Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a 5-year survival rate of 12%. It has two major molecular subtypes: classical and basal, regulated by the master transcription factors (MTFs) GATA6 and ΔNp63, respectively. Objective This study sought to uncover the transcriptional regulatory mechanisms controlling PDAC subtype identity. Design We integrated primary tumour single-cell RNA-seq, patient-derived xenograft RNA-seq and multispectral imaging to identify MTF-dependent, subtype-specific markers. We created subtype-specific fluorescent reporter systems and conducted drug screenings to find actionable targets. We analysed chromatin accessibility (ATAC-seq), genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac), MTFs (GATA6, ΔNp63), RNA polymerase II (Pol II), H3K4me3-anchored chromatin topology (HiChIP) and nascent RNA capture sequencing (PRO-seq). Additionally, we used nuclease-dead Cas9 (dCas9) to manipulate transcriptional regulatory mechanisms. Results Our approach identified glucocorticoid receptor (GR) agonists as agents that suppress the classical transcriptional programme by interacting with GATA6. GATA6 regulates classical-specific transcription through promoter-proximal pause release. Depletion of GATA6 increased Pol II occupancy at GATA6-bound enhancers and transcriptional start sites, stabilising enhancer–promoter interactions. Artificially inducing pausing at GATA6-bound enhancers with dCas9 abrogated target gene expression and induced pausing at both the enhancer and target gene promoter. Conversely, in basal PDAC ΔNp63 promotes Pol II recruitment and stabilises enhancer–promoter interactions. Conclusion This study provides new insights into the transcriptional control and role of GR agonists in controlling PDAC molecular subtype identity.

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 in CCA treatment, we investigated cell-targeted nanovesicles as a delivery platform for transcriptome-targeting therapeutics. Approach & Results: Milk-derived nanovesicles (MNVs) were loaded with short interfering RNAs targeting 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 (tMNVs) were decorated with a lipid-coupled RNA aptamer to epithelial cell adhesion molecule (EpCAM), including a tracking fluorophore. In vitro studies were conducted using multiple CCA cell lines. In vivo studies were performed using C57BL/6 and NOD/SCID mice to evaluate delivery and efficacy in both an immunocompetent syngeneic murine and a patient-derived xenograft (PDX) model. We demonstrated that tMNVs were selectively taken up by liver tumor cells, which was augmented by the incorporation of a targeting aptamer, and that MNVs loaded with siRNA 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 tMNVs targeting YAP, LCK, and tafazzin inhibited CCA growth and further synergized with chemotherapy in two preclinical CCA models. Conclusions: Herein, we show that aptamer-directed, nanovesicle-mediated targeting of YAP, LCK, and tafazzin potentiates chemosensitivity in two CCA models when delivered using aptamer-guided milk-derived nanovesicles.

Although EPHA2 is a receptor tyrosine kinase widely expressed in many cancers, it exhibits a uniquely high frequency of coding sequence mutations in cholangiocarcinoma, a cancer of the biliary tract with dismal prognosis. EPHA2 is extensively studied, but very little is known about the role of EPHA2 cancer mutations. To define the functional significance of EPHA2 mutations in biliary tract cancers, we generated representative EPHA2 mutants and monitored major receptor autophosphorylation sites as indicators of kinase activity-dependent signal transduction (known as forward signaling). We found that missense mutations in the ligand-binding domain abrogate ephrinA ligand binding, while missense mutations in the kinase domain abrogate kinase activity. The effects of missense mutations in other domains were less pronounced and varied depending on the phosphosite. The majority of the EPHA2 mutations are nonsense or frame-shift mutations that introduce early stop codons. They generate EPHA2 truncated forms that lack an intact kinase domain or, in some cases, most of the coding sequence. Several EPHA2 mutants tested inhibited tyrosine phosphorylation of co-expressed EPHA2 wild-type, indicating the ability to exert dominant negative effects. We show that EPHA2 forward signaling in cholangiocytes inhibits the ERK oncogenic pathway and cell proliferation, suggesting that loss-of-function mutations facilitate tumor development in the biliary tract. Indeed, an EPHA2 kinase-inactive mutant, but not EPHA2 wild-type, induced proliferative masses consistent with well differentiated cholangiocarcinoma in a validated mouse model of cholangiocarcinogenesis. Thus, EPHA2 has the attributes of a driver gene with tumor suppressor activity in biliary tract cancers.

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.

Background & Aims: 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. Methods: 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). Results: A genetic mouse model of intrahepatic CCA (iCCA) driven by intrabiliary transduction of Fbxw7 D F/ Akt that mimics human iCCA was generated. From the Fbxw7 D F/ Akt tumors, a murine cell line (FAC) and syngeneic model with genetic and phenotypic characteristics of human iCCA were developed. Established SB1 ( YAP S127A / Akt ) and KPPC ( Kras G12D p53 L/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 a preponderance 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. Conclusions: 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. (c) 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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 Background Cholangiocarcinoma (CCA) is a heterogeneous malignancy. Platinum-based chemotherapy (Gemcitabine and Cisplatin) has been the standard treatment for over a decade and is the base for the current immunotherapy combinations. Response rates are typically between 20-30%. Herein, we present outcomes of preclinical efficacy trials on CCA PDX models in correlation with baseline multiomics profiling of CCA patient-derived xenograft (PDX) models to identify molecular features associated with response. Methods Tumor samples from twenty-eight CCA PDX were used to isolate RNA and protein to be included in multiomics analysis (Phosphoproteomics, Proteomics, and Transcriptomics). A subset of PDX models were treated with various doses of Gemcitabine and Cisplatin. The Median Efficacy Index (MEI) was calculated as the ratio of the difference in median of delta in tumor volume at the last day of the study between the treatment and the control arms divided by the median of delta in tumor volume of the control at the last day of the treatment (positive= growth, negative=inhibition, and 0=no efficacy). A Hierarchical All-Against-All algorithm was utilized to compare multiomics features to the MEI and pathway analysis completed for identified molecular features. Results After conducting preclinical efficacy trials, the five tested CCA PDX models were classified into sensitive, moderately sensitive, and resistant models based on the MEI on each treatment arm. The top up-regulated genes were identified for Gemcitabine response (SRPK2, GLK2, and RIOK2) and Cisplatin response (NTRK1, PRPF4B, and SRPK2). The top up-regulated genes related to Gemcitabine resistance were NTRK1, PIP5K1C, and CSNK2A2, while those related to Cisplatin resistance were SRPK1, PRPF4B, and CSNK2A1. Conclusion Cholangiocarcinoma multiomics signatures can predict therapeutic response to standard-of-care chemotherapy in preclinical models. Novel mechanisms of Gemcitabine and Cisplatin resistance were identified related to NTRK1, SRPK1 and SRPK2. Resistant signatures will be challenged with the appropriate targeted therapies on the resistant CCA PDX models. Citation Format: Amro M. Abdelrahman, Danielle M. Carlson, Erik Jessen, Dong-Gi Mun, Isaac Lynch, Alessandro Fogliati, Stella Konadu Adjei Antwi, Aushinie Abeynayake, Akhilesh Pandey, Mark J. Truty, Gregory J. Gores, Rory L. Smoot. Pretreatment multiomics in conjunction with preclinical trials on patient-derived xenograft models of cholangiocarcinoma can identify chemotherapy response signatures [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 6912.

BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal malignancy. Surgical resection is the only curative modality combined with neoadjuvant chemotherapy to improve survival. Given the limitations of traditional responses such as cross-sectional imaging (CT/MRI) or tumor markers, carbohydrate antigen 19-9 (CA19-9), the 2023 National Comprehensive Cancer Network guidelines included 18 F-fluorodeoxyglucose (FDG)-PET as an adjunct to assess response to neoadjuvant chemotherapy. There are common misconceptions on the metabolic activity (tumor avidity) in PDAC so we aimed to describe the baseline characteristics and use of FDG-PET in a cohort of treatment-naive patients with PDAC.STUDY DESIGN:A single-center retrospective study was conducted capturing all biopsy-proven, treatment-naive patients with PDAC who underwent either baseline FDG-PET/CT or FDG-PET/MRI imaging between 2008 and 2023. Baseline FDG-PET characteristics were collected, including primary tumors' maximum standardized uptake value defined as metabolic activity (FDG uptake) of tumor compared with surrounding pancreatic parenchymal background, and the identification of extrapancreatic metastatic disease.RESULTS:We identified 1,095 treatment-naive patients with PDAC who underwent baseline FDG-PET imaging at diagnosis. CA19-9 was elevated in 76% of patients. Overall, 96.3% (1,054) of patients had FDG-avid tumors with a median maximum standardized uptake value of 6.4. FDG-PET also identified suspicious extrapancreatic metastatic lesions in 50% of patients, with a higher proportion (p < 0.001) in PET/MRI (59.9%) vs PET/CT (44.3%). After controlling for CA19-9 elevation, PET/MRI was superior in detection of extrapancreatic lesions compared with PET/CT.CONCLUSIONS:FDG-PET has significant use in PDAC as a baseline imaging modality earlier neoadjuvant therapy given the majority of tumors are FDG-avid. FDG-PET can identify additional extrapancreatic suspicious lesions allowing for optimal initial staging, with PET/MRI having increased sensitivity over PET/CT.

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 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 <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.

Introduction: Cholangiocarcinoma (CCA) is a highly lethal, and biologically heterogenous malignancy arising from the biliary tree. With only moderate responses to cytotoxic chemotherapy and 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 heat shock factor 1 (HSF1) small-molecule inhibitor with activity previously noted in ovarian cancers with ARID1A mutations. ARID1A mutations are common, well-known players in cholangiocarcinoma. In preclinical exploratory studies, we examined treatment responses in validated cholangiocarcinoma patient derived xenograft (PDX) models. Methods: In our preliminary studies we evaluated response to HSF1 inhibition in a single, ARID1A wildtype PDX model. Xenografts were expanded into the flank of NOD/SCID mice and treatment begun when tumors averaged 120mm3. 16 tumor bearing mice were randomized and 8 mice were grouped into both the treatment and control arms. Mice were then treated with either NXP800 (35 mg/kg), in the treatment arm, or vehicle, in the control arm, via oral gavage 5 days on, 2 days off, for 28 days. Tumor volume and animal weight were collected twice weekly. Results: After screening of our Human CCA PDX library of 57 patients, we identified an established and histologically validated PDX from a patient with intrahepatic cholangiocarcinoma obtained at surgical resection. Molecular characterization of the tumor identified NRAS Q61 mutation, FGFR1 and FGFR2 overexpression, androgen receptor overexpression, microsatellite stability, and a total tumor mutational burden of 2.6. RNA sequencing was completed and an evaluation of a 21-gene signature of YAP activity had identified this PDX as having an elevated activity signature compared to a cohort of 27 additional PDX tumors. Treatment with NXP800 was associated with a statistically significant decrease in tumor size compared to control tumor bearing mice, with a mean difference of the delta in tumor volume (852.6 mm3; 95% CI: 321 - 1384; P-value = 0.0057). Conclusion: NXP800, a HSF1 inhibitor, demonstrated significant therapeutic activity in a cholangiocarcinoma PDX. Further studies are needed and being performed to determine the role of HSF1 inhibition in human cholangiocarcinoma. Citation Format: Danielle M. Carlson, Amro Abdelrahman, Alessandro Fogliati, Isaac Lynch, Jennifer Tomlinson, Mark Truty, Rory Smoot. Inhibition of HSF1 demonstrates therapeutic efficacy in preclinical models of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2237.

Background: Cholangiocarcinoma (CCA) is a heterogeneous malignancy arising from the biliary epithelium. Its diverse molecular landscape and aggressive biology render many anti-cancer therapies ineffective. Nanovesicle technology provides an opportunity for therapeutic inhibition of oncogenic targets that have been previously classified as undruggable. EpCAM is an epithelial-specific, transmembrane glycoprotein with increased expression in human and murine CCA which can be used for nanovesicle targeting. As a proof of concept study, we designed and validated a novel strategy to direct therapeutic milk-derived nanovesicles (tMNVs) to CCA tumors. Methods: tMNVs were decorated with RNA nanoparticles containing a validated aptamer (EpDT3) against EpCAM conjugated to a cholesterol-triethylene-glycol (TEG) scaffold containing an Alexa647 fluorophore. Human and murine CCA cell lines were treated with aptamer directed tMNVs and assessed for nanovesicle uptake by fluorescent microscopy. CCA tumor tissue, derived from orthotopic implantation of a syngeneic CCA cell line, SB1, into a C57BL/6 mouse, was collected and treated with either aptamer-directed or bare tMNVs ex vivo, and compared with adjacent normal liver tissue. Flow cytometry was utilized to characterize tMNVs absorption profile. C57BL/6 mice who had previously undergone SB1 orthotopic and flank implantation were treated with aptamer-directed tMNVs by tail-vein injection and subsequently euthanized. Tissue was collected for biodistribution analyses by fluorescent microscopy. The experiment was repeated in NOD-scid mice following orthotopic implantation of patient derived xenograft (PDX) CCA tumor. Results: Both human and murine CCA cells treated with aptamer-directed tMNVs demonstrated high fluorescent signal consistent with tMNV absorption within 12 hours of application. Flow cytometry analysis showed aptamer-directed tMNVs were absorbed at a higher proportion by CCA tumors than bare tMNVs ex vivo. Aptamer-directed tMNVs also had better absorption by CCA tumors compared to adjacent normal liver tissue. Following treatment with aptamer-directed or bare tMNVs in vivo, fluorescent microscopy demonstrated that aptamer-directed tMNVs were significantly better absorbed in the orthotopic SB1 tumors, followed by the subcutaneous tumors. Minimal fluorescent signal was noted in the normal adjacent liver. Orthotopically implanted PDX tumors also demonstrated high fluorescent signals following intravenous treatment with aptamer-directed tMNVs. Conclusions: Utilizing a novel targeting strategy, we were able to design tMNVs capable of reliably and specifically targeting CCA in preclinical models. This work is foundational to the future application of nanovesicle technology in the CCA treatment paradigm. Citation Format: Mincheng Yu, Jennifer L. Tomlinson, Emilien J. Loeuillard, Ryan D. Watkins, Caitlin B. Conboy, Shohei Takaichi, Nathan W. Werneburg, Roberto Alva-Ruiz, Amro Abdelrahman, Danielle M. Carlson, Jingchun Yang, Sumera I. Ilyas, Gregory J. Gores, Tushar Patel, Rory L. Smoot. Novel strategy for aptamer-directed nanovesicle targeting in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 814.

Abstract Genes encoding SWI/SNF chromatin remodeling complex subunits are recurrently mutated across human cancers. In cholangiocarcinoma (CCA), AT-rich interaction domain-1A (ARID1A) and polybromo-1 (PBRM1) loss of function mutations occur in approximately 18 and 10% of cases respectively. Yet our mechanistic understanding and ability to therapeutically exploit ARID1A and PBRM1 deficiency are immature. Genetically accurate preclinical models are needed to define the role of ARID1A and PBRM1 and test therapeutic approaches in CCA. Analysis of human CCA tumors showed that loss of function mutations in ARID1A and PBRM1 frequently co-occur with each other and with PI3K/AKT pathway activation. Therefore, we developed novel murine models of cholangiocarcinoma initiated by Arid1a knockout or dual Arid1a/Pbrm1 knockout in the context of AKT activation, employing a surgical biliary transfection method for somatic gene editing with CRISPR/Cas9. Mice with cholangiocyte Arid1a knockout and AKT activation (AAC) or dual Arid1a/Pbrm1 knockout and AKT activation (APAC) formed numerous liver tumors within 8 months. Histologically, tumors were predominantly CCA and mixed HCC/CCA, with a similar spectrum of tumors across AAC and APAC mice. Gene editing with CRISPR/Cas9 was confirmed at the DNA level, and loss of protein expression was confirmed in tumor cells by immunohistochemistry. Similar to human CCA, many tumors were densely infiltrated with cancer-associated fibroblasts and a variable degree of CD45+ immune cells. To generate scalable models for preclinical testing, tumors from AAC and APAC mice were dissociated to create cell lines for in vitro studies and propagated by orthotopic transplantation for in vivo studies. Cell lines and orthotopic tumors were validated and retained the genetic features and histologic characteristics of the parental tumors. Collectively these studies have produced a suite of novel, well-characterized, genetically relevant mouse models of epigenetic dysregulation in CCA due to ARID1A and PBRM1 deficiency, which can be used broadly for discovery research and preclinical testing of novel treatment approaches. Citation Format: Caitlin B. Conboy, Jennifer L. Tomlinson, Ryan D. Watkins, Jayla T. Millender, Danielle M. Carlson, Rory L. Smoot, Keith D. Robertson, Greg J. Gores. Novel murine models of ARID1A and PBRM1-deficient cholangiocarcinoma for preclinical discovery and development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A048.

how various treatment modalities work to improve survival outcomes, insight into how socioeconomic factors impact survival is lacking.Disparities in income, insurance status, and education has greatly affected outcomes in various other cancers.In this study, the primary objective was to determine if similar socioeconomic factors affected the overall survival of patients with gallbladder cancer.Methods: We performed a retrospective analysis of the National Cancer Database (NCDB) from 2010 to 2017 to evaluate the relationship between the socioeconomic factors (insurance, income, education) and survival among patients with gallbladder cancer.Survival analysis was done for each of the different stages of gallbladder cancer based on the seventh edition of the American Joint Committee on Cancer (AJCC) staging system.Kaplan Meier analysis was performed for the survival curves.Results: Insurance status was found to be a statistically significant factor affecting overall survival for all stages of gallbladder cancer except for stage 4b.This effect was most noticeable in stage 2, where the survival of patients with private insurance was approximately 18 months longer than in patients with public insurance (ie Medicare and Medicaid).Education and income were not statistically significant factors in overall survival for gallbladder cancer in any stage.Conclusion: Disparities in socioeconomic factors have a major impact on the overall survival for patients with gallbladder cancer.Increasing access to affordable, comprehensive insurance in particular has the potential to enhance long term survival for those afflicted by the disease.

Abstract Background: Cholangiocarcinoma (CCA) is a lethal disease with limited therapeutic options. We have previously discovered LCK driving signaling through AXL, a TAM receptor tyrosine kinase (RTK), by phosphoproteomic analysis of CCA (J Hepatol 2022). AXL mediates acquired drug resistance in solid cancers. However, the exact role of AXL in CCA still remains to be elucidated. Here, we investigated the significance of AXL expression as a potential therapeutic target in CCA and the role of phosphorylated AXL Y866. Methods: We first evaluated the expression levels of AXL in CCA and its associations with patient outcomes using The Cancer Genome Atlas (TCGA) database. Next, to evaluate whether AXL inhibition sensitizes CCA cells to gemcitabine and cisplatin (GemCis) therapy, AXL downregulation was achieved via the siRNA approach and the selective inhibitor bemcentinib. We examined the 50% inhibitory concentration (IC50) value of HuCCT1, a well-characterized CCA cell line, on GemCis therapy with or without AXL knockdown using cell viability assay. Then we assessed the efficacy of the combinatorial therapy of GemCis and bemcentinib utilizing Calcusyn software. Apoptosis was evaluated by Annexin V assay. In vivo efficacy was assessed using an SB-1 (murine CCA cell line) and a syngeneic murine model of CCA treated with vehicle, GemCis, bemcentinib, and the combination of GemCis and bemcentinib. Finally, to investigate the role of AXL Y866 in CCA, we performed BioID to identify and compare the interactomes of AXL WT and AXL Y866F in HuCCT1 cells. Results: In the TCGA cohorts, AXL is significantly expressed in CCA (P < 0.01), and disease-free survival and overall survival in the low AXL expression group are significantly longer than those in the high AXL expression group (P = 0.04, 0.01). In in vitro study, the IC50 value of GemCis was decreased from 685nM to 129nM after AXL knockdown. A synergistic effect was observed with CI = 0.17 and Fa = 0.50 in the combinatorial therapy. The combinatorial therapy caused significantly increased apoptosis compared to GemCis or bemcentinib alone (P < 0.01, P < 0.01). In in vivo study, the combinatorial therapy significantly suppressed the tumor growth compared to GemCis or bemcentinib alone (P = 0.04, 0.01), and the expression levels of Ki67 and the phosphorylation levels of PEAK1 decreased in the combinatorial therapy group compared to other groups in immunostaining examination. In BioID experiments, among the 222 proteins detected in both AXL WT and AXL Y866F interactomes, PEAK1 kinase, which localizes to actin cytoskeleton and focal adhesions, was the kinase most affected by AXL Y866. In gene ontology analysis to characterize the interactome of AXL Y866, cell-cell adhesion and focal adhesion-related proteins were enriched. Conclusions: AXL inhibition sensitizes CCA cells to cytotoxic chemotherapy in the preclinical model. AXL Y866-PEAK1 signaling axis is a potential target for the treatment of CCA. Citation Format: Shohei Takaichi, Dong-Gi Mun, Jennifer L. Tomlinson, Amro M. Abdelrahman, Danielle M. Carlson, Alaa Abou Daher, Nathan W. Werneburg, Xinyan Wu, Akhilesh Pandey, Gregory J. Gores, Rory L. Smoot. Axl inhibition sensitizes cholangiocarcinoma cells to cytotoxic chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4996.

Mixed acinar neuroendocrine carcinoma of the pancreas (MANEC-P) is an extremely rare malignancy with a poor prognosis. However, epidemiological estimates of MANEC-P remain unknown. This study aimed to estimate and compare the incidence, prevalence, and cancer-specific survival (CSS) of MANEC-P in the United States (US). Patients with MANEC-P were identified through the Surveillance, Epidemiology, and End Results (SEER) and National Program of Cancer Registries databases between 2000-2017. The primary outcomes included age-adjusted incidence rate, limited-duration prevalence, and CSS. A total of 630 patients were identified for the incidence analysis and 149 for the prevalence and CSS analyses. The MANEC-P incidence rate was 0.011 per 100,000 individuals, which was the lowest among pancreatic cancer histologic subtypes. The incidence rate was significantly higher in men and Black races and peaked at 75-79 years of age. The incidence rate was the lowest in the midwestern region (0.009) and the highest in the northeastern US (0.013). The 17-year prevalence was 0.00005%, indicating that 189 patients were alive in the United States at the beginning of 2018. The median CSS of MANEC-P was estimated to be 41 (23, 69) months. In conclusion, MANEC-P is very rare, and its incidence rate has been steady in the US over the last two decades. MANEC-P has a poor prognosis and is the 5th leading cause of pancreatic cancer-related death in the US.

27 Background: Cholangiocarcinoma (CCA) is a devastating cancer with few effective treatment options. We have previously discovered LCK can activate AXL, a TAM receptor tyrosine kinase (RTK), by phosphorylation of AXL-Y866 ( J Hepatol 2022). AXL is known to confer therapeutic resistance to solid cancers. However, the role of AXL in CCA, and the impact of AXL-Y866 activation by LCK still remains to be elucidated. Here, we investigated the significance of AXL expression, signaling, and activation by LCK on Y866. Methods: We first evaluated the expression levels of AXL in CCA and its association with patient outcome using The Cancer Genome Atlas (TCGA) dataset. Next, to evaluate whether AXL inactivation sensitizes CCA cells to gemcitabine and cisplatin (GC), AXL downregulation was achieved via siRNA approach and the selective AXL inhibitor bemcentinib (Bem) using human CCA cell lines, HuCCT1 and RBE, and murine SB-1. We examined 50% inhibitory concentration (IC50) value on GC with or without AXL knockdown (KD) using cell viability assay. Then we assessed the efficacy of the combination of GC and Bem utilizing Calcusyn software and apoptosis by Annexin V assay. I n vivo efficacy was assessed using both a patient derived CCA xenograft and SB-1 a syngeneic model of CCA treated with vehicle, GC, Bem, or the combination. Next, we defined the interactome of wildtype AXL- Y866 compared to AXL-Y866F using biotin proximity labelling methods (BioID), validation of downstream target proteins using Western blot (WB), and a functional study with AXL-/- cells reconstituted with either AXL WT, or AXL Y866F. Results: In TCGA cohorts, AXL transcripts are more abundant than in normal adjacent liver, and higher levels are correlated with worse clinical outcome. In in vitro study, IC50 values of GC decreased after AXL KD. Synergistic effects were observed in the combination. The combination caused increased apoptosis compared to other treatments. In the in vivo studies, the combination suppressed tumor growth and the Ki67 levels, and the phosphorylation levels of PEAK1 decreased in the combination group compared to other groups. In BioID experiments, among the 222 proteins which were detected in both AXL WT and AXL Y866F interactomes, PEAK1 kinase, which localizes to actin cytoskeleton and focal adhesions, was most altered when Y866 was inactivated by the Y866F mutation. In gene ontology analysis, cell-cell adhesion and focal adhesion related proteins were enriched. In WB, we validated that the phosphorylation of PEAK1 decreased after AXL or LCK knockout (KO) and after Bem treatment in above in vivo study. In cell viability assay, reconstitution of AXL KO cells with WT AXL increased the IC50 to GC while reconstitution with AXL-Y866F did not. Conclusions: AXL inhibition sensitizes CCA cells to cytotoxic chemotherapy in preclinical model. LCK-AXL Y866-PEAK1 signaling axis is a potential target for the treatment of CCA.