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ObjectiveThe diversity of the tumour microenvironment (TME) of intrahepatic cholangiocarcinoma (iCCA) has not been comprehensively assessed. We aimed to generate a novel molecular iCCA classifier that incorporates elements of the stroma, tumour and immune microenvironment (‘STIM’ classification).DesignWe applied virtual deconvolution to transcriptomic data from ~900 iCCAs, enabling us to devise a novel classification by selecting for the most relevant TME components. Murine models were generated through hydrodynamic tail vein injection and compared with the human disease.ResultsiCCA is composed of five robust STIM classes encompassing both inflamed (35%) and non-inflamed profiles (65%). The inflamed classes, named immune classical (~10%) and inflammatory stroma (~25%), differ in oncogenic pathways and extent of desmoplasia, with the inflammatory stroma showing T cell exhaustion, abundant stroma and KRAS mutations (p<0.001). Analysis of cell–cell interactions highlights cancer-associated fibroblast subtypes as potential mediators of immune evasion. Among the non-inflamed classes, the desert-like class (~20%) harbours the lowest immune infiltration with abundant regulatory T cells (p<0.001), whereas the hepatic stem-like class (~35%) is enriched in ‘M2-like’ macrophages, mutations in IDH1/2 and BAP1, and FGFR2 fusions. The remaining class (tumour classical: ~10%) is defined by cell cycle pathways and poor prognosis. Comparative analysis unveils high similarity between a KRAS/p19 murine model and the inflammatory stroma class (p=0.02). The KRAS-SOS inhibitor, BI3406, sensitises a KRAS-mutant iCCA murine model to anti-PD1 therapy.ConclusionsWe describe a comprehensive TME-based stratification of iCCA. Cross-species analysis establishes murine models that align closely to human iCCA for the preclinical testing of combination strategies.

Tyrosine kinase inhibition of gastrointestinal stromal tumors (GIST) is effective but typically culminates in resistance and is rarely curative. Immunotherapy has potential application to GIST, as we previously showed that T-cell checkpoint blockade increases the antitumor effects of imatinib. Here, we showed that ligation of CD40 using an agonistic antibody (anti-CD40) activated tumor-associated macrophages (TAMs) in vivo in a knock-in mouse model of GIST harboring a germline mutation in Kit exon 11. Activated TAMs had greater TNF production and NFκB signaling and directly inhibited tumor cells in vitro. Anti-CD40 required concomitant therapy with imatinib for efficacy and depended on TAMs, and to a lesser extent CD8+ T cells, but not on CD4+ T cells or B cells. In an analysis of 50 human GIST specimens by flow cytometry, we found that CD40 was expressed on human TAMs and tumor cells yet was downregulated after response to imatinib. CD40 ligation did not have a direct inhibitory effect on human GIST cells. Our findings provide the rationale for combining anti-CD40 and tyrosine kinase inhibition to treat human GIST.

Imatinib dramatically reduces gastrointestinal stromal tumor (GIST) F-FDG uptake, providing an early indicator of treatment response. Despite decreased glucose internalization, many GIST cells persist, suggesting that alternative metabolic pathways are used for survival. The role of mitochondria in imatinib-treated GIST is largely unknown. We quantified the metabolic activity of several human GIST cell lines. We treated human GIST xenografts and genetically engineered mice with the mitochondrial oxidative phosphorylation inhibitor VLX600 in combination with imatinib and analyzed tumor volume, weight, histology, molecular signaling, and cell cycle activity. assays on human GIST cell lines were also performed. Imatinib therapy decreased glucose uptake and downstream glycolytic activity in GIST-T1 and HG129 cells by approximately half and upregulated mitochondrial enzymes and improved mitochondrial respiratory capacity. Mitochondrial inhibition with VLX600 had a direct antitumor effect while appearing to promote glycolysis through increased AKT signaling and glucose transporter expression. When combined with imatinib, VLX600 prevented imatinib-induced cell cycle escape and reduced p27 expression, leading to increased apoptosis when compared to imatinib alone. In mice, VLX600 alone did not induce tumor cell death, but had a profound antitumor effect when combined with imatinib. Our findings show that imatinib alters the metabolic phenotype of GIST, and this may contribute to imatinib resistance. Our work offers preclinical proof of concept of metabolic targeting as an effective strategy for the treatment of GIST. .

Gastrointestinal stromal tumor (GIST) is the most common sarcoma, often resulting from a KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutation. The lineage transcription factor ETV1 is expressed similarly in GISTs regardless of malignant potential. Although the related transcription factor ETV4 has been associated with metastasis and tumor progression in other cancers, its role in GIST is unknown. In this study, we found that ETV4 levels were high in a subset of human GISTs and correlated with high mitotic rate. Through Gene Set Enrichment Analysis in selected human GISTs, we identified a relationship between ETV4 levels and β-catenin signaling, especially in advanced GISTs. GIST specimens with high ETV4 levels overexpressed cell cycle regulating genes and had aberrant activation of the canonical Wnt pathway. In human GIST cell lines, ETV4 RNA interference suppressed cell cycle genes and Wnt/β-catenin signaling. ETV4 knockdown also reduced tumor cell proliferation, invasion, and tumor growth in vivo. Conversely, ETV4 overexpression increased cyclin D1 expression and Wnt/β-catenin signaling. Moreover, we determined that ETV4 knockdown destabilized nuclear β-catenin and increased its degradation via COP1, an E3 ligase involved in both ETV4 and β-catenin turnover. Aberrant accumulation of ETV4 and nuclear β-catenin was found in patient derived xenografts created from metastatic GISTs that became resistant to tyrosine kinase inhibitors. Collectively, our findings highlight the significance of ETV4 expression in GIST and identify ETV4 as a biomarker in human GISTs.