Neoadjuvant therapy leads to objective response in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is the second most common hepatic malignancy and has a poor prognosis. Surgical resection is the standard of care for patients with resectable disease, representing 30-40% of cases. Increasingly, neoadjuvant systemic therapy is being utilized in patients due to high-risk anatomic or biologic considerations. However, data on the clinical effect of this approach are limited. We performed a cohort study to evaluate the effect of neoadjuvant therapy in patients with oncologically high-risk iCCA. METHODS: iCCA patients (n = 181) between the years 2014-2020 were reviewed for clinical, histopathologic, treatment, and outcome-related data. Tumor regression grade was scored per CAP criteria for gastrointestinal carcinomas. RESULTS: 47 iCCA patients received neoadjuvant therapy and 72 did not. Neoadjuvant treatment led to objective response and tumor regression by CAP score. After adjustment for age, clinical stage, and tumor size, the outcomes of patients who had neoadjuvant therapy followed by surgery were not significantly different from those patients who had surgery first. DISCUSSION: In conclusion, neoadjuvant therapy in iCCA facilitated surgical care. The progression-free and overall survival for surgical patients with and without neoadjuvant therapy were not significantly different suggesting this approach needs further exploration as an effective treatment paradigm.

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

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 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 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. IMPACT AND IMPLICATIONS: 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.

PTEN deficiency induces an extrahepatic cholangitis-cholangiocarcinoma continuum via aurora kinase A in mice.

BACKGROUND & AIMS: The PTEN-AKT pathway is frequently altered in extrahepatic cholangiocarcinoma (eCCA). We aimed to evaluate the role of PTEN in the pathogenesis of eCCA and identify novel therapeutic targets for this disease. METHODS: The Pten gene was genetically deleted using the Cre-loxp system in biliary epithelial cells. The pathologies were evaluated both macroscopically and histologically. The characteristics were further analyzed by immunohistochemistry, reverse-transcription PCR, cell culture, and RNA sequencing. Some features were compared to those in human eCCA samples. Further mechanistic studies utilized the conditional knockout of Trp53 and Aurora kinase A (Aurka) genes. We also tested the effectiveness of an Aurka inhibitor. RESULTS: We observed that genetic deletion of the Pten gene in the extrahepatic biliary epithelium and peri-ductal glands initiated sclerosing cholangitis-like lesions in mice, resulting in enlarged and distorted extrahepatic bile ducts in mice as early as 1 month after birth. Histologically, these lesions exhibited increased epithelial proliferation, inflammatory cell infiltration, and fibrosis. With aging, the lesions progressed from low-grade dysplasia to invasive carcinoma. Trp53 inactivation further accelerated disease progression, potentially by downregulating senescence. Further mechanistic studies showed that both human and mouse eCCA showed high expression of AURKA. Notably, the genetic deletion of Aurka completely eliminated Pten deficiency-induced extrahepatic bile duct lesions. Furthermore, pharmacological inhibition of Aurka alleviated disease progression. CONCLUSIONS: Pten deficiency in extrahepatic cholangiocytes and peribiliary glands led to a cholangitis-to-cholangiocarcinoma continuum that was dependent on Aurka. These findings offer new insights into preventive and therapeutic interventions for extrahepatic CCA. IMPACT AND IMPLICATIONS: The aberrant PTEN-PI3K-AKT signaling pathway is commonly observed in human extrahepatic cholangiocarcinoma (eCCA), a disease with a poor prognosis. In our study, we developed a mouse model mimicking cholangitis to eCCA progression by conditionally deleting the Pten gene via Pdx1-Cre in epithelial cells and peribiliary glands of the extrahepatic biliary duct. The conditional Pten deletion in these cells led to cholangitis, which gradually advanced to dysplasia, ultimately resulting in eCCA. The loss of Pten heightened Akt signaling, cell proliferation, inflammation, fibrosis, DNA damage, epigenetic signaling, epithelial-mesenchymal transition, cell dysplasia, and cellular senescence. Genetic deletion or pharmacological inhibition of Aurka successfully halted disease progression. This model will be valuable for testing novel therapies and unraveling the mechanisms of eCCA tumorigenesis.

Macrophage RAGE activation is proinflammatory in NASH.

Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte-derived macrophages. The receptor for advanced glycation endproducts (RAGE) is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that RAGE-expressing macrophages are proinflammatory and mediate liver inflammation in NASH. Compared with healthy controls, RAGE expression was increased in liver biopsies from patients with NASH. In a high-fat, -fructose, and -cholesterol-induced (FFC)-induced murine model of NASH, RAGE expression was increased, specifically on recruited macrophages. FFC mice that received a pharmacological inhibitor of RAGE (TTP488), and myeloid-specific RAGE KO mice (RAGE-MKO) had attenuated liver injury associated with a reduced accumulation of RAGE+ recruited macrophages. Transcriptomics analysis suggested that pathways of macrophage and T cell activation were upregulated by FFC diet, inhibited by TTP488 treatment, and reduced in RAGE-MKO mice. Correspondingly, the secretome of ligand-stimulated BM-derived macrophages from RAGE-MKO mice had an attenuated capacity to activate CD8+ T cells. Our data implicate RAGE as what we propose to be a novel and potentially targetable mediator of the proinflammatory signaling of recruited macrophages in NASH.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Growth in Cholangiocarcinoma.

BACKGROUND & AIMS: Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in human beings, challenging the concept of TRAIL as a potent anticancer agent. Herein, we aimed to define mechanisms by which TRAIL+ cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). METHODS: Multiple immunocompetent syngeneic, orthotopic models of CCA were used. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of CD45+ cells in murine tumors from the different CCA models was conducted. RESULTS: In multiple immunocompetent murine models of CCA, implantation of TRAIL+ murine cancer cells into Trail-r-/- mice resulted in a significant reduction in tumor volumes compared with wild-type mice. Tumor-bearing Trail-r-/- mice had a significant decrease in the abundance of MDSCs owing to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent nuclear factor-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of immune cells from murine tumors showed enrichment of a nuclear factor-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis owing to enhanced expression of cellular FLICE inhibitory protein, an inhibitor of proapoptotic TRAIL signaling. Accordingly, cellular FLICE inhibitory protein knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. CONCLUSIONS: Our findings highlight the therapeutic potential of targeting TRAIL+ cancer cells for treatment of a poorly immunogenic cancer.

IL-17 Signaling in Primary Sclerosing Cholangitis Patient-Derived Organoids.

The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study we aimed to investigate the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Cholangiocytes obtained from PSC and non-PSC patients by endoscopic retrograde cholangiography (ERC) were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing (GS). Unsupervised clustering of all integrated scRNA-seq data identified 8 cholangiocyte clusters which did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, noted by an increased number of differentially expressed genes (DEG) by transcriptomics, and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, GS identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. In conclusion, PSC and non-PSC patient derived ECO respond differently to IL-17 stimulation implicating this pathway in the pathogenesis of PSC.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Progression in Cholangiocarcinoma.

Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in humans, challenging the concept of TRAIL as a potent anticancer agent. Herein, we demonstrate that TRAIL + cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). In multiple immunocompetent syngeneic, orthotopic murine models of CCA, implantation of TRAIL + murine cancer cells into Trail-r -/- mice resulted in a significant reduction in tumor volumes compared to wild type mice. Tumor bearing Trail-r -/- mice had a significant decrease in the abundance of MDSCs due to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent NF-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) of CD45 + cells in murine tumors from three distinct immunocompetent CCA models demonstrated a significant enrichment of an NF-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis due to enhanced expression of cellular FLICE inhibitory protein (cFLIP), an inhibitor of proapoptotic TRAIL signaling. Accordingly, cFLIP knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. In summary, our findings define a noncanonical TRAIL signal in MDSCs and highlight the therapeutic potential of targeting TRAIL + cancer cells for the treatment of a poorly immunogenic cancer.

Cholangiocarcinoma - novel biological insights and therapeutic strategies.

In the past 5 years, important advances have been made in the scientific understanding and clinical management of cholangiocarcinoma (CCA). The cellular immune landscape of CCA has been characterized and tumour subsets with distinct immune microenvironments have been defined using molecular approaches. Among these subsets, the identification of 'immune-desert' tumours that are relatively devoid of immune cells emphasizes the need to consider the tumour immune microenvironment in the development of immunotherapy approaches. Progress has also made in identifying the complex heterogeneity and diverse functions of cancer-associated fibroblasts in this desmoplastic cancer. Assays measuring circulating cell-free DNA and cell-free tumour DNA are emerging as clinical tools for detection and monitoring of the disease. Molecularly targeted therapy for CCA has now become a reality, with three drugs targeting oncogenic fibroblast growth factor receptor 2 (FGFR2) fusions and one targeting neomorphic, gain-of-function variants of isocitrate dehydrogenase 1 (IDH1) obtaining regulatory approval. By contrast, immunotherapy using immune-checkpoint inhibitors has produced disappointing results in patients with CCA, underscoring the requirement for novel immune-based treatment strategies. Finally, liver transplantation for early stage intrahepatic CCA under research protocols is emerging as a viable therapeutic option in selected patients. This Review highlights and provides in-depth information on these advances.

Immunobiology of cholangiocarcinoma.

Recent literature has significantly advanced our knowledge and understanding of the tumour immune microenvironment of cholangiocarcinoma. Detailed characterisation of the immune landscape has defined new patient subtypes. While not utilised in clinical practice yet, these novel classifications will help inform decisions regarding immunotherapeutic approaches. Suppressive immune cells, such as tumour-associated macrophages and myeloid-derived suppressor cells, form a barrier that shields tumour cells from immune surveillance. The presence of this immunosuppressive barrier in combination with a variety of immune escape mechanisms employed by tumour cells leads to poor tumour immunogenicity. Broad strategies to re-equip the immune system include blockade of suppressive immune cell recruitment to priming cytotoxic effector cells against tumour antigens. While immunotherapeutic strategies are gaining traction for the treatment of cholangiocarcinoma, there is a long road of discovery ahead in order to make meaningful contributions to patient therapy and survival.

LCK inhibition downregulates YAP activity and is therapeutic in patient-derived models of cholangiocarcinoma.

BACKGROUND & AIMS: There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, Yes-associated protein (YAP), is oncogenic in CCA, but has historically been difficult to target therapeutically. Recently, we described a novel role for the LCK proto-oncogene, Src family tyrosine kinase (LCK) in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK is a viable therapeutic target in CCA via regulation of YAP activity. METHODS: A novel tyrosine kinase inhibitor with relative selectivity for LCK, NTRC 0652-0, was pharmacodynamically profiled in vitro and in CCA cells. A panel of eight CCA patient-derived organoids were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft models bearing fibroblast growth factor receptor 2 (FGFR2)-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. RESULTS: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in CCA cells. LCK inhibition with NTRC 0652-0 led to decreased tyrosine phosphorylation, nuclear localization, and co-transcriptional activity of YAP, and resulted in apoptotic cell death in CCA cell lines. A subset of tested patient-derived organoids demonstrated sensitivity to NTRC 0652-0. CCAs with FGFR2 fusions were identified as a potentially susceptible and clinically relevant genetic subset. In patient-derived xenograft models of FGFR2 fusion-positive CCA, daily oral treatment with NTRC 0652-0 resulted in stable plasma and tumor drug levels, acceptable toxicity, decreased YAP tyrosine phosphorylation, and significantly decreased tumor growth. CONCLUSIONS: A novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell lines, and patient-derived organoid and xenograft models. IMPACT AND IMPLICATIONS: Although aberrant YAP activation is frequently seen in CCA, YAP targeted therapies are not yet clinically available. Herein we show that a novel LCK-selective tyrosine kinase inhibitor (NTRC 0652-0) effectively inhibits YAP tyrosine phosphorylation and cotranscriptional activity and is well tolerated and cytotoxic in multiple preclinical models. The data suggest this approach may be effective in CCA with YAP dependence or FGFR2 fusions, and these findings warrant further investigation in phase I clinical trials.

Potential Role of Inflammation-Promoting Biliary Microbiome in Primary Sclerosing Cholangitis and Cholangiocarcinoma.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a major risk factor for cholangiocarcinoma (CCA). We investigated biliary and fecal microbiota to determine whether specific microbes in the bile or stool are associated with PSC or CCA. METHODS: Bile was obtained from 32 patients with PSC, 23 with CCA with PSC, 26 with CCA without PSC, and 17 controls. Over 90% of bile samples were from patients with perihilar CCA. Stool was obtained from 31 patients with PSC (11 were matched to bile), 16 with CCA with PSC (10 matched to bile), and 11 with CCA without PSC (6 matched to bile). Microbiota composition was assessed using 16SrRNA-marker-based sequencing and was compared between groups. RESULTS: Bile has a unique microbiota distinguished from negative DNA controls and stool. Increased species richness and abundance of Fusobacteria correlated with duration of PSC and characterized the biliary microbiota in CCA. Stool microbiota composition showed no significant differences between groups. CONCLUSIONS: We identified a unique microbial signature in the bile of patients with increased duration of PSC or with CCA, suggesting a role for microbiota-driven inflammation in the pathogenesis and or progression to perihilar CCA. Further studies are needed to test this hypothesis.