Fig. 4

AC004540.4 promotes cell survival pathways and can be targeted to induce apoptosis. a) A volcano plot from RNAseq derived data highlights the differentially expressed (DE) genes from D04 cells treated with either AC004540.4 ASO, or Control ASO. Cut-off for significance was p < 0.05 and either < -1.5 (downregulated) or > 1.5 (upregulated) log2fold expression level change (n = 3). DAVID functional annotation clustering unveiled that “PI3K-AKT signaling pathway” (downregulated), and “protein tyrosine kinase activity” and “Ras guanyl-nucleotide exchange factor activity” (upregulated) were Gene Ontology (GO) terms and pathways found in the top enriched clusters. b) Peptide-associated phosphorylation profiles of melanoma cell lines treated with AC004540.4 ASO, or Control ASO. Unsupervised clustering was applied (using uncentered correlation and average linkage for both peptides/horizontal and samples/vertical). The profile of each sample is the average of two independent assay repeats. c) Unsupervised clustering of kinase activity signatures from results shown in previous panel. Kinases for which ≥ 3 biological peptides are available, are shown. AC004540.4 inhibition induced a conserved response across cell lines of either activity downregulation (Block A, mainly blue) and upregulation (Block B, mainly yellow). d) Kinase activity profiles of a subset of kinases known to promote cell survival by preventing apoptosis is specifically downregulated by AC004540.4 ASO treatment, as shown by a side-by-side comparison to the effects of inhibiting the distinct pro-oncogenic lncRNA MALAT1 with MALAT1 ASO treatment. e) The specificity of the effects of AC004540.4 ASO treatment on the kinase activity signatures of melanoma cells is assessed in comparison to MALAT1 ASO treatment using Pearson correlation. f) As measured by activity levels of the apoptosis markers Caspase-3 & -7, AC004540.4 ASO treatment induces apoptosis to a significantly stronger extent than MALAT1 ASO treatment in the D04 (3-fold VS 1.6-fold, p < 0.002) and MM415 (3.5-fold VS 1.1-fold, p < 0.006) cell-line, when compared to Control ASO treatment (treatment with 50 nM ASO concentration for 1 day, n = 4). Significance is shown as p-values calculated by Student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Error bars represent the standard deviation. g) Dot plot graph of flow cytometric analysis of PI and Annexin V staining after 1 day of ASO-treatment shows increased apoptotic cell death in D04-cells treated with AC004540.4 ASO compared to Control ASO treatment. Numbers in quadrants (red) show the percentage of vital (bottom left), early apoptotic (bottom right), late apoptotic (top right) and dead (top left) cells relative to the overall population. h) Schematic summarizing of the molecular impact of AC004540.4 (T-RECS) ASO-treatment, inducing apoptosis and inhibiting pro-oncogenic kinases in melanoma