Figure 5

Shared signaling alterations of the short-term persistent (early adaption) and fully resistant (late adaption) cell populations include JUN upregulation and a PKC isoform switch. (a) Selection of shared phosphorylation events based on summed effect ranking. Phosphorylation sites were ranked by their summed signed log t-test values for the two resistant/parental and the two persistent/parental pairs (data collapsed on proteins by largest absolute sum). Among the top proteins, JUN and PKC were selected for follow-up investigations. Note that phospho-JUN upregulation was initially only detected for the resistant pairs, but evidence for its upregulation was obtained through a targeted analysis of the mass-spectrometry data set (Supplementary Figure S9). (b) Transcription factor (TF) activity analysis shows upregulation of JUN activity in receptor tyrosine kinase (RTK)-resistance mechanism cells. Genes were ranked by their fold change expression difference in parental vs resistant melanoma cells. Gene-set enrichment analysis (GSEA) was conducted to identify enriched transcription factor binding sites (MSigDB database c3). The results were filtered for binding sites specifically enriched for M229P/R5 and M238P/R1 (false discovery rate (FDR) <0.1), but not for M249P/R4 (FDR>0.2). Normalized enrichment scores (NES) for significance filtered TF binding sites are shown (sorted by the sum of the NES for M229 and M238). See Supplementary Table S8. (c) Kinase enrichment analysis points to upregulated kinase activities in RTK-resistance mechanism cells. These include Akt1, ILK and PKC-alpha (PKCA). Phospho-peptides were ranked by their mean fold change in resistant to parental cells and upregulated kinase activities were identified (filtered for a FDR (BH-adjusted P-value) <0.2). See Supplementary Table S2. (d) Western blot confirmation of (phospho-) JUN upregulation and the PKC isoform switch in RTK-resistance mechanism cells (M229R, M238R, SKMEL28R), but not in an NRAS-mutated resistant cell line (M249R). (e) Short-term vemurafenib-treated, persisting cells share alterations in JUN and protein kinase C (PKC) signaling with fully resistant melanoma cells. M229P and M238P were left untreated (−) or treated with 1μm vemurafenib/PLX4032 (vem) for 3 h, 16 h and 6 days and compared by western blot with the fully resistant cells (M229R and M238R). Short-term vemurafenib-treated cells share the upregulation of (phospho-) JUN and a PKC isoform switch with the fully resistant state. (f) An extended set of five parental melanoma cell lines demonstrates a persistent cell population after a 6- day treatment with 1 μm vemurafenib. The percent viable cells vs number of cells initially plated (dashed line) and the percent viable cells vs cells left untreated and cultured for 3 days (bars) is shown (n=3). (g) Western blot shows shared signaling alterations upon 6-day 1 μm vemurafenib treatment in these melanoma cell lines. (Phospho-) JUN upregulation is shared by all cell lines; the PKC isoform switch is most prominent for vemurafenib-treated M229P, M395P and M397P cells. (h) Vemurafenib (PLX) and dabrafenib (Dabra) treatment induce similar alterations of JUN and PKC signaling. Western blot of M238P and M229P melanoma cells treated for 6 days with vemurafenib or dabrafenib. (i) BRAF, but not NRAS-mutated melanoma cells show (phospho-) JUN upregulation upon 6-day vemurafenib (vem) treatment.