Roberts, Lewis R.

Abstract Background and Aims: Cholangiocarcinomas (CCA) are highly desmoplastic tumors containing abundant cancer associated myofibroblasts (CAF). An inverse association between the abundance of CAF and survival has been demonstrated in CCA, suggesting that CAF play a pivotal role in CCA development and progression. The CAF phenotype represents an ‘activated cell state’ and as such may be uniquely susceptible to apoptosis. BH3 mimetics induce apoptosis in cells ‘primed’ for cell death, representing a therapeutic strategy for cancer. Herein we examine whether CAF display a selective sensitivity to the BH3 mimetic ABT-263 and whether ABT-263 induced ablation of CAF reduces tumor growth and progression. Methods: We employed human and rat CAF, myofibroblastic human LX2 cells, human and rat control fibroblasts, the rat cholangiocarcinoma cell line BDEneu and human CCA cell lines for these studies. In-vivo experiments were conducted using a syngeneic, orthotopic rat CCA model. Results: We observed a significant and dose dependent induction of apoptosis by ABT-263 in human and rat primary CAF as well as activated LX2 cells as compared to rat BDEneu and human CCA cell lines. Comparison of Bcl-2 protein expression in quiescent fibroblasts versus CAF demonstrated a consistent upregulation of Bax in the CAF, likely explaining their enhanced sensitivity to pro-death stimuli. Indeed, ABT-263 treatment of CAF was associated with Bax activation as compared to human CCA cell lines. Finally, administration of ABT-263 decreased tumor size and metastasis in a human-like in-vivo rat model of CCA. Analysis of the rat tumors revealed a decrease in Tenascin C positive tumor stroma and an increase of TUNEL-positive apoptotic stromal cells. Conclusions: CCA associated CAF appear to be ‘primed’ for cell death, possibly due to overexpression of the pro-apoptotic Bcl-2 protein Bax. Consequently CAF display increased sensitivity to the BH3 mimetic ABT-263, which selectively depletes them from a rat in-vivo model of CCA, thereby reducing tumor growth and metastasis. Thus targeting CCA tumor stroma by inducing apoptosis in CAF may represent a new therapeutic approach in human CCA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4905. doi:1538-7445.AM2012-4905

Abstract Cholangiocarcinomas are rare but aggressive cancers, with an increasing incidence and a dismal 5-year survival rate of less than 5%. Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in ∼70% of gliomas, ∼20% of myeloid leukemias, ∼56% of chondrosarcomas, and ∼10% of melanomas. We discovered IDH1 and IDH2 mutations in 29 of 305 (9%) intrahepatic cholangiocarcinomas. Tumors with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels, as well as increased dimethylation of histone H3K79. Mutations in IDH1 or IDH2 were independently associated with longer time to recurrence of cholangiocarcinoma in multivariate analysis (p = 0.046). We identified 2,267 genes that were significantly hypermethylated in tumors with mutations in IDH1 or IDH2. Hypermethylated CpG sites were significantly enriched in CpG shores and in the 1500 bp upstream of annotated transcription start sites, suggesting a global regulation of transcriptional potential. Gene expression profiling of 7 IDH1/2-mutant and 20 IDH1/2-wildtype tumors revealed increased expression of citric acid cycle and oxidative phosphorylation enzymes, as well as decreased expression of cytoskeleton organization genes. Integrated analyses of DNA methylation and gene expression identified interleukin-6 and several of its downstream targets as candidates for silencing by DNA methylation. Conclusions: Cholangiocarcinomas with mutations in IDH1 or IDH2 represent a molecular subclass with distinct signaling pathway alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1058. doi:1538-7445.AM2012-1058