Citations

Deregulated inflammatory signaling via STAT family transcription factors, particularly STAT1, underlies a variety of immune-related diseases, including inflammatory bowel disease. Whereas activation of STATs by JAKs via canonical receptor-driven JAK–STAT signaling is well understood, little is known about JAK-independent mechanisms of STAT activation. Here, we identify the understudied nonreceptor tyrosine kinase TNK1 as a therapeutically targetable, JAK-independent activator of STAT signaling. Using a multiomics approach, we mapped a network of TNK1 substrates associated with protein condensates and proinflammatory signaling, including STAT1. We found that TNK1, but not its sister kinase ACK1, directly phosphorylates STATs at well described STAT-activating JAK sites. In cells, TNK1-mediated STAT1 phosphorylation and activation occurs independently of JAKs. Imaging and interactomics data suggest that TNK1 interacts with STAT1 in cytosolic condensates, which likely compartmentalize TNK1-substrate interactions. We show that an intrinsically disordered proline-rich region in TNK1, which includes a 14-3-3 docking phosphorylation site, is required for the formation of kinase-active TNK1 condensates and STAT1 phosphorylation. Mutations within the proline-rich region that eliminate 14-3-3 binding increase formation of TNK1 condensates, suggesting a model in which 14-3-3 acts as a clamp that constrains the flexible PRR to inhibit condensate formation and STAT1 activation. Finally, we show that TNK1 is a targetable driver of STAT1-mediated inflammation in the gut as inhibition of TNK1 reduces active STAT1 in the colon and ameliorates colitis symptoms in mice.

Abstract Cholangiocarcinoma (CCA) is an aggressive biliary malignancy with poor prognosis and limited treatment options, underscoring the need for targeted RNA-based therapeutic strategies. Though DNA- aptamers could be one of the promising targeted ligands, conventional enrichment-driven selection often fails to reliably identify high-affinity binders due to PCR bias, stochastic drift, and sequencing errors. In this study, we combined cell-SELEX with a data-driven artificial intelligence framework to identify DNA aptamers targeting CCA cells. Eleven rounds of selection were performed on a library exceeding 6×1014 sequences, with iterative negative selection against normal hepatocytes and cholangiocytes to enhance tumor specificity. Given the limitations of enrichment-based ranking in prioritizing functional binders, we developed and evaluated four deep learning architectures—MLP, CNN, BiLSTM, and a hybrid CNN–BiLSTM—using aptamer pools derived from the final SELEX round. These models were built by integrating one-hot sequential features and DNA structural descriptors of the dataset along with their enrichment patterns across SELEX rounds. Among them, the hybrid CNN–BiLSTM model demonstrated superior performance by capturing both local sequence motifs and long-range dependencies. Model performance was evaluated using AUC-ROC and complementary classification metrics on an independent test set. The hybrid CNN–BiLSTM model achieved the best overall performance with an AUC-ROC of 0.97 and a precision of 68%, indicating strong discriminative power under class imbalance (strong: non-binder=1:68). The model exhibited high specificity (99.5%; 29,855/30,005 true negatives) and moderate sensitivity (71.7%; 312/435 true positives), reflecting its ability to reliably filter non-binding sequences while retaining true functional binders. Model-prioritized candidates were experimentally validated using qPCR-based aptamer–cell binding assays. Notably, two low-abundance/high-prediction aptamers (Read counts: 4) exhibited 2–3-fold higher binding than the reference aptamer (Positive Control) derived from traditional SELEX enrichment. A high-read/high-prediction candidate showed ∼2-fold greater binding than the positive control, highlighting the model’s ability to recover overlooked functional binders. To assess translational relevance, selected aptamers were used to functionalize the lactosome-based nanovesicles for siRNA delivery targeting PD-L1, resulting in measurable gene silencing in HuCCT1 cells after 48 hours. Collectively, this integrative framework enables accurate identification and prioritization of high-affinity, functionally relevant aptamers beyond enrichment alone. This approach enhances SELEX efficiency, supports rational ligand design, and provides a scalable strategy for developing aptamer-guided RNA therapeutics for any target. Citation Format: Sourav Pal, Julia Driscoll, Brandon Wilbanks, Ayano Kabashima, Rory Smoot, Tushar Patel. Integrating cell-SELEX and deep learning method for discovery of high-affinity aptamers targeting cholangiocarcinoma [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A033.

Hepatic resection (HR) and liver transplantation (LT) are curative-intent treatments for hepatocellular carcinoma (HCC). Patients without cirrhosis are typically analyzed as a single group, obscuring potentially important differences between those with histologically normal liver and those with non-cirrhotic parenchymal disease. We aimed to characterize long-term outcomes following HR stratified by underlying parenchymal quality, with a contemporary LT cohort as an external benchmark. Retrospective cohort study of 662 adults undergoing curative-intent surgery for HCC at 2 tertiary centers (2010–2024). Resection patients (n=380) were stratified by non-tumor parenchymal histology into HR-Normal (n=84), HR-Diseased non-cirrhotic (n=159), and HR-Cirrhotic (n=137). A total of 282 LT recipients served as an external reference but were not entered into comparative models. Overall survival (OS) was analyzed by Kaplan–Meier and multivariable Cox regression, and recurrence by Fine–Gray competing-risks regression. Ninety-day mortality was low across resection strata (1.2%–3.2%). OS differed significantly across parenchymal groups (log-rank p =0.01), driven by superior outcomes in HR-Normal patients compared with both HR-Cirrhotic ( p =0.008) and HR-Diseased ( p =0.005). HR-Cirrhotic and HR-Diseased did not differ ( p =0.97). Five-year OS was 74.4%, 59.8% and 57.2%, respectively. On multivariable Cox regression, both cirrhosis (HR 2.03, 95% CI 1.16–3.54, p =0.01) and diseased non-cirrhotic parenchyma (HR 2.04, 95% CI 1.21–3.43, p =0.008) independently predicted death versus normal liver. Fine–Gray analysis confirmed higher recurrence risk in cirrhotic (sHR 2.09, p =0.01) and diseased non-cirrhotic (sHR 1.80, p =0.04) groups. The LT benchmark cohort achieved a 5-year OS of 82.4%. Histologically normal liver identifies a distinct HR subgroup with favorable oncologic outcomes. The conventional cirrhotic/non-cirrhotic dichotomy underestimates the prognostic impact of underlying parenchymal disease.

PURPOSE Deficient mismatch repair/microsatellite instability (dMMR, MSI-H) in biliary tract cancers (BTCs) is observed in 1%-5% of tumors. MSI-H across solid tumors has demonstrated durable response to immune checkpoint inhibitors (ICIs) regardless of the anatomic location. Here, we describe clinical outcomes and surgical conversion of MSI-H BTC treated with ICIs. Patients and Methods We conducted a multicenter, retrospective analysis of BTC patients with dMMR/MSI-H between 2017 and 2024 at Mayo Clinic, University of Michigan, and University Hospitals Seidman Cancer Center. Outcomes of interest were surgical resection rate, rate of pathologic response, 2-year overall survival (OS; time from initiation of treatment to death/last follow-up), and time to treatment discontinuation (TTD; time from initiation of treatment to cessation because of any cause). Analyses were descriptive. RESULTS Thirty patients with MSI-H BTC were identified: 19 with cholangiocarcinoma, three with gallbladder cancer, and 8 with BTC undefined. The median age at diagnosis was 61.5 years (range, 26-86). Twenty-six (86.7%) patients had advanced stage at diagnosis. Twenty-seven patients (90%) received first-line systemic therapy, and 13 (48.1%) continued onto second line. Six patients (23.1%) were restaged to resectable from advanced disease after ICI therapy and underwent resection; all remained recurrence-free at last follow-up. Of these six patients, 3 (50%) achieved a pathologic complete response. Among those receiving first-line therapy, the median TTD was 9.9 months (95% CI, 5.7 to 14.5 months) and the 2-year OS was 73% (95% CI, 56% to 94%). CONCLUSION ICI therapy in patients with MSI-H BTC demonstrated durable response and downstaged some BTC to a resectable stage. dMMR/MSI-H should be performed up-front to guide treatment decisions.

Abstract Carcinoid heart disease (CHD) is associated with advanced neuroendocrine tumor liver metastases (NETLM) and may preclude surgical cytoreduction. We assessed perioperative and long‐term outcomes of hepatectomy in patients with CHD. We retrospectively analyzed 311 patients undergoing cytoreductive hepatectomy for intestinal NETLM: non‐functional ( n = 163), carcinoid syndrome (CS) without CHD ( n = 110), and CHD ( n = 38), including patients undergoing pre‐hepatectomy valve replacement. CHD patients more frequently had >10 liver metastases (78%) and larger lesions (median 9.5 cm) and required major hepatectomy more often (58%). Major morbidity was higher in CHD (up to 47%), yet 90‐day mortality remained low (≤4%). Median overall survival after hepatectomy was comparable across groups (12.5 vs. 9.1 vs. 11.4 years; p = .19), including matched analyses. With optimal cardiac management, cytoreductive hepatectomy in CHD is feasible and provides long‐term survival comparable to patients without CHD.

ABSTRACT Cholangiocarcinoma (CCA) is a lethal biliary cancer in which chemoresistance is nearly universal, and its tumor immune microenvironment is dominated by immunosuppressive tumor-associated macrophages (TAMs) that exclude cytotoxic CD8 + T cells. How tumor cells sustain this immunosuppressive state during chemotherapy is undefined. Here we demonstrate that therapy-induced senescent-like (Sen-L) cancer cells accumulate after gemcitabine/cisplatin in human and murine CCA, are predominantly cancer cells, and predict shorter survival. Genetic elimination of Sen-L cancer cells reduces tumor burden, lowers TAM abundance, and restores intratumoral CD8 + T cells, establishing them as causal drivers. Growth differentiation factor 15 (GDF-15) is the dominant Sen-L-secreted factor and reprograms macrophages to suppress CD8 + T cells through the non-canonical receptor TGFBR2 and STAT6, and p16-restricted Gdf15 silencing phenocopies Sen-L elimination. Combined with chemotherapy, Sen-L elimination improves survival beyond chemotherapy alone. These findings establish Sen-L cancer cells and their GDF-15 output as causal, targetable drivers of macrophage-mediated immune evasion in CCA. SIGNIFICANCE STATEMENT Therapy-induced senescent-like cancer cells, not stromal cells, are the dominant senescent-like and immunosuppressive population in cholangiocarcinoma, and their elimination restores antitumor immunity. GDF-15 is their dominant secreted effector and engages a non-canonical macrophage receptor, TGFBR2, identifying a cancer-cell-to-macrophage axis and a Sen-L-elimination strategy to restore chemosensitivity. Abstract Figure Graphical abstract. Senescent-like CCA cells promote tumor immunosuppression through TAMs polarization by GDF-15

Abstract Background and Objectives Cholangiocarcinoma (CCA) is an aggressive, highly lethal cancer of the biliary tract epithelium for which tumor resection is the only curative treatment. However, approximately half of patients present with nonresectable disease and median survival of these patients is just 8-12 months. A significant need exists for tumor-targeted therapeutics for patients with advanced disease who currently have limited treatment options. We aimed to address this need by developing an aptamer-targeted therapy for delivery of siRNAs inhibiting undruggable components of the Hippo pathway, a key driver of cholangiocarcinoma. Method We applied cell-SELEX against human CCA model cell line HuCCTl with negative selection against normal hepatocytes and cholangiocytes to identify an aptamer that selectively bound tumor cells. This aptamer, termed MC7122, was chemically modified to include fluorescent labels that enabled whole-animal biodistribution studies to assess in vivo selectivity. MC7122 was then deployed onto the surface of siRNA-loaded nanovesicles to drive tumor-targeted nucleic acid delivery. Tumor-bearing mice were treated with siRNAs three times per week for two weeks alone or in combination with standard of care chemotherapy. Results Combined treatment with siRNAs and standard of care chemotherapy reduced tumor burden in mice versus vehicle controls and slowed the spread of metastasis. Importantly, we found tumor-specific target gene knockdown without corresponding off-target knockdown in healthy surrounding liver tissue. Further, we demonstrated that sites in the lung, the primary location of metastasis in this model, were reduced not because of global target knockdown in lung but due to tumor-specific knockdown at the site of liver implantation. Conclusions Aptamer-guided vesicles represent a promising platform for targeted therapy delivery for previously undruggable targets. With this platform, tumor-selective therapies can be designed to exhibit exceptionally potent cytotoxicity, as healthy tissues remain unaffected by off-target effects. Future work will focus on refinement of therapeutic siRNA target selection and modification of nanovesicle components for enhanced levels of tumor specificity.

Abstract Background Cholangiocarcinoma (CCA) is an aggressive hepatobiliary malignancy with marked molecular heterogeneity, limited systemic treatment options, and poor prognosis. Chemoresistance and KRAS mutations remain major therapeutic challenges. Dysregulation of the Hippo pathway, particularly YAP/TAZ hyperactivation, drives CCA progression and resistance. TEAD, the essential transcriptional partner of YAP/TAZ, represents a promising target. IAG933 is a novel oral pan-TEAD inhibitor designed to disrupt YAP/TAZ–TEAD interactions. RMC-6236, a pan-KRAS inhibitor, has also gained attention. Combining TEAD and KRAS inhibition may overcome resistance and improve efficacy in CCA. Methods We evaluated IAG933 in human (HuCCT1, RBE) and murine (SB1, FAC) CCA cell lines. Cells were treated with IAG933 alone or in combination with GemCis or RMC-6236. Cell viability assays were used to determine IC50 values and calculate combination index (CI). TEAD activity was assessed by luciferase reporter and qRT-PCR of YAP/TAZ target genes (CTGF, CYR61, NUAK2). In vivo efficacy was evaluated in syngeneic orthotopic and patient-derived xenograft (PDX) models. Results IAG933 suppressed TEAD-dependent transcription (IC50≈500 nM) and downregulated YAP/TAZ target genes. Combination with GemCis or RMC-6236 produced synergistic effects (CI < 1) in all CCA lines. In vivo, IAG933 monotherapy suppressed tumor growth similarly to GemCis. Dual TEAD and KRAS inhibition achieved marked tumor regression and significantly delayed tumor regrowth after treatment cessation in KRAS-mutant PDX models. Conclusion IAG933 effectively suppresses YAP/TAZ–TEAD signaling and inhibits CCA growth. While the combination with GemCis showed limited additional benefit due to strong single-agent efficacy, synergy with RMC-6236 produced durable antitumor effects, particularly in run-out studies, delaying regrowth in KRAS-mutant models. These findings highlight IAG933 as a promising therapeutic candidate targeting the Hippo–TEAD pathway, with potential benefit in KRAS-mutant CCA.

Abstract Background Cholangiocarcinoma (CCA) is a deadly cancer of the hepatic bile duct epithelial cells. Loss-of-function mutations of epigenetic regulator ubiquitin C-terminal hydrolase deubiquitinase BRCA1associated protein 1 (BAP1) occur in 26-32% of human CCA and are associated with worse prognosis and resistance to cytotoxic therapy. The goal of this study is to identify how BAP1 loss promotes cell death evasion through metabolic rewiring. Materials and methods Isogenic wild-type and BAP1-deficient human and murine CCA cell lines were generated with shRNA/siRNA or CRISPR/Cas9 and were used to assess ferroptosis sensitivity by treatment with ferroptosis inducers. In vitro, ferroptosis was assessed by phospholipid peroxidation (C-11BODIPY), mitochondrial phospholipid peroxidation (MitoPeDPP, MitoCLox), iron overload (MitoFerro green) and cell death (CellTiter-Glo). Phospholipidomics and proteomics were performed in isogenic cells by LC-MS/MS. Syngeneic liver orthotopic CCA murine models and patient-derived xenografts (PDX) were used for in vivo pre-clinical studies. Results Our findings indicate that BAP1 loss confers CCA ferroptosis resistance. BAP1 loss protects CCA against mitochondrial iron overload, ROS accumulation, and phospholipid peroxidation. BAP1 loss protects from mitochondrial phospholipid peroxidation and cardiolipin oxidation. Unbiased phospholipidomic profiling revealed that BAP1 loss remodels membrane phospholipid composition, enriching for ferroptosis blocking monounsaturated phospholipids (MUFA-PLs) at the expense of ferroptosis-inducing polyunsaturated (PUFA-PLs). We found that BAP1 loss upregulates TLCD1, a phosphatidylethanolamine acyltransferase that incorporates MUFAs into membrane phospholipids, thereby stabilizing membranes against peroxidation. TLCD1 knockdown in ferroptosis-resistant BAP1-deficient CCA cells, restored ferroptosis sensitivity. Notably, TLCD1 is significantly upregulated in human BAP1-mutant CCA as compared to BAP1-expressing tumors. While not a mitochondrial protein, TLCD1 localizes in the ER and by the mitochondrial outer membrane, as demonstrated by our immunofluorescence analysis in human CCA cells. From a therapeutic perspective, we developed EpCAM (adhesion molecule highly expressed in CCA cells)-aptamer coated lactosomes packaged with siRNA against glutathione peroxidase 4 (GPX4), as a proof-of-concept strategy to safely induce ferroptosis selectively in CCA in vitro and in vivo. Administration of siGPX4-lactosomes in pre-clinical syngeneic liver orthotopic CCA murine models demonstrated a favorable safety profile, achieved robust intratumoral GPX4 knockdown (>50%), exhibited optimal biodistribution, induced significant tumor lipid peroxidation, and resulted in reduced tumor burden. Lastly, ferroptosis inducer JKE-1674, combined with gemcitabine/cisplatin (Gem/Cis) significantly blocked tumor growth of Gem/Cis-resistant patient-derived xenografts. Conclusions Our findings reveal that BAP1 loss reprograms mitochondrial phospholipid metabolism thus conferring CCA resistance to ferroptosis, identifying TLCD1 as a targetable metabolic vulnerability in BAP1-mutant CCA. Additionally, our findings support ferroptosis inducing compounds as promising therapeutic approaches for CCA.

Abstract Background SWI/SNF chromatin remodeling complexes are frequently mutated across human cancers, including biliary tract cancers (BTC). Within the SWI/SNF complexes, loss-of-function (LOF) mutations in ARID1A and PBRM1 are the most common and frequently co-occur alongside PI3K/AKT pathway activation. Yet, how those mutations affect the intracellular signaling landscape and transcriptomic programs is underexplored and may yield novel therapeutic opportunities. Methods Clinicodemographic parameters and mutation profiles of 250 patients with BTC were retrospectively collected through electronic medical records. Advanced overall survival (OS) was assessed in a multivariable analysis. Transcriptomic data were obtained and differentially expressed genes (DEGs) and pathways associated with SWI/SNF mutation status were analyzed. Changes in the transcriptomic landscape were compared to a murine syngeneic model of intrahepatic cholangiocarcinoma (CCA) initiated by loss of ARID1A and/or PBRM1 for cross-species validation. Results After adjusting for age, sex, and anatomical subtype, patients with SWI/SNF LOF mutations were found to have a 30% reduction in mortality risk (HR = 0.70, 95% CI: 0.50-0.98, p = 0.037). The transcriptomic cohort of 233 BTC patients included 71 (30%) with SWI/SNF LOF mutations, 21 (9%) with variants of uncertain significance, and 141 (61%) with SWI/SNF wild-type tumors. Differential gene expression and pathway analysis revealed changes in adaptive immune response, extracellular matrix, and cancer-associated proteoglycans. RNA-sequencing of murine tumors revealed the dominant effect of ARID1A loss in single and double knock-out tumors and conserved pathway alterations between mouse and human tumors. Conclusion Both murine and human primary tumors with either ARID1A alone or ARID1A/PBRM1 co-mutations exhibited altered expression of critical genes and pathways associated with proteoglycan synthesis and extracellular matrix. Ongoing research is investigating how CCA cells depend on those pathways and their canonical regulators for tumor formation and progression.