The most recent reeasearch includes (1) Intrahepatic cholangiocarcinoma (iCCA) is a treatment-refractory disease with heterogeneous underlying pathobiology, no approved therapy and rising incidence. This emphasizes the urgency for the development of therapeutic options for select patient subsets. In a pursuit for novel treatment options, we analyzed 15 iCCA patients with paired tumor and surrounding liver samples using whole-exome sequencing at a depth of 250X. In the prevalence screen of 144 cases, which involved the analysis of 48 cancer-related genes, we confirmed the deregulation of multiple potential causal pathways i.e., FGFR2, PI3K/mTOR, IDH1, TP53, CDKN2A and Notch1 in iCCA. Next, we examined the consequence of targeting Notch by blocking the receptor activation via the gamma-secretase (GS) complex both in a panel of 13 well-characterized CCA cell lines and in murine xenografts. A comprehensive analysis of the notch-signaling network revealed four distinct prognostic patient groups. Notch1 expression is a strong independent prognostic predictor of survival (?2=17.2, P 0.0007) with a 17% 5-year survival rate. Immunostaining confirmed a preferential Notch1 expression and receptor activation in patients with poor prognosis. Multiple notch-related genes, which include the receptor ligands, target-genes and key regulators of biliary differentiation, were significantly deregulated in tumors compared to matched liver samples. Also, we found a significant deregulation of all components of the GS complex. The effect of targeting Notch1 activation was evaluated using two GS inhibitors (R04929097 and YO-01027), which ranged from 0% to 100% sensitivity, and enabled the classification of cells based on drug-response. Importantly, the effect of blocking the GS activity on tumor growth was assessed in two xenograft models, which only demonstrated a beneficial drug-response on reduced tumor burden in GS inhibitor-sensitive iCCA. Our study revealed multiple putative drug targets, and highlights the effect of blocking the Notch receptor activation in iCCA. These data emphasize the usefulness of genomics-based medicine in selecting optimal therapy for defined subsets of patients. (2) Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL-2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPAR-gamma signaling and other regulators of glycolysis (ChREBP-alpha, GLUT4) and lipogenesis (SREBP1C, FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer. (3) We have previously demonstrated therapeutic effects of lipid nanoparticles (LNP) loaded with single siRNA targeting CSN5 or WEE1 against human HCC cell lines in an orthotropic mouse models.To test the safety and the efficacy of a combinatorial versus single siRNA therapy in the orthotopic mouse model and to identify molecular mechanism(s) involved in therapeutic responses by global transcriptome analyses. LNP formulations of chemically modified siRNAs targeting CSN5 and WEE1 were produced by Tekmira Pharmaceuticals. Safety was assessed in ICR mice after 9 injections. SCID-beige mice were used for intra-hepatic (Huh7-luciferase) tumor transplantation. Mice with established tumors were treated intravenously with 2 mg/kg of a single siRNA + 2 mg/kg ?gal siRNA or 2 mg/kg each of siCSN5:siWEE1 siRNA co-encapsulated in the same LNP. Tumors were assayed following 1 to 9 injected doses. Tumor progression in the Huh7 orthotopic model was monitored by bioluminescence imaging and metastases were evaluated at endpoint. Safety data show that combinatorial siRNA is well tolerated compared to single or control siRNA. We observed significant inhibition of tumor growth and metastases in mice treated with active siRNAs compared to LNP containing a non-targeting control siRNA. Significant targeted silencing was observed in tumors after single or repeat administration with no interference between the siRNAs for the CSN5:WEE1 combination. Potency was not lost with siCSN5:siWEE1 LNP, relative to the most efficacious single agent. Microarray analyses of the tumors demonstrate an extensive difference of gene expression between treatment groups. Interestingly, the microarray analyses of the surrounding liver show a minimal modification of gene expression in this non-tumor tissue. In conclusion, we have demonstrated that LNP-based combinatorial siRNA therapy is safe and effective in a human mouse model of HCC, with a significant decrease of tumor size associated with a massive downregulation of the targeted genes. Global gene expression of the surrounding liver is minimally affected by this therapy compared with what seen in the tumor.
