RNA-seq analysis of combination effect of H3B-6527 and Lenvatinib treatment in the Hep3B in vivo model

To investigate the underlying mechanism of the enhanced combination effect of H3B-6527 and Lenvatinib, we conducted RNA-seq analysis of Hep3B cell line xenograft tumors following treatment of tumor bearing mice with one dose of H3B-6527 or Lenvatinib as single agents or in combination. Principal component analysis and hierarchical clustering of differentially expressed genes (differentially expressed genes with adjusted p value < 0.05) showed two largely separated groups in which vehicle and Lenvatinib samples are close to each other whereas the H3B-6527 and the combination samples show similar pattern (Fig. 4A and 4B), suggesting Lenvatinib at 10mg/kg does not drastically alter transcriptional profiles of tumor cells. We then performed pathway enrichment analysis of differentially expressed genes, which showed bile acid metabolism as the top upregulated pathway in H3B-6527 single-agent group, a well-known selective effect to the FGFR4 signaling pathway (Fig. 4C). E2F targets and MYC targets scored as the top downregulated pathways; an effect that is common among many receptor tyrosine kinases including FGFR4. Combination of H3B-6527 and Lenvatinib showed significant upregulation of hypoxia and glycolysis pathways, consistent with anti-angiogenic effect resulting in lack of oxygen and nutrients in tumor microenvironment (Fig. 4C). Moreover, the combination groups showed stronger effects on E2F targets as compared with the single agent groups, and explains the increased antitumor activity observed in efficacy experiments (Fig. 4D). In summary, the RNAseq analysis provided direct mechanistic evidence for the combined anti-angiogenic and anti-tumorigenic effects resulting in enhanced efficacy. Overall design: 4 study groups including Vehicle, H3B-6527, Lenvatinib and H3B-6527+Lenvatinib. 3 tumor samples collected for each group for RNAseq analysis