Citations

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 18F-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.

Upamostat is an orally available small-molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2, trypsin 3 (PRSS1/2/3), and the urokinase-type plasminogen activator (uPA). These enzymes are expressed in many cancers, especially during tissue remodeling and subsequent tumor cell invasion. Opaganib (ABC294640), a novel, orally available small molecule is a selective inhibitor of the phosphorylation of sphingosine to sphingosine-1-phosphate (S-1-P) by sphingosine kinase 2 (SPHK2). Both sphingosine kinase 1 (SPHK1) and SPHK2 are known to regulate the proliferation-inducing compound S-1-P. However, SPHK2 is more critical in cancer pathogenesis. The goal of this project was to investigate the potential antitumor effects of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) xenografts in nude mice. PAX165, a patient-derived xenograft (PDX) from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2, and PRSS3. Four groups of 18 mice each were treated with upamostat, opaganib, both, or vehicle. Mouse weights and PAX165 tumor volumes were measured. Tumor volumes in the upamostat, opaganib, and upamostat plus opaganib groups were significantly decreased compared to the control group.

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.

BACKGROUND: Pancreaticobiliary (PB) cancers are a diverse group of cancers with poor prognoses and high rates of recurrence after resection. Patient-derived xenografts (PDX), created from surgical specimens, provide a reliable preclinical research platform and high-fidelity cancer model from which to study these malignancies with consistent recapitulation of their original patient tumors in vivo. However, the relationship between PDX engraftment success (growth or no growth) and patient oncologic outcomes has not been well studied. We sought to evaluate the correlation between successful PDX engraftment and survival in several PB exocrine carcinomas, including the pancreatic and biliary tract. STUDY DESIGN: In accordance with IRB and Institutional Animal Care and Use Committee protocols and with appropriate consent and approval, excess tumor tissue obtained from surgical patients was implanted into immunocompromised mice. Mice were monitored for tumor growth to determine engraftment success. PDX tumors were verified to recapitulate their tumors of origin by a hepatobiliary pathologist. Xenograft growth was correlated with clinical recurrence and overall survival data. RESULTS: A total of 384 PB xenografts were implanted. The successful engraftment rate was 41% (158/384). We found that successful PDX engraftment was highly associated with both recurrence-free survival (p < 0.001) and overall survival (p < 0.001) outcomes. Successful PDX tumor generation occurs significantly in advance of clinical recurrences in their corresponding patients (p < 0.001). CONCLUSIONS: Successful PB cancer PDX models predict recurrence and survival across tumor types and may provide critical lead time to alter patients’ surveillance or treatment plans before cancer recurrence.

Abstract A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material. Implications: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

BACKGROUND: Portal or superior mesenteric vein (PV-SMV) resection and reconstruction is sometimes required during pancreatic tumor resection. In patients requiring segmental venous resection with interposition grafting, the left renal vein (LRV) is an accessible autologous solution. However, long-term patency outcomes of the LRV as an interposition conduit in this setting have not been analyzed. STUDY DESIGN: We conducted a retrospective analysis of patients undergoing pancreatic resection with PV-SMV reconstruction using LRV between 2002 and 2022. The primary outcome was PV-SMV patency at last follow-up, assessed with postoperative CT scans and analyzed using Kaplan-Meier survival methods that account for variation in follow-up duration. Development of any postoperative acute kidney injury within 7 days of surgery and morbidity were secondary outcomes. RESULTS: The study cohort includes 65 patients who underwent LRV harvest; 60 (92%) ultimately underwent successful reconstruction with harvested LRV graft. Kaplan-Meier 2-year estimated patency rate of the LRV graft was 88%, with no cases of complete occlusion. Six (10%) patients experienced graft stenosis. Nine of 61 (15%) patients experienced grade II or III acute kidney injury, 6 of 9 returning to normal renal function before discharge. No difference in median serum creatinine was observed at baseline, 6 and 12 months from surgery. LRV remnant thrombosis was seen in 7 of 65 (11%) patients. Only 3 of 61 (5%) patients had persistent acute kidney injury caused by complications unrelated to LRV harvesting. CONCLUSIONS: Autologous LRV graft was a reliable conduit for segmental PV-SMV reconstruction, resulting in a high patency rate and marginal impact on renal function. LRV harvest is a safe and potentially ideal surgical option for PV-SMV reconstruction in pancreatic surgery.

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 &lt; 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 &lt; 0.01, P &lt; 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.

Abstract 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.

Abstract 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.

Objective Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. Design We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. Results iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. Conclusion Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.