Human CCA Research Articles

Notch-Driven Cholangiocarcinogenesis Involves the Hippo Pathway Effector TAZ via METTL3-m6A-YTHDF1

Background and AimsNotch and TAZ are implicated in cholangiocarcinogenesis, but whether and how these oncogenic molecules interact remain unknown. MethodsThe development of CCA was induced by hydrodynamic tail vein (HDTV) injection of oncogenes (NICD/AKT) to the FVB/NJ mice. CCA xenograft was developed by inoculation of human CCA cells into the livers of SCID mice. Tissues and cells were analyzed using qRT-PCR, Western blotting analyses, Immunohistochemistry, ChIP-qPCR and WST-1 cell proliferation Assay. ResultsOur experimental findings show that TAZ is indispensable in NICD-driven cholangiocarcinogenesis. Notch activation induces the expression of METTL3 (Methyltransferase like-3) which catalyzes N6-methyladenosine (m6A) modification of TAZ mRNA and that this mechanism plays a central role in the crosstalk between Notch and TAZ in CCA cells. Mechanistically, Notch regulates the expression of METTL3 through the binding of NICD to its downstream transcription factor CSL in the promoter region of METTL3. METTL3 in turn mediates m6A modification of TAZ mRNA which is recognized by the m6A reader YTHDF1 to enhance TAZ protein translation. We observed that inhibition of Notch signaling decreased the protein levels of both MELLT3 and TAZ. Depletion of METTL3 by shRNAs or by the next generation GapmeR antisense oligonucleotides (ASOs) decreased the level of TAZ protein and inhibited the growth of human CCA cells in vitro and in mice. ConclusionThis study describes a novel Notch-METTL3-TAZ signaling cascade which is important in CCA development and progression. Our experimental results provide new insight into how the Notch pathway cooperates with TAZ signaling in CCA, and the findings may have important therapeutic implications.

Identification of PRMT5 as a therapeutic target in cholangiocarcinoma

BackgroundCholangiocarcinoma (CCA) is a very difficult-to-treat cancer. Chemotherapies are little effective and response to immune checkpoint inhibitors is limited. Therefore, new therapeutic strategies need to be identified.ObjectiveWe characterised the enzyme...

RBM39 Enhances Cholangiocarcinoma Growth Through EZH2-mediated WNT7B/β-catenin Pathway

Background and aimsThe RNA-binding motif protein 39 (RBM39) functions as both an RNA-binding protein and a splicing factor in a variety of cancer types. However, the function of RBM39 in cholangiocarcinoma (CCA) remains undefined. In this study, we aimed to investigate the role of RBM39 in CCA and explore its potential as a therapeutic target. MethodsThe expression of RBM39 in CCA was investigated by analyzing human CCA tumor specimens. CRISPR/Cas9 or shRNA-mediated depletion of RBM39 was performed in vitro and in vivo to document the oncogenic role of RBM39 in CCA. The anti-tumor effect of the RBM39 inhibitor Indisulam in combination with the EZH2 degrader MS177 was assessed in vitro and in vivo. ResultsRBM39 is significantly increased in human CCA tissues and associated with a poor prognosis in CCA patients. Depletion of RBM39 by CRISPR/Cas9 or shRNA inhibited CCA cell proliferation in vitro and prevented CCA development and tumor growth in mice. Mechanistically, our results showed that depletion of RBM39 suppressed EZH2 expression via disrupting its mRNA splicing. RBM39-regulated EZH2 controls WNT7B/β-catenin activity. Pharmacological co-targeting of RBM39 (with Indisulam) and EZH2 (with MS177) resulted in a synergistic antitumor effect, both in vitro and in vivo. ConclusionThis study discloses a novel RBM39-EZH2-β-catenin signaling axis that is crucial for CCA growth. Our findings suggest that simultaneous inhibition of RBM39 and EZH2 presents a promising therapeutic strategy for CCA treatment.

Hypoxia Stimulates PYGB Enzymatic Activity to Promote Glycogen Metabolism and Cholangiocarcinoma Progression.

Cholangiocarcinoma (CCA) displays enhanced glycolysis, pivotal for fulfilling the heightened energy demands intrinsic to its malignant progression. Recent research has indicated that endogenous glycogen rather than exogenous glucose acts as the major carbon source for glycolysis, highlighting the need to better understand the regulation of glycogen homeostasis in CCA. Here, through comprehensive integrative analysis, we identified that glycogen phosphorylase brain form (PYGB), the main enzyme involved in glycogen homeostasis, was markedly upregulated in CCA tissues, serving as an independent prognostic indicator for human CCA patients. Moreover, elevated PYGB expression potentiated cholangiocarcinogenesis and augmented CCA cell proliferation in both organoid and xenograft models. Hypoxia stimulated PYGB activity in a phosphoglycerate kinase 1 (PGK1)-dependent manner, leading to glycogenolysis and the subsequent release of glucose-6-phosphate (G6P) and thereby facilitating aerobic glycolysis. Notably, a virtual screening pinpointed the beta-blocker carvedilol as a potent pharmacological inhibitor of PYGB that could attenuate CCA progression. Collectively, these findings position PYGB as a promising prognostic biomarker and therapeutic target for CCA.

Novel Adiponectin Receptor Agonist Inhibits Cholangiocarcinoma via Adenosine Monophosphate-activated Protein Kinase.

Cholangiocarcinoma (CCA) has a poor prognosis and only limited palliative treatment options. The deficiency of adiponectin and adenosine monophosphate-activated protein kinase (AMPK) signaling was reported in several malignancies, but the alteration of these proteins in CCA is still unclear. This study aimed to assess the role of adiponectin and AMPK signaling in CCA. Furthermore, AdipoRon, a novel adiponectin receptor (AdipoR) agonist, was evaluated in vitro and in vivo as a new anti-tumor therapy for CCA. The expression of AdipoR1 and p-AMPKα in human tissue microarrays (TMAs) was evaluated by immunohistochemistry staining (IHC). The effect of 2-(4-Benzoylphenoxy)-N-[1-(phenylmethyl)-4-piperidinyl]-acetamide (AdipoRon) was investigated in vitro with proliferation, crystal violet, migration, invasion, colony formation, senescence, cell cycle and apoptosis assays and in vivo using a CCA engineered mouse model (AlbCre/LSL-KRASG12D/p53L/L). RT-qPCR and western blot methods were applied to study molecular alterations in murine tissues. AdipoR1 and p-AMPKα were impaired in human CCA tissues, compared to adjacent non-tumor tissue. There was a positive correlation between the AdipoR1 and p-AMPKα levels in CCA tissues. Treatment with AdipoRon inhibited proliferation, migration, invasion and colony formation and induced apoptosis in a time- and dose-dependent manner in vitro (p<0.05). In addition, AdipoRon reduced the number of CCA and tumor volume, prolonged survival, and decreased metastasis and ascites in the treated group compared to the control group (p<0.05). AdipoR1 and p-AMPKα are impaired in CCA tissues, and AdipoRon effectively inhibits CCA in vitro and in vivo. Thus, AdipoRon may be considered as a potential anti-tumor therapy in CCA.

Huaier inhibits cholangiocarcinoma cells through the twist1/FBP1/Wnt/β-catenin axis.

Although Huaier granules can be used as prospective anti-cholangiocarcinoma drugs, the mechanism of action of Huaier granules in cholangiocarcinoma is not clear. The anti-cholangiocarcinoma effect of Huaier granules was validated in cell line research. In vitro experiments were conducted to investigate the signalling pathways affected by Huaier in CCA cells. Real-time quantitative PCR (RT‒qPCR) and Western blot analysis were performed to analyse gene expression in CCA cells. MTT assays, scratch tests, and Transwell assays were used to explore the effects on the proliferation and metastasis of CCA cells. Chromatin immunoprecipitation assays were performed to reveal the potential underlying mechanisms involved. Twist1 was upregulated in human CCA tissues. In addition, its expression levels were negatively related to FBP1 expression levels. Mechanistically, Twist1 can bind to the region of the FBP1 promoter to reduce its expression. Huaier plays an indispensable role in suppressing Twist1 expression to inhibit the Twist1/FBP1/Wnt/β-catenin axis. Then, we verified the effect of Huaier in vitro. These findings suggested that Huaier granules were capable of inhibiting CCA development through regulating the Twist1/FBP1/Wnt/β-catenin signalling axis and provided a novel orientation for the development of novel anti-CCA drugs.

Probe-guided endoscopic system for 5-aminolevulinic acid-based photodynamic diagnosis in cholangiocarcinoma

Background and AimThe diagnostic accuracy for cholangiocarcinoma (CCA) is inadequate, necessitating the exploration of novel diagnostic approaches. Protoporphyrin IX (Pp IX), a metabolic product of 5-aminolevulinic acid (5-ALA), emits red fluorescence upon blue light exposure. Because it accumulates selectively in cancer cells, photodynamic diagnosis using 5-ALA (5-ALA-PDD) has been integrated into clinical practice for diverse cancer types. Nevertheless, there is currently no device capable of capturing Pp IX-derived fluorescence for real-time 5-ALA-PDD within the biliary tract, largely due to challenges in device miniaturization. MethodsTo investigate the feasibility of real-time 5ALA-PDD in CCA, we developed two essential components of the cholangioscopy system: a small-diameter flexible camera and a light guide for emitting blue light. We evaluated the detectability of Pp IX fluorescence using these devices in experimental gels and animal models. ResultsOur camera and light guide were smoothly inserted into the lumen of existing cholangioscopes. Incorporating a long-pass filter at the camera tip enabled efficient detection of red fluorescence without significantly impacting white-light observation. The integration of these devices facilitated clear visualization of red fluorescence from gels containing Pp IX at concentrations of 5 μM or higher. Additionally, when observing subcutaneous human CCA tumor models in nude mice treated with 5-ALA, we successfully demonstrated distinct red fluorescence from Pp IX accumulation in tumors compared to peritumoral subcutaneous areas. ConclusionThe integration of our device combination holds promise for real-time 5-ALA-PDD in human CCA, potentially enhancing the diagnostic accuracy for this complex condition.

Transcription factor EHF drives cholangiocarcinoma development through transcriptional activation of glioma-associated oncogene homolog 1 and chemokine CCL2.

Cholangiocarcinoma (CCA) is characterized by rapid onset and high chance of metastasis. Therefore, identification of novel therapeutic targets is imperative. E26 transformation-specific homologous factor (EHF), a member of the E26 transformation-specific transcription factor family, plays a pivotal role in epithelial cell differentiation and cancer progression. However, its precise role in CCA remains unclear. In this study, through in vitro and in vivo experiments, we demonstrated that EHF plays a profound role in promoting CCA by transcriptional activation of glioma-associated oncogene homolog 1 (GLI1). Moreover, EHF significantly recruited and activated tumor-associated macrophages (TAMs) through the C-C motif chemokine 2/C-C chemokine receptor type 2 (CCL2/CCR2) axis, thereby remodeling the tumor microenvironment. In human CCA tissues, EHF expression was positively correlated with GLI1 and CCL2 expression, and patients with co-expression of EHF/GLI1 or EHF/CCL2 had the most adverse prognosis. Furthermore, the combination of the GLI1 inhibitor, GANT58, and CCR2 inhibitor, INCB3344, substantially reduced the occurrence of EHF-mediated CCA. In summary, our findings suggest that EHF is a potential prognostic biomarker for patients with CCA, while also advocating the therapeutic approach of combined targeting of GLI1 and CCL2/CCR2-TAMs to inhibit EHF-driven CCA development.

The deubiquitinase USP5 promotes cholangiocarcinoma progression by stabilizing YBX1

AimsUbiquitin specific peptidase 5 (USP5), a member of deubiquitinating enzymes, has garnered significant attention for its crucial role in cancer progression. This study aims to explore the role of USP5 and its potential molecular mechanisms in cholangiocarcinoma (CCA). Main methodsTo explore the effect of USP5 on CCA, gain-of-function and loss-of-function assays were conducted in human CCA cell lines RBE and HCCC9810. The CCK8, colony-forming assay, EDU, flow cytometry, transwell assay and xenografts were used to assess cell proliferation, migration and tumorigenesis. Western blot and immunohistochemistry were performed to measure the expression of related proteins. Immunoprecipitation and immunofluorescence were applied to identify the interaction between USP5 and Y box-binding protein 1 (YBX1). Ubiquitination assays and cycloheximide chase assays were carried out to confirm the effect of USP5 on YBX1. Key findingsWe found USP5 is highly expressed in CCA tissues, and upregulated USP5 is required for the cancer progression. Knockdown of USP5 inhibited cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro, along with suppressed xenograft tumor growth and metastasis in vivo. Mechanistically, USP5 could interact with YBX1 and stabilize YBX1 by deubiquitination in CCA cells. Additionally, silencing of USP5 hindered the phosphorylation of YBX1 at serine 102 and its subsequent translocation to the nucleus. Notably, the effect induced by USP5 overexpression in CCA cells was reversed by YBX1 silencing. SignificanceOur findings reveal that USP5 is required for cell proliferation, migration and EMT in CCA by stabilizing YBX1, suggesting USP5-YBX1 axis as a promising therapeutic target for CCA.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling

BackgroundCholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy.MethodsThe expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA.ResultsWe identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth.ConclusionsThis study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.

Cannabidiol suppresses proliferation and induces cell death, autophagy and senescence in human cholangiocarcinoma cells via the PI3K/AKT/mTOR pathway

Background and aimCholangiocarcinoma (CCA) is usually diagnosed at a late stage, leading to treatment failure. Cannabidiol (CBD), exhibits diverse anti-cancer effects in various cancers, offering avenues for improving CCA treatment. This study investigated the effects of CBD on human CCA cells and the underlying mechanisms in vitro and in vivo. Experimental procedureThe effects of CBD on three CCA cell lines (KKU-213B, KKU-100, KKU-055) were assessed using the SRB assay, clonogenic assay, cell cycle arrest, and 3D holotomography. Morphological changes were examined using transmission electron microscopy, while mitochondrial ROS levels and mitochondrial membrane potential were studied using MitoSOX, JC-1, and DCFH-DA. Cellular senescence induction was evaluated via SA-β-gal staining. Protein associatedwith autophagy and cellular senescence were analyzed using Western blot and/or immunofluorescent assays. A xenograft model demonstrated the anti-tumor activity of CBD and the induction of cellular senescence through immunohistochemistry targeting PCNA, β-gal, and p21. Results and conclusionCBD effectively inhibited CCA cell proliferation, suppressed colony formation and induced G0/G1 phase cell cycle arrest. Morphological examination revealed lipid droplets/vesicles in CCA cell lines. CBD induced autophagy by upregulating LC3BII, downregulating p62, and inhibiting the p-PI3K, p-AKT, and p-mTOR pathways. Additionally, CBD disrupted mitochondrial homeostasis by elevating ROS, reducing membrane potential, and induced cellular senescence by increasing the expression of p53 and p21. In-vitro results were confirmed by xenograft models. Overall, CBD suppresses proliferation and induces cell death, autophagy and senescence in CCA cells via the PI3K/AKT/mTOR pathway, which indicates a therapeutic option for CCA treatment.

Abstract 440: BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis

Abstract Background: Cholangiocarcinoma (CCA) is a highly lethal and aggressive epithelial cell malignancy of the liver and biliary tract. CCA survival remains poor and response to chemotherapy or immunotherapy is limited. We AIM to identify metabolic vulnerabilities that render CCA susceptible to ferroptosis, an iron-dependent and caspase-independent cell death driven by lipid peroxidation. Materials and Methods: We treated human and mouse CCA cell lines with ferroptosis inducers (RSL3, Erastin), inhibitors (Fer-1, Lip-1), necroptosis inhibitor and a pan-caspase inhibitor. Cell viability, intracellular and mitochondrial iron, lipid peroxidation, mitochondrial structure, and function by cell-based and biochemical assays. Gain- and loss-of-function strategies, TURBO proximity-dependent biotin identification, and metabolic techniques were used for mechanistic studies. In vivo, orthotopically implanted C57B/6 mice were treated with RSL3 or vehicle intraperitoneally, followed by assessment of tumor burden and immunophenotyping analysis. Epcam-aptamer coated nanoparticles (tMNPs) packaged with siRNA against GPX4 were used for selective ferroptosis induction. Results: We identified ferroptosis-sensitive and -resistant human and mouse CCA cells. Ferroptosis-sensitive cells, rescued by ferroptosis inhibitors but not by necroptosis or pan-caspase inhibitors, exhibited elevated intracellular free iron and mitochondrial iron overload. This led to significant mitochondrial dysfunction and structural changes associated with ferroptosis. Mitochondrial free iron triggered reactive oxygen species generation and lipid peroxidation. We identified BRCA-associated protein 1 (BAP1) as a potential driver of ferroptosis sensitivity. BAP1, mutated in 22-25% of human CCAs, is a tumor suppressor and deubiquitinase (DUB). BAP1 knockdown by siRNA protected cells from ferroptosis, while re-expression of BAP1 sensitized cells to ferroptosis. Subcellular fractionation identified BAP1 in extranuclear compartments, including mitochondria and cytosol. We found that BAP1 interacts and deubiquitinates ATP citrate lyase (ACLY), which converts iron chelator citrate to oxaloacetate and acetyl-CoA. Inhibition of mitochondrial citrate export, protected cells from iron overload and ferroptosis. In vivo, RSL3-induced ferroptosis significantly reduced tumor burden, as compared to vehicle-treated mice. RSL3-induced ferroptosis promoted an immunogenic tumor immune microenvironment. Lastly, tMNPs with siGPX4 induced marked ferroptosis of mouse CCA cells and showed maximal enrichment in liver orthotopic tumors in vivo. Conclusions: Our findings indicate that BAP1 deubiquitination of ACLY predisposes CCA cells to iron overload by exhausting citrate to support lipogenesis. CCA vulnerability to ferroptosis opens up novel therapeutic opportunities for CCA. Citation Format: Peyton Classon, Sophia Jaramillo, Danielle Carlson, Irene Yan, Tushar Patel, Sumera I. Ilyas, Rory L. Smoot, Gregory J. Gores, Davide Povero. BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 440.

Abstract 6819: Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma

Abstract Intrahepatic cholangiocarcinoma (CCA) is an aggressive biliary tract cancer that carries unfavorable prognosis. 10-20% of intrahepatic CCAs harbor a mutation in IDH1 that can be targeted with mutant IDH1-specific inhibitors. However, objective and durable responses to treatment with mutant IDH1 inhibitors are rare in CCA. Mutant IDH1 has neomorphic enzymatic activity that produces the oncometabolite D-2-hydroxyglutarate (D-2-HG). D-2-HG promotes biliary tumor formation through cancer cell-intrinsic mechanisms but is also a paracrine factor in the tumor microenvironment (TME). Our group and others have identified that IDH1-mutant CCAs are not responsive to immunotherapy treatment approaches in part due to decreased CD8+ T cell infiltration and an increase in immunosuppressive macrophage cells in the TME. To better understand the immunosuppressive features of the IDH1-mutant CCA TME, we generated an isogenic cell line panel of IDH1-mutant (MUT) and IDH1-wild type (WT) mouse cholangiocarcinoma cells in the SB1 cell line background through a CRISPR homology directed repair approach. Compared to IDH1-WT cells, IDH1-MUT cells produce high levels of D-2-HG and both cell line panels readily form tumors in immunocompetent C57BL/6 mice. Tumors formed from IDH1-mut cells have reduced CD8+ T cell infiltration compared to IDH1-WT tumors, a finding that correlates with analysis of human CCA samples. Using cytometry by time of flight (CyTOF), we identified that CD8+ T cells in IDH1-MUT tumors have decreased expression of key regulators of the antitumor immune response including EOMES, TBET, and GZMB compared to those isolated from IDH1-WT tumors. Analysis of secreted factors from these IDH1-MUT and IDH1-WT cells identified that IDH1-MUT cells release increased levels of CCL2, a chemokine shown to diminish the antitumor immune response across multiple cancer types, compared to IDH1-WT cells. Further, IDH1-MUT tumors grown in syngeneic, immunocompetent mice have increased CCL2 staining by IHC compared to IDH1-WT tumors. CCL2 expression is also increased in a preliminary analysis of IDH1-mutant human CCA primary tumors. Treatment of mice harboring orthotopic liver tumors formed from IDH1-MUT cells with a CCL2 neutralizing antibody resulted in decreased tumor size compared to isotype control. Together, our data suggest that CCL2 is a mediator of immunosuppression in the IDH1-mutant CCA TME and that modulation of CCL2 activity may improve outcomes in this rare disease subtype. Further work to understand the mechanisms through which IDH1 mutations regulate CCL2 levels in these tumors is underway. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Kiyoko Yoshima, Daniel J. Zabransky, Mark Yarchoan. Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6819.

Abstract 2098: A novel GCN2 kinase activator demonstrates therapeutic efficacy in preclinical PDX models of human cholangiocarcinoma

Abstract Introduction Cholangiocarcinoma (CCA) is a highly lethal, heterogenous biliary malignancy. Despite the development of targeted therapies, treatment success has been hindered by primary or acquired resistance. New therapeutic strategies are necessary to improve outcomes in these patients. NXP800 is a GCN2 small-molecule activator that phosphorylates eIF2a inducing selective ATF4 transcription which stimulates stress-induced genes leading to apoptosis. NXP800 has been previously validated in ovarian cancers with ARID1A mutations, well-known players in cholangiocarcinoma. We sought to determine the treatment efficacy of novel NXP800 in CCA utilizing patient derived xenograft (PDX) preclinical models. Methods In our preliminary studies, four human and two murine CCA cell lines were tested for cell viability using CellTiter-Glo to determine the maximal inhibitory concentration (IC50). Immunoblot analysis of HuCCT-1 cells incubated with 1 mM of NXP800 for 6 hours vs. vehicle was performed. We also evaluated response to GCN2 activation in multiple RNA sequenced PDX models. Xenografts were expanded into the flank of NOD/SCID mice and treatment begun when tumors averaged 120mm3. Tumor bearing mice were treated with NXP800 (35 mg/kg) or vehicle via oral gavage five days on, two days off, for 28 days. Tumor volume and animal weight were collected twice weekly. Predicted sensitive signatures were determined using multi-omics. Results IC50 dose response curves showed nanomolar activity in all six cell lines, ranging from 5-170 nM. Immunoblotting demonstrated an upregulation of phosphorylated eIF2a and in turn upregulation of ATF4 with treatment of NXP800 compared to vehicle. Five PDX tumors from patients with intrahepatic or distal CCA was established and validated by histologic analysis. Molecular characterization of the tumor identified tumor mutation and a total tumor mutational burden. RNA sequencing was completed and identified 21-gene signatures related to YAP activity, which were cross-referenced against our existing PDX model tumor bank. Treatment with NXP800 was associated with a statistically significant decrease in tumor size in three of five PDX models tested, which included an ARID1A, FGFR1, and ATM mutants. Preliminary multi-omics on the PDX models revealed that sensitivity mechanisms were centered around EGFR signaling. Conclusion The novel GCN2 kinase activator, NXP800, has nanomolar efficacy in both human and murine CCA cell lines in vitro. Additionally, NXP800 demonstrated therapeutic activity in multiple cholangiocarcinoma PDX models in vivo. We are opening a Phase 1b clinical trial investigating the effects of NXP800 use in patients with advanced CCA. Future studies determining the cellular effect of GCN2 activation utilizing NXP800 are being conducted. Citation Format: Danielle Marie Carlson, Hendrien Kuipers, Amro Abdelrahman, Erik Jessen, Joshua Roldan Kalil, Jack Sample, Jennifer Tomlinson, Mark Truty, Rory Smoot. A novel GCN2 kinase activator demonstrates therapeutic efficacy in preclinical PDX models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2098.

Abstract 5753: Milk-derived extracellular nanovesicles: A novel delivery strategy for transcriptome targeting therapies in cholangiocarcinoma

Abstract Introduction: Cholangiocarcinoma (CCA) is an aggressive and heterogenous biliary malignancy. Previous therapeutic shortcomings have encouraged the investigation of new strategies, such as targeted therapeutics. YAP/TAZ, transcriptional coactivators of the hippo pathway, have been implicated in the tumorigenesis of CCA along with the Src familiar kinase, LCK. Milk-derived nanovesicles (MNVs) have emerged as a promising biologic delivery apparatus for hepatobiliary malignancy due to their predilection for hepatic uptake. We sought to investigate the utility of aptamer guided MNVs loaded with short interfering RNA (siRNA) targeting YAP, TAZ, and LCK. Methods: MNVs were prepared and loaded with species-specific siRNA prior to decoration with an epithelial cellular adhesion molecule (EpCAM) targeting aptamer. In vitro studies were performed using murine and human CCA cell lines, SB1 and HuCCT-1, respectively. In vivo studies were carried out using C57BL/6 mice following Institutional Animal Care and Use Committee protocols. Results: We previously validated EpCAM as an effective aptamer demonstrated by increased levels of expression in CCA cell lines using immunoblot, RT-PCR and immunofluorescence. Additionally, in vivo experiments demonstrated excellent biodistribution and affinity for tumor-selective uptake. To evaluate knockdown efficacy, CCA cell lines were incubated with EpCAM aptamer guided MNVs individually targeting YAP, TAZ, and LCK which resulted in downregulation of these targets on immunoblot and RT-PCR. MNVs were then loaded with equal concentrations of siRNA targeting YAP, TAZ, and LCK in combination which resulted in similar degrees of gene downregulation compared with single transcriptome targeting MNVs. Due to the role of YAP/TAZ activation in CCA systemic therapy resistance, we investigated the effect of MNVs loaded with either single target or combinatory target siRNA alongside and in combination with gemcitabine and cisplatin (GemCis). Incubation with both combinatory siRNA and GemCis significantly intensified cell death on Pi/Hoechst staining compared with single target knockdown alone. Similar findings were found using separate assays of cell viability, including ATP luminescent quantitation and caspase 3/7 activity. These data suggest MNV transcriptome targeting may induce a state of cell stress which allows for amplification of chemotherapy treatment effect. Conclusion: Aptamer guided, nanovesicle mediated transcriptome targeting is a promising therapeutic strategy in preclinical CCA models that may potentiate chemotherapy response. Our future direction involves further investigating the role of CCA target downregulation and its effect on the tumor immune microenvironment utilizing aptamer guided nanovesicle delivery and inducible knockdown cell lines. Citation Format: Jack W. Sample, Mincheng Yu, Hendrien Kuipers, Danielle M. Carlson, Nathan W. Werneberg, Jennifer L. Tomlinson, Gregory J. Gores, Rory L. Smoot. Milk-derived extracellular nanovesicles: A novel delivery strategy for transcriptome targeting therapies in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5753.

Abstract 614: Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA) is a lethal and heterogenous malignancy of the biliary tree characterized by perineural invasion. About 85% of CCA show activation of YAP signaling, which promotes proliferation, anti-apoptosis, and therapeutic resistance. YAP signaling is mediated by Src family kinases (SFK) by phosphorylation of YAP (pYAPy357). NXP900 is a first-in-class, highly selective, SFK inhibitor with a novel mechanism of action which locks SFK in its closed and inhibited conformation, in contrast to other Src inhibitors such as dasatinib. A phase 1a study of NXP900 in patients with advanced solid tumors was recently initiated. Here, we examined treatment responses of CCA to NXP900 in vitro and in vivo. Methods: Cell viability was determined in seven CCA cell lines by CellTiter-Glo. Cell death and apoptosis were assayed by PI/Hoechst and Caspase-Glo 3/7 assay. CalcuSyn software was used to determine synergistic drug effects. Immunoblot analysis, RT-PCR, and IF staining of YAP localization were used to evaluate YAP knockdown. Clones of human CCA cell lines resistant to NXP900 were generated through escalating exposure to NXP900 over 6 months. Five patient-derived xenograft (PDX) tumors were expanded into flanks of NOD/SCID mice. Tumor bearing mice were randomized 1:1 with 5 mice per arm and treated with vehicle or NXP900 (40 mg/kg) once daily for up to 4 weeks. To determine drivers of sensitivity and resistance, predicted sensitivity scores based on tumor growth were linked to multi-omics (RNA seq and [phospho-]proteomics) of our PDX models. Results: All cell lines were sensitive to NXP900 with IC50 values between 7nM-15µM; IC50’s of resistant clones were 1000 times higher. NXP900 induced more cell death compared to vehicle, which appeared to be through apoptosis. NXP900 inhibited pSrc, decreased pYAPy357, and upregulated inactive pYAPs127. Correspondingly, decreased YAP target gene (Cyr61, NUAK, and CTGF) levels and a nuclear-to-cytoplasmic translocation were observed. NXP900/GemCis combination therapy increased cell death demonstrated a synergistic effect (Combination Indices &amp;lt;1) at all concentrations. In our PDX models, treatment was associated with a significant decrease in tumor growth in 3 models (mean fold change 3.1 vs. 14.2; 1.0 vs. 7.6; 1.4 vs. 2.6). The major resistant signature was the TRK signaling network, involved in nerve growth factor binding activity, while drivers of sensitivity included the SRC network. Conclusion: NXP900 demonstrated therapeutic activity in vitro and in human PDX models. We are currently performing multi-omic approaches in NXP900 sensitive and resistant cell lines to unravel determinants of activity and resistance. Additional in vivo studies will be performed to determine effects of NXP900/GemCis and NXP900/anti-PDL-1 combination therapy. Citation Format: Hendrien Kuipers, Jennifer L. Tomlinson, Danielle M. Carlson, Amro M. Abdelrahman, Erik Jessen, Jack W. Sample, Nathan W. Werneburg, Hannah E. Stumpf, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 614.

Syngeneic murine models with distinct immune microenvironments represent subsets of human intrahepatic cholangiocarcinoma

Cholangiocarcinoma (CCA) is a poorly immunogenic malignancy associated with limited survival. Syngeneic immunocompetent mouse models of CCA are an essential tool to elucidate the tumor immune microenvironment (TIME), understand mechanisms of tumor immune evasion, and test novel immunotherapeutic strategies. The scope of this study was to develop and characterize immunocompetent CCA models with distinct genetic drivers, and correlate tumor genomics, immunobiology, and therapeutic response. A multifaceted approach including scRNA-seq, CITE-seq, whole exome and bulk RNA sequencing was employed. FDA-approved PD-1/PD-L1 antibodies were tested in humanized PD-1/PD-L1 mice (HuPD-H1). A genetic mouse model of intrahepatic CCA (iCCA) driven by intrabiliary transduction of Fbxw7ΔF/Akt that mimics human iCCA was generated. From the Fbxw7ΔF/Akt tumors, a murine cell line (FAC) and syngeneic model with genetic and phenotypic characteristics of human iCCA were developed. Established SB1 (YAPS127A/Akt) and KPPC (KrasG12Dp53L/L) models were compared to the FAC model. Although the models had transcriptomic similarities, they had substantial differences as well. Mutation patterns of FAC, SB1, and KPPC cells matched different mutational signatures in Western and Japanese CCA patient cohorts. KPPC tumors had a high tumor mutation burden. FAC tumors had a T cell-infiltrated TIME, while SB1 tumors had apreponderance of suppressive myeloid cells. FAC, SB1, and KPPC tumors matched different immune signatures in human iCCA cohorts. Moreover, FAC, SB1, and KPPC tumor-bearing HuPD-H1 mice displayed differential responses to nivolumab or durvalumab. Syngeneic iCCA models display a correlation between tumor genotype and TIME phenotype, with differential responses to FDA-approved immunotherapies. This study underscores the importance of leveraging multiple preclinical models to understand responses to immunotherapy in different genetic subsets of human CCA. Understanding the relationship between tumor genotype and the phenotype of the immune microenvironment is an unmet need in cholangiocarcinoma (CCA). Herein, we use syngeneic murine models of intrahepatic CCA with different genetic drivers to demonstrate a correlation between tumor genotype and immune microenvironment phenotype in murine models, which is associated with differential responses to FDA-approved immunotherapies. This information will help guide other preclinical studies. Additionally, it emphasizes that immune checkpoint inhibition in patients with CCA is not a "one-size-fits-all" approach. Our observations suggest that, as for targeted therapies, patients should be stratified and selected for treatment according to their tumor genetics.

High expression of autotaxin is associated with poor recurrence-free survival in cholangiocarcinoma.

Autotaxin (ATX) is an extracellular lysophospholipase D that catalyzes the hydrolysis of lysophosphatidylcholine into lysophosphatidic acid (LPA). Recent accumulating evidence indicates the biological roles of ATX in malignant tumors. However, the expression and clinical implications of ATX in human cholangiocarcinoma (CCA) remain elusive. In this study, the expression of ATX in 97 human CCA tissues was evaluated by immunohistochemistry. Serum ATX levels were determined in CCA patients (n=26) and healthy subjects (n=8). Autotaxin expression in cell types within the tumor microenvironment was characterized by immunofluorescence staining. High ATX expression in CCA tissue was significantly associated with a higher frequency of lymph node metastasis (p=0.050). High ATX expression was correlated with shorter overall survival (p=0.032) and recurrence-free survival (RFS) (p=0.001) than low ATX expression. In multivariate Cox analysis, high ATX expression (p=0.019) was an independent factor for shorter RFS. Compared with low ATX expression, high ATX expression was significantly associated with higher Ki-67-positive cell counts (p<0.001). Serum ATX levels were significantly higher in male CCA patients than in healthy male subjects (p=0.030). In the tumor microenvironment of CCA, ATX protein was predominantly expressed in tumor cells, cancer-associated fibroblasts, plasma cells, and biliary epithelial cells. Our study highlights the clinical evidence and independent prognostic value of ATX in human CCA.

The importance of preclinical models in cholangiocarcinoma

Cholangiocarcinoma (CCA) is an adenocarcinoma of the hepatobiliary system with a grim prognosis. Incidence is rising globally and surgery is currently the only curative treatment, but is only available for patients who are fit and diagnosed in an early-stage of disease progression. Great importance has been placed on developing preclinical models to help further our understanding of CCA and potential treatments to improve therapeutic outcomes. Preclinical models of varying complexity and cost have been established, ranging from more simplistic in vitro 2D CCA cell lines in culture, to more complex in vivo genetically engineered mouse models. Currently there is no single model that faithfully recaptures the complexities of human CCA and the in vivo tumour microenvironment. Instead a multi-model approach should be used when designing preclinical trials to study CCA and potential therapies.

Role of Wisteria floribunda agglutinin binding glycans in carcinogenesis and metastasis of cholangiocarcinoma.

Aberrant glycosylation is an important factor in facilitating tumor progression and therapeutic resistance. In this study, using Wisteria floribunda agglutinin (WFA), we examined the expression of WFA-binding glycans (WFAG) in cholangiocarcinoma (CCA). The results showed that WFAG was highly detected in precancerous and cancerous lesions of human CCA tissues, although it was rarely detected in normal bile ducts. The positive signal of WFAG in the cancerous lesion accounted for 96.2% (50/52) of the cases. Overexpression of WFAG was significantly associated with lymph node and distant metastasis (P < 0.05). The study using the CCA hamster model showed that WFAG is elevated in preneoplastic and neoplastic bile ducts as early as 1month after being infected with liver fluke and exposed to N-nitrosodimethylamine. Functional analysis was performed to reveal the role of WFAG in CCA. The CCA cell lines KKU-213A and KKU-213B were treated with WFA, followed by migration assay. Our data suggested that WFAG facilitates the migration of CCA cells via the activation of the Akt and ERK signaling pathways. In conclusion, we have demonstrated the association of WFAG with carcinogenesis and metastasis of CCA, suggesting its potential as a target for the treatment of the disease.