Werneburg, Nathan W.

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 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. Combined 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 indexes (CI). TEAD activity was assessed by luciferase reporter and qRT-PCR of YAP/TAZ target genes (CTGF, CYR61, NUAK2). Transcriptomic alterations were analyzed by RNA sequencing. 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. Transcriptomic analysis revealed activation of oxidative stress response pathways, particularly SOD2 upregulation. MitoSOX staining confirmed increased mitochondrial ROS after treatment. 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. Citation Format: Hidemi Nishi,Erik Jessen,Danielle M. Carlson,Amro M. Abdelrahman,Enis H. Ozmert,J. Pedro Fincatto Safi,Brandon A. Wilbanks,Shelby K. Yee,Jack W. Sample,Nathan W. Werneburg,Rory L. Smoot. TEAD inhibition by IAG933 as a novel therapeutic strategy for cholangiocarcinoma [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 3081.

Abstract Introduction: Src Family Kinases (SFKs) have a crucial role in tumor proliferation, survival, and metastasis. NXP900 is a highly selective SFK inhibitor with a novel mechanism of action that locks SFKs in a closed, inhibited conformation, providing sustained suppression of catalytic and non-catalytic functions, which is distinct from other SFK inhibitors like dasatinib. A phase I clinical trial of NXP900 in patients with advanced solid tumors is ongoing. Our preclinical studies showed therapeutic efficacy of NXP900 in cholangiocarcinoma (CCA) through decreased YAP, RAF-MAP and PI3K-AKT signaling. Multiomic profiling (RNA-seq, proteomics, phosphoproteomics) of CCA patient-derived xenografts implicated AKT pathway activation in primary NXP900 resistance. NXP900-resistant CCA cell lines generated via prolonged sublethal dose escalation showed a strong IL13RA2-AKT signature on multiomic analysis. Thus, we hypothesized that IL13RA2-AKT signaling was a mechanism of NXP900 resistance. We aimed to define this mechanism and demonstrate that the combinatorial targeting of IL13RA2 or AKT resensitizes to NXP900 treatment. Methods: HuCCT1 or HuCCT1-Resisant (HuCCT1-R) cells were injected bilaterally into the flanks of NOD/SCID mice and were allowed to grow for 3 months prior to tumor collection and analysis. Tumor engraftment and growth between HuCCT1 and HuCCT1-R groups were compared. To delineate the mechanisms for acquired NXP900 resistance, in-vitro treatment studies were performed using siRNAs targeting IL13RA2 as well as combination treatment with capivasertib, a pan-AKT inhibitor. Results: The HuCCT1-R cell line exhibited a higher rate of tumor engraftment and formed significantly larger tumors compared to the parental HuCCT1 cell line, indicating enhanced tumorigenic potential associated with acquired resistance. Silencing of IL13RA2 in HuCCT1-R cells resulted in pronounced reduction of cell proliferation and partially restored sensitivity to NXP900 treatment. Immunoblot analysis demonstrated a corresponding reduction in phosphorylated AKT (Thr308), consistent with suppression of downstream PI3K-AKT signaling. Furthermore, combination treatment of HuCCT1-R cells with the AKT inhibitor capivasertib, and NXP900 led to a significant increase in cell death and a greater inhibition in cell proliferation compared to either agent alone, indicating enhanced responsiveness and improved therapeutic efficacy. Conclusion: These findings confirm the central role of IL13RA2 signaling and the PI3K-AKT pathway in mediating acquired resistance to NXP900, consistent with our previous multiomics analysis. Importantly, this study identifies combination strategies that may be able to overcome NXP900 resistance, supporting the development of combinatorial approaches to restore therapeutic efficacy in resistant tumors. Citation Format: Shelby K. Yee, Hendrien Kuipers, Danielle M. Carlson, Jack W. Sample, Enis Ozmert, Hidemi Nishi, Erik Jessen, Dong-Gi Mun, Aushinie M. Abeynayake, Jennifer L. Tomlinson, Amro M. Abdelrahman, Nathan W. Werneburg, Binbin Li, Mitesh J. Borad, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Resistance to the SFK inhibitor NXP900 in cholangiocarcinoma is characterized by IL13RA2-AKT signaling and can be overcome by combination therapy [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 7124.

ABSTRACT The limited efficacy of current therapies against cholangiocarcinoma (CCA) necessitates the development of novel treatment strategies. Src family kinases (SFKs) contribute significantly to tumor progression and resistance in CCA. Therefore, we investigated the novel, first-in-class SFK ‘OFF’ inhibitor NXP900 in diverse preclinical CCA models, including those with acquired resistance. This study evaluated the therapeutic effects of NXP900 and detailed adaptive molecular responses to SFK inhibitor therapy. We also aimed to identify biomarkers predictive of drug sensitivity using integrated multiomic profiling and develop strategies to overcome resistance. NXP900 inhibited YAP activity through direct inhibition of tyrosine phosphorylation and indirect activation of the Hippo pathway via LATS. These effects were associated with decreased tumor cell viability in CCA cell lines and several in vivo models. Notably, IDH -mutant patient-derived xenograft CCA models were particularly sensitive to NXP900. NXP900 also synergized with gemcitabine/cisplatin chemotherapy, enhancing antitumor efficacy in both in vitro and in vivo models. Multiomic analyses combining transcriptomics, global proteomics, and phosphoproteomics identified molecular features associated with primary response and acquired resistance. IL13RA-AKT signaling was upregulated in resistant models; NXP900 sensitivity could be restored with AKT or IL13RA2 inhibition. Together, these findings demonstrate the therapeutic potential of NXP900 as a novel YAP inhibitor in CCA and support further investigation in a clinical trial. Graphical Abstract

Lacking effective therapeutics, cholangiocarcinoma (CCA) remains a deadly malignancy of the biliary tract. The Hippo pathway effector protein Yes-associated protein (YAP) is implicated in CCA pathogenesis and chemotherapeutic resistance; however, the oncogenic mechanisms underlying YAP regulation remain incompletely understood. An enhanced understanding of YAP and its role in CCA may uncover novel therapeutic targets and better define resistance pathways. Human CCA cells and murine syngeneic CCA models were utilized to explore the molecular relationship of YAP and protein tyrosine phosphatase 1B (PTP1B). Previous work in CCA has demonstrated that YAP interacts with multiple protein tyrosine phosphatases, including SHP2 and PTP1B. We observed that PTP1B pharmacologic inhibition was associated with increased cell proliferation and YAP target gene expression, while genetically enforced overexpression of PTP1B was associated with a decrease in YAP activation. Treatment of CCA cells in vitro and syngeneic, orthotopically implanted CCA murine tumors in vivo with standard cytotoxic chemotherapy, gemcitabine/cisplatin, had enhanced efficacy in the setting of PTP1B overexpression. These findings demonstrate that pYAPY357 can be modulated through protein tyrosine 1B phosphatase activity, and reducing pYAPY357 through enhanced phosphatase levels can sensitize CCA to chemotherapy.

Abstract Background: The transcriptional cofactor Yes-associated protein (YAP) is activated in CCA pathophysiology. While canonical regulation by the Hippo pathway resulting in phosphorylation at serine 127 (S127) is inhibitory, SRC-family kinases (SFKs) phosphorylate YAP at tyrosine 357 (Y357), causing activation. Previously, cholangiocytes, hepatic progenitor cells (HPCs), and even mature hepatocytes were suggested as potential cells of origin of CCA, which highlights the plasticity in the liver. However, the role of YAP activation in selecting the cell of origin and lineage commitment during hepatobiliary oncogenesis is unknown. Aim: To determine if YAP Y357 phosphorylation is required for CCA tumorigenesis, we utilized an in vivo model. Unexpectedly, a shift in tumor phenotype to hepatocellular carcinoma (HCC) was identified. Subsequently, we further explored cell signaling and cell of origin contributions to this phenotypic switch. Method: C57BL/6 mice underwent biliary transfection with Sleeping Beauty transposase and transposons encoding myr-AKT and either YAP S127A (YAP-S) or YAP S127A/Y357F (YAP-SY), followed by IL-33 treatment. Tumor nodule count, histologic classification, and liver-to-body ratio were assessed at necropsy after 10 weeks. RNA sequencing and ingenuity pathway analysis (IPA) were performed on HCC and CCA nodules. To determine the cell of origin, tumors were generated in lineage tracing mice (Rosa26 mT/mG mice treated with AAV8-TBG-Cre). Finally, two tumor-derived cell lines with YAP-SY and constitutive AKT expression were isolated. Results: Both YAP-S and YAP-SY models developed similar tumor numbers and bulk. YAP-S mice generated frequent CCA (25.3%) in addition to combined HCC-CCA (cHCC-CCA) tumors (10.7%) and HCC (64%), whereas YAP-SY mice developed predominantly HCC (86.7%), with rare CCA (8.8%) and mixed tumors (4.5%). Transcriptome analysis identified 3,367 differentially expressed genes (DEGs) between HCC from YAP-SY mice and CCA from YAP-S mice. Notably, YAP-regulated genes had decreased expression in the HCC from YAP-SY mice. IPA revealed key upstream regulators, including cytokines and growth factors (e.g., TGFB1, TNF, INFG, CSF2, IL6, IL1B, IL4) and transcription factors (e.g., TP53, JUN, SP1, NFKBIA, STAT3). Lineage tracing demonstrated HCC originated from hepatocytes and CCA from cholangiocytes in both models. Finally, gene expression analysis of novel syngeneic cancer cell lines from YAP-SY mice showed mixed features of both HCC and CCA. Conclusion: In this murine model, we found that sub-maximal YAP activation is associated with increased formation of HCC from hepatocytes, while a higher level of YAP activation due to YAP Y357 phosphorylation is associated with efficient formation of CCA. We identified cytokines and growth factors that may orchestrate this phenotypic switch. Finally, we developed novel syngeneic cancer cell lines with cHCC-CCA characteristics. Citation Format: Muhammed D. Aksu,Jayla T. Millender,Ryan D. Watkins,Jennifer A. Yonkus,EeeLN H. Buckarma,Nathan W. Werneburg,Daniel R. O'Brien,Chantal E. McCabe,Rondell P. Graham,Gregory J. Gores,Rory L. Smoot,Caitlin B. Conboy. YAP tyrosine phosphorylation regulates lineage commitment in liver tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1457.

Abstract Introduction: Src Family Kinases (SFKs) have a crucial role in tumor proliferation, survival, and metastasis. NXP900 is a highly selective SFK inhibitor a novel mechanism of action that locks SFKs in a closed, inhibited conformation, preventing interaction with substrate proteins, unlike other SFK inhibitors like dasatinib. A phase 1 study of NXP900 in patients with advanced solid tumors was recently initiated. Our preclinical studies showed therapeutic efficacy of NXP900 in cholangiocarcinoma (CCA) through decreased YAP, RAF-MAP and PI3K-AKT signaling. Here, we examined mechanisms of sensitivity and resistance to SFK inhibition. Methods: Secondary resistance was explored by generating a NXP900-resistant human cell line (HuCCT1-R) via prolonged exposure to NXP900, followed by multiomic profiling. Primary resistance was studied in vivo using nine heterogeneous patient-derived xenograft (PDX) tumors implanted in the flanks of NOD/SCID mice. Tumor-bearing mice were treated with vehicle or 40 mg/kg NXP900 daily for up to 4 weeks. To identify factors associated with response, a multiomic approach was used, integrating RNA-Seq, global proteomics, and phosphoproteomics. A hierarchical-all-against-all algorithm (HallA) assessed associations between multiomic features and drug response in tumor growth. Kinase enrichment analysis (KEA3) identified features linked to sensitivity or resistance. Results: HuCCT1-R cells showed increased pTyr abundancy of TNK2, a protein known to increase PIP3 levels leading to AKT activation, and of DYRK3 and LAMTOR1, both of which can sustain AKT phosphorylation via mTORC, indicating acquired resistance through bypassing inhibited PI3K-AKT signaling to rescue AKT. NXP900 reduced tumor growth rates in 5 of 9 PDX models. Kinases associated with sensitivity in the PDX models included SFKs (SRC, LYN, and FYN), and AKT, PTK2, PRKCZ, EGFR, SYK, and GSK3B, suggesting involvement of the PI3K-AKT pathway in responsiveness to NXP900. Based on tumor inhibition responses in the tested models, we estimated NXP900 response for our additional PDX models, predicting 39% (11/28) of patient tumors may respond. Conclusion: This study identified molecular markers linked to response to SFK inhibition, which could be utilized for patient selection for NXP900 treatment. Further studies are validating these findings and exploring combination therapies to address resistance mechanisms. Citation Format: Hendrien Kuipers, Danielle M. Carlson, Jack W. Sample, Erik Jessen, Dong-Gi Mun, Aushinie M. Abeynayake, Jennifer L. Tomlinson, Amro M. Abdelrahman, Nathan W. Werneburg, Binbin Li, Mitesh J. Borad, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Multiomic profiling identified biomarkers of response to selective Src family kinase inhibition in preclinical models of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5595.

Abstract SWI/SNF chromatin remodeling complexes are frequently mutated across human cancers, including cholangiocarcinoma (CCA). Dysregulation of this complex due to loss of function mutations in AT-rich interaction domain-1A (ARID1A) and polybromo-1 (PBRM1) occur in up to 18% and 10% of cases respectively. SWI/SNF subunit mutations frequently co-occur alongside PI3K/AKT pathway activation. Preexisting literature suggests altered gene expression and protein synthesis contribute to CCA oncogenesis, but our understanding of these mechanisms remains immature. Genetically accurate preclinical models are needed to define the roles of ARID1A and PBRM1 in CCA and identify therapeutic targets. We developed novel murine models of CCA with either ARID1A, PBRM1 or dual ARID1A/PBRM1 (APAC) knockout in the context of AKT activation, utilizing a surgical biliary transfection method for somatic gene editing with CRISPR/Cas9. Within 8 months, mice formed numerous liver tumors, predominantly CCA and mixed CCA/HCC. Knockout of ARID1A and PBRM1 was confirmed through genomic PCR, qRT-PCR, and immunoblot assays. Histologically, murine APAC tumors displayed phenotypic similarities to human CCA, including dense cancer-associated fibroblast infiltration and variable CD45+ immune cell infiltration. A novel, syngeneic CCA cell line was isolated from a dual ARID1A/PBRM1 knockout tumor (APAC-2). RNA-sequencing of primary tumors and the APAC-2 cell line versus a non-invasive cholangiocyte cell line with AKT activation only (AC), was performed to identify differentially expressed genes (DEGs) of interest in ARID1A and PBRM1-deficient CCA. Periostin (Postn) was among the top upregulated genes in the APAC-2 cell line. POSTN is a secreted glycoprotein that is upregulated in human CCA and may be therapeutically targetable. Knockdown of periostin inhibited the in vitro growth of APAC-2 cells and a human CCA cell line with ARID1A mutation and periostin overexpression (HuH-28). These studies serve to characterize and validate a genetically relevant murine model of epigenetic dysregulation in ARID1A and PBRM1-deficient CCA. This model can be used for identification and validation of potential drug targets. Ongoing work focuses on studying multiple dysregulated pathways and testing dependence on genes of interest, including periostin, as potential treatment strategies for CCA. Citation Format: Jayla Millender, Jennifer Tomlinson, Ryan Watkins, Danielle Carlson, Jack Sample, Nathan Werneburg, Rory Smoot, Keith Robertson, Gregory Gores, Caitlin Conboy. Novel murine models of epigenetic dysregulation in SWI/SNF-mutated cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2741.

Background and Aims: Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the biliary epithelium with limited therapeutic options and poor long-term survival rates. To address the limitations of CCA treatment, we investigated cell-targeted nanovesicles as a delivery platform for transcriptome-targeting therapeutics. Approach and Results: Milk-derived nanovesicles were loaded with short interfering RNAs targeting Yes-associated protein (YAP), the downstream effector of the Hippo pathway; LCK, an upstream regulator of YAP; and tafazzin, a protein critical for the integrity of the inner mitochondrial membrane. These transcriptome-targeting nanovesicles were decorated with a lipid-coupled RNA aptamer to epithelial cell adhesion molecule, including a tracking fluorophore. In vitro studies were conducted using multiple CCA cell lines. In vivo studies were performed using C57BL/6 and non-obese diabetic/severe combined immunodeficient mice to evaluate delivery and efficacy in both an immunocompetent syngeneic murine and a patient-derived xenograft model. We demonstrated that transcriptome-targeting nanovesicles were selectively taken up by liver tumor cells, which was augmented by the incorporation of a targeting aptamer, and that milk-derived nanovesicles loaded with short interfering RNA effectively downregulated target gene expression, both in vitro and in vivo. Downstream effects of target gene inhibition were observed, including downregulation of YAP-TEAD target genes and an increase in reactive oxygen species production at the mitochondrial level. Administration of transcriptome-targeting nanovesicles targeting YAP, LCK, and tafazzin inhibited CCA growth and further synergized with chemotherapy in 2 preclinical CCA models. Conclusions: Herein, we show that aptamer-directed, nanovesicle-mediated targeting of YAP, LCK, and tafazzin potentiates chemosensitivity in 2 CCA models when delivered using aptamer-guided milk-derived nanovesicles.

Cholangiocarcinoma (CCA) is a highly aggressive form of liver cancer and is an increasing cause of cancer‐related death worldwide. Despite its increasing incidence globally and alarming mortality, treatment options for CCA have largely remained unchanged, stressing the importance of developing new effective therapies. YAP activation is common in CCA, and its major transcriptional signaling partners are the TEAD proteins. CA3 is a small‐molecule YAP‐TEAD disrupter discovered utilizing a TEAD reporter assay. Utilizing CCA, gastric cancer cell lines, and patient‐derived xenograft models (PDX), we demonstrate that CA3 is effective in inducing cell death and delaying tumor growth in both FGFR2 fusion and wild‐type models. CA3 was associated with on‐target decreases in YAP‐TEAD target gene expression, TEAD reporter activity, and overall TEAD levels. Hippo pathway signaling was not altered as there was no change in YAP phosphorylation status in the cells exposed to CA3. RNA sequencing of gastric cancer and CCA models demonstrated upregulation of an androgen receptor–mediated transcriptional program following exposure to CA3 in five unique models tested. Consistent with this upstream regulator analysis, CA3 exposure in CCA cells was associated with increased AR protein levels, and combinatorial therapy with CA3 and androgen receptor blockade was associated with increased cancer cell death. CA3 behaves functionally as a YAP‐TEAD disrupter in the models tested and demonstrated therapeutic efficacy. Exposure to CA3 was associated with compensatory androgen receptor signaling and dual inhibition improved the therapeutic effect.