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Figure 6. Dose-response curves of selected kinase inhibitors in the presence or absence of BRAF inhibitors in healthy cells. A) NHEM and B) NHDF cells were subjected to 3-fold serial dilutions of each kinase inhibitor in the presence or absence of constant amounts of Vemurafenib (5 ÂľM) or Dabrafenib (100 nM). Cell viability was assessed 72h after treatment by measuring cell viability. One representative curve of at least 3 biological replicates is depicted. NHEM: normal human epidermal melanocytes; NHDF: normal human dermal fibroblasts. (PDF 238 kb)

Figure S5. Chou-Talalay analysis: A combination of Wee1 and Chk inhibitors synergistically inhibits proliferation of sensitive and resistant melanoma cells. A) Parental and BRAFi-resistant A375 cells were treated with MK-1775 (Wee1i) alone or in combination with either AZD7762 (left panel) or CHIR-124 (right panel) (both Chki). B) Parental and BRAFi-resistant IGR37 (left panel), 501Mel (right panel) cells and C) intrinsically resistant IGR39 cells were treated with MK-1775 (Wee1i) alone or in combination with AZD7762 (Chki). After 72â h, cell viability was determined. A dose-effect analysis of the drug combination was performed using the Compusyn software. CI values shown above the bars were mostly â 1, indicating antagonism. White bars show Wee1i (MK-1775) treatment alone, grey bars show Chki (AZD7762 or CHIR-124) treatment alone and black bars show the combined drugs. Red arrows pinpoint the most effective combinations. One representative experiment of at least 3 is shown here. A375/IGR37/501Mel-XP: resistant to Vemurafenib; A375/IGR37/501Mel-GP: resistant to Dabrafenib. (PDF 294 kb)

Figure S1. Dose-response curves of selected kinase inhibitors in parental and BRAFi-resistant A375 cells. Response to 3-fold serial dilutions of each kinase inhibitor was assessed 72â h after treatment by measuring cell viability. Interesting candidates further tested in combination treatments in A375 cells are highlighted by a red frame (see also Table 1). One representative curve of at least 3 biological replicates is depicted here. _XP: cells resistant to Vemurafenib, _GP: cells resistant to Dabrafenib. (PDF 1030 kb)

Figure S2. Dose-response curves of selected kinase inhibitors in parental and BRAFi-resistant IGR37 and 501Mel cells. Response to 3-fold serial dilutions of each kinase inhibitor was assessed 72â h after treatment by measuring cell viability in IGR37 (A) and 501Mel (B) cells. The values depicted in the different graphs indicate the half-maximal inhibitory concentrations (IC50) of inhibitors for which IC50 values could be determined (as explained in Methods). Values represent the mean of at least three biological replicates; one representative curve of at least 3 biological replicates is depicted. _XP: cells resistant to Vemurafenib (red), _GP: cells resistant to Dabrafenib (green). (PDF 304 kb)

Table S2. Mutation status of genes related to this study. Data are based on own sequencing experiments (WES and Sanger sequencing), online available data from the “COSMIC cell lines project” ( https://cancer.sanger.ac.uk/cell_lines ) and on literature (Halaban et al., Pigm Cell Mel Res, 2010). Synonymous mutations or mutations in non-coding sequences were not taken into account here. wt: no mutation detected; ni: no information available. Genomic profiles (exome sequencing) of the cell lines (A375, -XP and –GP, IGR37, -XP and –GP and IGR39) are available upon request. (PDF 63 kb)

Figure S4. Western blot analysis for selected drug treatments and apoptosis assays in healthy and melanoma cells. A) Western Blot analysis of A375, A375-XP and A375-GP cells treated with the BRAFi Vemurafenib (PLX), Chki AZD7762 (AZD), Wee1i MK-1775 (MK), FAKi TAE226 (TAE) or combinations thereof. Cells were treated for 3 h with indicated concentrations of inhibitors. Actin staining was used as loading control. B) The combination of MK-1775 and AZD7762 efficiently induced apoptosis in primary melanoma cells (M45), but not so much in healthy cells. Cells were treated for 72 h with the indicated concentrations of MK-1775 (Wee1i) or AZD7762 (Chki) or a combination thereof. Etoposide (Eto) treatment was used as positive apoptosis control. Resulting caspase-3 activity was normalized to the untreated control. 1 representative experiment out of 3 is shown. C) Western blot analysis of NHEM, NHDF and M45 primary melanoma cells after treatment for 3 or 24 h with indicated amounts of drugs. P-cdc2 (CDK1), cdc2 (CDK1), p-Chk1 and Chk1 were detected after 3 h drug treatment, while PARP cleavage was detected after 24 h treatment. Vinculin and α-tubulin were used as loading controls. AZD: AZD7762, MK: MK-1775; NHEM. Normal human epidermal melanocytes, NHDF: normal human dermal fibroblasts. (PDF 306 kb)

Table S1. List of Primer sequences. Table S2. ALK siRNA sequences. (PDF 288 kb)

Supplementary methods. (PDF 269 kb)

In my thesis, I elucidated several aspects of melanoma biology, all related to the influence of targeted therapies in both responding and resistant melanoma cells. To better understand the MAPK signalling pathway, the impact of BRAF inhibitors on metabolic alterations as well as the connection between BRAF inhibitors and the onset of drug resistance was investigated. This introduction is focused on four topics: i) melanoma, ii) cancer metabolism, iii) miRNAs and iv) extracellular vesicles. First, melanoma biology including incidence rates, etiology, canonical and altered signalling pathways, therapies and resistance mechanisms will be introduced. The second part of the introduction will concentrate on metabolic alterations in the context of cancer and their implication on proliferation and survival. Thirdly, miRNAs and extracellular vesicles will be illustrated providing insights into their role in cancer development and especially drug resistance

PLX4032 and GSK2118436 do not induce up-regulation of HIF-1α protein in BRAFV600E melanoma cells. Western blot analysis of A375, IGR37 and 501Mel cells (BRAFV600E) treated with 1 μM of PLX4032 and 100 nM of GSK2118432 for the indicated time points. HIF-1α protein was not detectable in all three cell lines. Positive control: A375 short term hypoxia. (PDF 155 kb)

BRAF inhibitors do not induce phosphorylation of PDH in resistant melanoma cells. Western blot analysis of untreated A375, A375 cells resistant to Vemurafenib (A375-R) and under constant presence of 1 μM of PLX4032 and A375 cells stimulated with 1 μM of PLX4032 for 24 h. α-Tubulin was used as loading control; representative blots of three biological replicates are shown. (PDF 102 kb)