n = 87), consistent with the expected effect of PRMT5 inhibition; a trend toward deeper modulation of PRMT5-regulated pathways was observed at higher doses. No proteins at baseline (n = 94) were found to be significantly associated with ORR (false discovery rate > 0.05). Responses in clinically evaluable pts with NSCLC (n = 33) or PDAC (n = 35) were observed regardless of EGFR , KRAS , or TP53 status. In pts with NSCLC, STK11 and SMARCA4 alterations were numerically higher in nonresponders. No gene alterations were associated with response in PDAC, except GABRA6 alterations (n = 2, both responders). A trend toward higher ORRs was seen in pts with alterations in DDR genes ( CHEK2, FANCD2, XRCC4 , and/or MSH2 ), consistent with the role of PRMT5 in DDR via regulation of mRNA splicing. ctDNA analysis will be presented. Conclusions: BMS-986504 demonstrated robust dose-dependent PD effects in pts with advanced solid tumors in plasma SDMA and proteomic analyses. Downregulation of PRMT5-regulated pathways was observed with BMS-986504 treatment. Response was seen regardless of EGFR, KRAS , and TP53 alterations in NSCLC or PDAC, and DDR gene alterations were associated with improved response. These findings further support the MOA of BMS-986504 and its ongoing investigation in the phase 2/3 MountainTAP-9, -29, and -30 studies as a potential treatment option in pts with MTAP -del advanced solid tumors. Clinical trial information: NCT05245500 .

10522 Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality. While antiviral and metabolic therapies have reduced incidence, scalable pharmacologic strategies for primary prevention (PP) remain a critical unmet need. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have demonstrated preclinical antitumor activity and the potential to mitigate hepatic lipotoxicity while promoting metabolic reprogramming to stabilize cirrhosis. However, while chemoprevention signals are observed in metabolic dysfunction-associated steatotic liver disease (MASLD) and type 2 diabetes, the efficacy of GLP-1RAs for PP across diverse liver disease etiologies remains underexplored. To address this gap, we conducted a global, multicenter analysis on the efficacy and safety of GLP-1RAs for the PP of HCC in a pan-etiology high-risk cohort. Methods: A retrospective cohort study was conducted using the TriNetX database (150M patients across 108 health organizations). Adults 18–90 years at elevated HCC risk with ≥ 1 risk factor (chronic viral hepatitis, cirrhosis, MASLD, alcohol use disorder, or hereditary liver disorders) were identified. Cohort A (≥3 months of GLP-1RA) was propensity score-matched 1:1 with Cohort B (non-users) on demographics, HbA1c, BMI, and MELD-Na scores. The primary endpoint was incident HCC, and secondary endpoints were incidence of gastrointestinal (GI) side effects. We employed a 3-month lag period to mitigate immortal time bias. Time-to-event outcomes were assessed via Kaplan-Meier analysis and Cox proportional hazards models. Sensitivity & subgroup analyses were performed to confirm robustness across non-metabolic etiologies. Results: After matching, 592,718 patients were analyzed (296,359 per cohort). Median follow-up was 2,450 days for GLP-1RA users vs 2,114 days for non-users. GLP-1RA use was associated with a 73% reduction in incident HCC (140/296,345 events vs 432/296,198) [HR 0.271 (0.224-0.328)], (NNT=1,015). Notably, the protective effect was most pronounced in non-diabetics [HR 0.179 (0.140-0.229)] and lean patients (BMI < 29) [HR 0.161 (0.103-0.252)]. Risk reductions were sustained across all etiologies, including alcohol liver disease (HR 0.379), cirrhosis (HR 0.456), chronic viral hepatitis (HR 0.477), and MASLD (HR 0.605). GLP-1RA use was associated with a significant increase in GI adverse events. Conclusions: GLP-1RA use was associated with a 73% reduction in incident HCC, consistent across subgroups. The enhanced benefit in non-diabetic and lean cohorts suggests a direct anti-neoplastic mechanism independent of weight loss or glycemic control. The NNT of 1,015 highlights the substantial public health impact of GLP-1RA therapy in preventing a high-mortality malignancy. These findings warrant prospective validation.

Abstract TRIM29 (Tripartite Motif-Containing Protein 29), also known as ATDC (Ataxia-Telangiectasia Group D-Complementing Protein), is an E3 ubiquitin ligase frequently upregulated in muscle-invasive bladder cancer (MIBC). We have previously shown that TRIM29 overexpression in a genetically engineered mouse model (GEMM) is sufficient to drive development of MIBC. We have also shown that TRIM29 is enriched in basal bladder cancer subtypes and is part of a TP63-regulated program that promotes migration and invasive progression. Prior studies suggest that Keratin 5/14 (K5+/K14+) expressing basal-stem progenitor cells in the urothelium are the cells of origin for MIBC in mice. However, whether Trim29 expression in these basal urothelial cells is required for MIBC development has not been established. In this study, we find that Trim29 expression in K5+ basal cells is linked to the inflammatory response and is critical for bladder tumor formation and invasive progression. We show that urothelial TRIM29 expression is enhanced during inflammation in humans and under inflammatory and genotoxic stress in mice. Knockout (KO) of Trim29 in the K5+ basal urothelium of a GEMM exposed to a bladder-specific chemical carcinogen blocked MIBC development. Further, urothelial Trim29 KO enriches immune cell recruitment to the bladder and upregulates STING and inflammatory signaling in mouse and human bladder cancers. These data reveal a critical role for TRIM29 in immunomodulation of the bladder tumor microenvironment (TME), which may act in concert with pro-migratory basal gene programs to expedite MIBC development.