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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)

The pyruvate dehydrogenase complex (PDC) is an important gatekeeper enzyme connecting glycolysis to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Thereby, it has a strong impact on the glycolytic flux as well as the metabolic phenotype of a cell. PDC activity is regulated via reversible phosphorylation of three serine residues on the pyruvate dehydrogenase (PDH) E1α subunit. Phosphorylation of any of these residues by the PDH kinases (PDKs) leads to a strong decrease in PDC activity. Under hypoxia, the inactivation of the PDC has been described to be dependent on the hypoxia-inducible factor 1 (HIF-1)-induced PDK1 protein upregulation. In this study, we show in two hepatocellular carcinoma cell lines (HepG2 and JHH-4) that, during the adaptation to hypoxia, PDH is already phosphorylated at time points preceding HIF-1-mediated transcriptional events and PDK1 protein upregulation. Using siRNAs and small molecule inhibitor approaches, we show that this inactivation of PDC is independent of HIF-1α expression but that the PDKs need to be expressed and active. Furthermore, we show that reactive oxygen species might be important for the induction of this PDH phosphorylation since it correlates with the appearance of an altered redox state in the mitochondria and is also inducible by H2O2 treatment under normoxic conditions. Overall, these results show that neither HIF-1 expression nor PDK1 upregulation is necessary for the phosphorylation of PDH during the first hours of the adaptation to hypoxia.

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

Supplementary methods. (PDF 269 kb)

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)

The Janus tyrosine kinases JAK1-3 and tyrosine kinase-2 (TYK2) are frequently hyperactivated in tumors. In lung cancers JAK1 and JAK2 induce oncogenic signaling through STAT3. A putative role of TYK2 in these tumors has not been reported. Here, we show a previously not recognized TYK2-STAT3 signaling node in lung cancer cells. We reveal that the E3 ubiquitin ligase seven-in-absentia-2 (SIAH2) accelerates the proteasomal degradation of TYK2. This mechanism consequently suppresses the activation of STAT3. In agreement with these data the analysis of primary non-small-cell lung cancer (NSCLC) samples from three patient cohorts revealed that compared to lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) show significantly higher levels of SIAH2 and reduced STAT3 phosphorylation levels. Thus, SIAH2 is a novel molecular marker for SCC. We further demonstrate that an activation of the oncologically relevant transcription factor p53 in lung cancer cells induces SIAH2, depletes TYK2, and abrogates the tyrosine phosphorylation of STAT1 and STAT3. This mechanism appears to be different from the inhibition of phosphorylated JAKs through the suppressor of cytokine signaling (SOCS) proteins. Our study may help to identify molecular mechanisms affecting lung carcinogenesis and potential therapeutic targets.

Figure S3. Response of PH5CH1 and PH5CH7 non-neoplastic hepatocytes to OSM, hypoxia, and a combination of both stimuli. PH5CH1 and PH5CH7 cells were treated with OSM (50 ng/ml), hypoxia (1 % O2), or a combinatorial treatment for the indicated periods of time. (A, D) Quantitative RT-PCR of PDP2, PDK1, and HIF-1 α mRNA. Fold changes were calculated relative to the untreated control. (B, E) Western blot analysis for HIF-1 α, HIF-2 α, PDK1, and all three PDH phosphorylation sites (S232, S293, S300). Vinculin and α-tubulin were used as loading controls, and one representative blot is shown. (C, F) Quantification of PDK1 protein levels. (PDF 428 kb)

Table S2. Mass isotopomer distributions (MIDs) from [13C6]glucose in PH5CH8 immortalized human hepatocytes treated for 36 h with 50 ng/mL OSM or left untreated. MIDs were corrected for natural isotope abundance. (PDF 94.2 kb)

Table S3. Mass isotopomer distributions (MIDs) from [13C5]glutamine in PH5CH8 immortalized human hepatocytes treated for 36 h with 50 ng/mL OSM or left untreated. MIDs were corrected for natural isotope abundance. (PDF 94.2 kb)

Table S1. For GC/MS measurements, the detector was operated in single ion mode (SIM). Metabolite specific parameters (selected ions, dwell time, retention time) are described. (PDF 66.8 kb)

Figure S2. (A) Glucose uptake and lactate secretion rates in PH5CH8 immortalized hepatocytes in response to a 36 h treatment with 50 ng/mL OSM. Uptake and secretion rates were determined by GC/MS. (B) Îą-ketoglutarate to citrate ratios in PH5CH8 cells treated with 50ng/mL of OSM for 36 h in comparison to the respective untreated control. (PDF 42.2 kb)

Figure S1. Effects of OSM and hypoxia on HIF-1 α and HIF-2 α protein expression in hepatoma cells and hepatocytes. Eleven hepatoma cell lines and two immortalized hepatocyte cell lines (PH5CH8 and THLE-2) were treated with 20 ng/ml OSM (O) or grown at 1 % O2 (H) over 8 and 36 h. (A) HIF-1 α protein expression after OSM treatment represented in comparison to untreated cells (Ut). (B) HIF-1 α protein expression after OSM treatment represented in comparison to cells grown under hypoxia. (C) HIF-2 α protein expression after OSM treatment represented in comparison to untreated cells. (D) HIF-2 α protein expression after OSM treatment represented in comparison to cells grown under hypoxia. (E) HIF-1 α and HIF-2 α protein induction in non-neoplastic THLE-2 hepatocytes after OSM or hypoxia or a combinatorial treatment. (PDF 655 kb)