Fig. 5: TAK1 inhibitor sensitive GSCs are characterized by high cytokine/interferon signaling gene expression signatures.

A Heatmap showing % growth inhibition expressed as the relative reduction in cell numbers after 4 days of treatment with HS-276 relative to mean of DMSO-treated controls in 12 GSC lines. GSCs are classified as sensitive (red) or insensitive (blue) based on a significant difference between cell numbers in HS-276 and DMSO treatment conditions. Shown are representative results of 3 biological replicates. Heatmap to the right shows GSVA score of gene expression signature from cell line for mesenchymal, proneural, or classical GBM subtype. B Principal component analysis plot (PCA) of RNAseq data from GSC lines. HS-276 sensitive lines are shown in red, insensitive ones in blue. C Volcano plot of differentially expressed genes between sensitive and insensitive GSC lines (n = 6 in each group). Significantly higher expressed genes in sensitive GSCs are colored in red, lower expressed genes in blue, and unchanged in gray. Dotted lines indicate cut-off value used to determine deregulated genes (absolute log2 Fold Change of >1 and adjusted p-value of <0.1). D Barplot of the 12 most significantly enriched Hallmark gene signatures in GSCs sensitive to HS-276 treatment (gene set high, n = 513). E Box and wiskers plot of log2 normalized read counts of baseline expression of selected interferon-stimulated genes (ISGs) in sensitive (n = 6) and insensitive GSCs (n = 6) measured by RNAseq. Whiskers show minimum and maximum values within group. Boxes indicate median, upper, and lower quartiles. F ELISA of TNFα concentration in 7 days conditions GSCs supernatant (6 biological replicates). nd = not detected. G Barplot of IFNB1, IFNG, and TNF gene expression in GSCs measured by qPCR and normalized to RPLP0. H Barplot of fold cell expansion of U3013MG treated for 4 days with indicated drugs. I Heatmap of GSVA scores in GCGR-GSC lines. Samples were ranked based on sensitivity signature GSVA score. ID, GCGR patient ID. * indicates GSC lines selected for testing of responsiveness to HS-276 in vitro. J Scatter plot of % growth in 14 GCGR GSCs after 4 days of treatment with HS-276 relative to DMSO against the sensitivity signature GSVA score. Shown is the relative mean of 3 biological replicates (HS-276/DMSO treated). GSCs with significant reduction in cell numbers upon HS-276 treatment are indicated in red. Dotted line indicates separation based on GSVA score into predicted sensitive (positive score) and predicted insensitive (negative score) GSCs and 25% in growth reduction for sensitivity to TAK inhibition by HS-276 treatment. K Scatter plot of MAP3K7 gene knockout effect against sensitivity signature GSVA score from 59 DepMap glioma cell lines.