Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds

cancers Article Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds Veronika Palušová1,2, Tereza Renzová1, Amandine Verlande 1, Tereza Vaclová1, Michaela Medková1, Linda Cetlová1, Miroslava Sedláˇcková1, Hana Hˇríbková1, Iva Slaninová1, Miriama Krutá1, Vladimír Rotrekl 1,2 , Hana Uhlíˇrová3,4 , Aneta Kˇrížová4, Radim Chmelík3,4 , Pavel Veselý4, Michaela Krafˇcíková5, Lukáš Trantírek 6, Kay Oliver Schink 7,8 and Stjepan Uldrijan 1,2,* 1Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; ver[email protected] (V.P.); [email protected] (T.R.); [email protected] (A.V.); [email protected] (T.V.); [email protected] (M.M.); [email protected] (L.C.); [email protected] (M.S.); [email protected] (H.H.); [email protected] (I.S.); [email protected] (M.K.); vr[email protected] (V.R.) 2International Clinical Research Center, St. Anne’s University Hospital Brno, Pekaˇrská664/53, 656 91 Brno, Czech Republic 3Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, Technická2896/2, 616 69 Brno, Czech Republic; [email protected].cz (H.U.); [email protected].cz (R.C.) 4CEITEC—Central European Institute of Technology, Brno University of Technology, Purkyˇnova 656/123, 612 00 Brno, Czech Republic; [email protected].cz (A.K.); [email protected].cz (P.V.) 5National Centre for Biomolecular Research, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; [email protected] 6CEITEC—Central European Institute of Technology, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; [email protected] 7Centre for Cancer Cell Reprogramming, Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo, Norway; [email protected] 8 Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway *Correspondence: [email protected] Received: 29 February 2020; Accepted: 5 June 2020; Published: 10 June 2020   Abstract: BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 atcontactsitesoflarge vesiclesand delocalization of mTOR from thelysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core. Cancers 2020,12, 1516; doi:10.3390/cancers12061516 www.mdpi.com/journal/cancers Cancers 2020,12, 1516 2 of 24 Keywords: melanoma; BRAF V600E; BRAF inhibitor; small molecule drug; pyridinyl imidazole; endosome; lysosome; mTORC1; ER stress 1. Introduction Malignant melanoma is aggressive cancer affecting the skin and other tissues where pigment-producing melanocytes reside. Although melanoma accounts for less than 5% of all dermatologic tumors, it is responsible for approximately 80% of all deaths from skin cancers [ 1 ]. The main risk factor for the development of melanoma is overexposure to solar UV radiation. The UV light can induce cancer-promoting mutations that can stimulate melanocyte growth independently from external stimuli [ 2 , 3 ]. Most human melanomas can be placed into two subgroups based on the type of mutation driving the ERK MAPK signaling pathway and cell proliferation [4,5]. About a half of melanoma patients carry a potent activating mutation V600E in the BRAF kinase, which renders the normally dimeric BRAF kinase enzymatically active as a monomer, independent of upstream signaling. The second-largest group of melanoma patients bears activating mutations in the NRAS protein. The crucial role of BRAF-driven oncogenic ERK signaling in melanoma stimulated the preclinical and clinical development of a large number of structurally different RAF inhibitors [ 6 ]. Targeting mutant BRAF can lead to a strong response in melanoma patients. Still, unfortunately, the cancers commonly acquire other pro-survival mutations compensating for the depletion of BRAF activity [ 7 ] and causing resistance to treatment [ 8 ]. In BRAF inhibitor-resistant melanoma cells, a combination of MEK inhibitors with phosphatidylinositol 3-kinase (PI3K) or mechanistic target of rapamycin (mTOR) inhibitors can significantly decrease cancer cell survival [ 9 , 10 ]. Furthermore, in mouse models of BRAF-mutated melanoma, inhibition of PI3K cooperated with ERK pathway inhibition to forestall the onset of MAPK pathway inhibitor resistance [11]. Activation of receptor tyrosine kinases by growth factors stimulates both MAPK and PI3K/AKT/mTOR signaling pathways to coordinate cell growth, proliferation, and survival [ 12 ]. The mTOR serine/threonine protein kinase is a core component of two functionally distinct protein complexes—mTOR complex 1 (mTORC1) and 2 (mTORC2). Complex 1 balances the anabolic and catabolic processes in response to growth factors with ATP, oxygen, and nutrient availability. When active, mTORC1 stimulates proteosynthesis and inhibits catabolic processes such as autophagy [ 13 ]. Small GTPases Rheb (Ras homolog enriched in brain) and Rag mediate the activation of mTORC1 [ 14 ]. To activate the mTORC1 complex, Rheb must be in a GTP-bound state and proximity to mTORC1 [ 15 ]. Insufficient growth factor signaling, low glucose conditions, or low oxygen conditions promote the dissociation of the TSC1/2 protein complex. Released TSC2 causes a switch of Rheb into its inactive GDP-bound state, preventing the activation of mTORC1 [ 16 , 17 ]. Interestingly, TSC2 translocation to the lysosome appears to be a universal cellular response to stress stimuli [18]. Critical factors enabling the mTOR complex 1 anchoring to endomembranes are the Rag family of small GTPases and a scaffold protein complex called Ragulator [ 19 , 20 ]. Four related Rag GTPases, RagA, RagB, RagC, and RagD, are expressed in mammalian cells. They form RagA/B–RagC/D heterodimers, in which RagA or RagB physically interact with RagC or RagD [ 20 , 21 ]. The recruitment of mTORC1 to the lysosome depends on the nucleotide-bound state of Rags, which form a docking site for Raptor, an essential subunit of mTORC1 [ 22 ]. In amino acid-starved conditions, RagA/B are bound to GDP, which is rapidly exchanged with GTP when amino acid levels are restored [ 20 , 23 ]. The sensing of amino acid levels takes place in the lysosomal lumen, and individual amino acids have their specific sensors, which act upstream of Rag GTPases [24,25]. The PI3K/AKT/mTOR and RAF/MEK/ERK signaling cascades form numerous feedback loops and interconnect at multiple points of crosstalk. Inhibition of one pathway can be partially compensated by the enhanced activity of the other, implying that dual targeting of both pathways may improve treatment efficacy and lead to better clinical outcomes [ 26 ]. In the current study, we show that Cancers 2020,12, 1516 3 of 24 pyridinyl imidazole compounds are capable of simultaneously targeting the BRAF oncogene and mTORC1 signaling in human melanoma cells. They directly inhibit the BRAF kinase activity and, at the same time, interfere with the endolysosomal compartment, leading to a loss of mTORC1 lysosomal localization and activity. The pyridinyl imidazole compounds suppress the growth and proliferation of melanoma cells and sensitize them to additional stress stimuli. These findings provide a biological rationale for further development of pyridinyl imidazole anti-melanoma drugs. 2. Results 2.1. Pyridinyl Imidazole p38 MAPK Inhibitors Disrupt BRAF V600E-Driven ERK Signaling in Human Melanoma Cells Pyridinyl imidazole inhibitors SB202190 and SB203580 have been widely used in biomedical research as selective chemical probes for p38 MAPK biological activity, despite reports suggesting additional kinase targets and cell type-specific p38-independent effects of these compounds [ 27 – 30 ]. We analyzed the biological activity of SB202190 in A375 melanoma cells and found that SB202190, but not a structurally distinct p38 inhibitor SB239063, strongly affected the growth of melanoma cell cultures, suggesting a p38-independent antiproliferative effect of the pyridinyl imidazole compound in human melanoma cells (Figure 1A). ThemostcommondriversofmelanomaproliferationareNRASandBRAFmutations, constitutively activating the ERK MAPK pathway in about 80% of tumors [ 4 , 5 ]. Interestingly, some reports suggested that pyridinyl imidazole compounds could activate ERK signaling by promoting CRAF (RAF-1) activity [ 31 – 33 ]. A small-molecule library screen using bioluminescence resonance energy transfer-based biosensors identified SB202190 and SB203580 as potent activators of RAF dimerization, which might explain the reported ERK pathway activation in response to SB203580 [ 34 ]. We, therefore, tested the possibility that the pyridinyl imidazole p38 inhibitors could directly modulate RAF kinase activity and ERK signaling in melanoma cells. We analyzed ERK-dependent transcription in A375 cells, bearing the most common activating mutation of BRAF kinase (V600E), stably transfected with a recently developed ERK activity luciferase reporter construct [ 35 ]. Surprisingly, we found that SB202190 strongly inhibited ERK-driven luciferase activity in this system, as potently as MEK kinase inhibitors U0126 and PD184352 that were used as positive controls (Figure 1B). Next, we treated A375 cells with increasing concentrations of SB202190 or SB203580 and analyzed ERK pathway activity by Western blotting, using MEK and ERK phospho-specific antibodies. Specific MEK inhibitor PD184352 served as a positive control. Both pyridinyl imidazole compounds induced a dose-dependent decrease in the levels of active ERK and MEK kinases (Figure 1C). The experiment was repeated three times (additional Western blots are available in Figure S1), and we determined the relative P-MEK/MEK and P-ERK/ERK ratios between phosphorylated (active) and total MEK and ERK kinase levels. The results presented in Figure S2 indicate that both compounds could inhibit ERK pathway activity in A375 cells, but SB202190 affected the pathway more potently than SB203580. The fact that both MEK and ERK activity was decreased suggested that the pyridinyl imidazole compounds target the ERK signaling pathway upstream of MEK kinase. The inhibitory effect of SB202190 on ERK activity was observed in human melanoma cell lines carrying BRAF V600E mutation (A375, G361, Colo-800), but not in melanoma cells with NRAS mutations (MEL-JUSO, SK-MEL-30, IPC-298) (Figure 1D). This result indicated that pyridinyl imidazole p38 inhibitors might act as inhibitors of mutant BRAF, but not wild type CRAF kinase, which activates MEK in cells bearing mutated NRAS. Importantly, two structurally unrelated small-molecule p38 inhibitors SB239063 and BIRB796 did not affect ERK activity in melanoma cells (Figure 1D). The results of two additional independent replicates of this experiment are available in Figure S1. Cancers 2020,12, 1516 4 of 24 Cancers 2020, 12, x 4 of 25 (https://imagej.net/Fiji). The results presented in Figure 1E suggest that SB202190 could inhibit the activity of endogenous BRAF V600E protein immunoprecipitated from A375 melanoma cells. The possibility that the p38 MAPK inhibitors SB202190 and SB203580 might target mutant BRAF kinase was indirectly supported by the fact that a structurally related pyridinyl imidazole derivative SB590885 was developed as a BRAF-specific inhibitor [36]. When we compared in the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays the effect of SB202190 and SB590885 on the proliferation of a panel of melanoma cell lines, as expected, we observed that BRAFmutated melanoma cell lines were more sensitive to the compounds than NRAS-mutated melanoma cells (Figure S3). BRAF-inhibitor vemurafenib served as a positive control. Interestingly, higher concentrations of SB590885 also negatively affected the growth of NRAS-mutated cell lines, indicating the possibility of additional, BRAF-independent, cytotoxic activity of the pyridinyl imidazole compounds in melanoma cells (Figure S3). Figure 1. Pyridinyl imidazole p38 mitogen-activated protein kinase (MAPK) inhibitors SB202190 and SB203580 disrupt mutant BRAF kinase activity in human melanoma cells. ( A ) The proliferation of A375 cells was measured by flow cytometry during 4-day cultivation. The number of cells was compared between controls (DMSO-treated) and cells treated with SB202190 (SB202; 15 µ M) and SB239063 (SB239; 15 µ M). The presented data (mean +SD) were obtained in three independent experiments. ( B ) Relative ERK activity in A375 cells stably transfected with an ERK activity luciferase reporter plasmid. Cells were treated for 24 h with SB202190 (10 µ M) and MEK inhibitors U0126 (10 µ M) and PD184352 (PD; 1 µ M). Three independent experiments were performed. Results are presented as mean relative ERK activity +SD. ( C ) Levels of phosphorylated MEK and ERK kinase were analyzed by Western blot. A375 cells were treated for one hour with increasing concentrations of SB202190 (SB202) and SB203580 (SB203). MEK inhibitor PD184352 (PD; 100 nM) was used as a positive control. The relative ratio between P-ERK and the total ERK levels is also indicated for each sample. ( D ) ERK activity was analyzed by Western blot in melanoma cell lines bearing BRAF (A375, G361, COLO-800) or NRAS (MEL-JUSO, SK-MEL-30, IPC-298) mutations. Cells were treated for 24 h with the p38 inhibitors SB202190 (SB202; 10 µ M), SB239063 (SB239; 10 µ M), and BIRB796 (BIRB; 10 µ M). The relative ratio between P-ERK and the total ERK levels is also indicated for each sample. ( E ) In vitro BRAF kinase assay using kinase-dead MEK as a substrate and endogenous V600E BRAF kinase immunoprecipitated from A375 cells. SB202190 was used at 5 µ M. Data were obtained in three independent experiments. Results are presented as relative values, mean +SD. **** denotes p<0.0001. Cancers 2020,12, 1516 5 of 24 Next, we performed an in vitro BRAF kinase activity assay using a recombinant kinase-dead MEK protein as a substrate. Three independent experiments were performed, and the levels of MEK phosphorylation were determined by Western blotting and quantified using ImageJ/Fiji (https: //imagej.net/Fiji). The results presented in Figure 1E suggest that SB202190 could inhibit the activity of endogenous BRAF V600E protein immunoprecipitated from A375 melanoma cells. The possibility that the p38 MAPK inhibitors SB202190 and SB203580 might target mutant BRAF kinase was indirectly supported by the fact that a structurally related pyridinyl imidazole derivative SB590885 was developed as a BRAF-specific inhibitor [ 36 ]. When we compared in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays the effect of SB202190 and SB590885 on the proliferation of a panel of melanoma cell lines, as expected, we observed that BRAF-mutated melanoma cell lines were more sensitive to the compounds than NRAS-mutated melanoma cells (Figure S3). BRAF-inhibitor vemurafenib served as a positive control. Interestingly, higher concentrations of SB590885 also negatively affected the growth of NRAS-mutated cell lines, indicating the possibility of additional, BRAF-independent, cytotoxic activity of the pyridinyl imidazole compounds in melanoma cells (Figure S3). 2.2. SB202190-Induced Vacuoles in Melanoma Cells Have an Endocytic Origin Among the effects reported for the p38 MAPK inhibitors, SB202190 and SB203580, was the formation of large vacuole-like structures. Some reports linked the phenotype to the disruption of autophagy, which was later shown to be p38-independent [ 30 , 37 ]. In our experiments, both compounds induced strong cytoplasmic vacuolization in A375 melanoma cells (Figure 2A). We, therefore, analyzed in detail this phenotype and its possible contribution to the growth-inhibitory activity of the pyridinyl imidazole drugs in melanoma cells. Analysis of individual frames of a time-lapse recording from a phase-contrast microscope revealed that the large vacuoles induced by SB202190 in melanoma cells might form by the fusion of smaller vesicles (Figure S4). Electron microscopy analysis of SB202190-treated A375 cells showed that the large vacuoles are mostly devoid of dense structures (Figure 2B). The low mass density of the vacuole-like structures was also confirmed using quantitative phase imaging (Figure 2C). The above results indicated the possibility that SB202190-induced vacuole-like structures could form in response to a disruption of vesicular endocytic transport, a molecular pathway that is responsible for the active transport of membrane proteins, including receptors regulating growth signaling, and for the uptake of vital nutrients from the extracellular environment [ 38 ]. Small molecule fluorescent dyes such as acridine orange or LysoTracker Green DND-26 stain acidic compartments in living cells, including endosomes and lysosomes. Both dyes readily marked the SB202190-induced vacuoles in A375 melanoma cells, supporting the possibility of their endolysosomal origin (Figure 3A,B). Using confocal fluorescence microscopy, we observed that only a small proportion of SB202190-induced vacuoles with a diameter larger than 1 µ m express early endosomal marker RAB5 (Figure 3C). In contrast, most SB202190-induced vacuoles with a diameter larger than 1 µ m express late endosome marker RAB7 at their surface (Figure 3D), indicating that the majority of enlarged vacuoles originated from the late endosome. Moreover, sucrose-induced osmotic stress or the small molecule inhibitor 5-(N-Ethyl-Nisopropyl)amiloride (EIPA), which inhibits a form of endocytosis called macropinocytosis, prevented the formation of large vacuoles in SB202190-treated cells (Figure 3E). The negative impact of EIPA on RAB7-positive vacuole-like vesicles was also observed using fluorescence microscopy (Figure S5). These data further supported the endocytic origin of the enlarged vesicles. Furthermore, live-cell imaging indicated that most SB202190-induced vacuoles could not be recycled back to the plasma membrane, suggesting a block in later stages of endosomal trafficking (Video S1). Cancers 2020,12, 1516 6 of 24 Cancers 2020, 12, x 6 of 25 Figure 2. Pyridinyl imidazole compounds induce vacuolization of cytoplasm in BRAF-mutated human melanoma cells. A375 cells were treated with DMSO (control), SB202190 (SB202; 10 μM), and SB203580 (SB203; 10 μM). (A) Phase-contrast light microscopy of living cells after 12 h treatment with pyridinyl imidazole p38 MAPK inhibitors. Scale bar: 45 µm. The representative graph shows the mean number of vacuoles per cell quantified using ImageJ/Fiji (find maxima—bright spots above a certain threshold). Dying rounded cells were excluded from the analysis. Similar results were obtained in three independent experiments. (B) The content of vacuole-like vesicles was visualized by electron microscopy 24 h post-treatment with SB202190 (SB202). Control and SB202(1)—scale bar 5 µm. SB202(2)—scale bar 1 µm. Two independent experiments showed similar results. (C) Quantitative phase-imaging analysis of cellular dry mass in melanoma cells treated for 12 h with SB202190. The zero level of dry mass density was defined as the density of the observation medium. The areal density distribution of the dry mass was quantified in profiles (right panels) indicated by dark lines. Red arrows highlight the position of vacuoles. Two independent digital holography microscopy experiments showed similar results. The above results indicated the possibility that SB202190-induced vacuole-like structures could form in response to a disruption of vesicular endocytic transport, a molecular pathway that is Figure 2. Pyridinyl imidazole compounds induce vacuolization of cytoplasm in BRAF-mutated human melanoma cells. A375 cells were treated with DMSO (control), SB202190 (SB202; 10 µ M), and SB203580 (SB203; 10 µ M). ( A ) Phase-contrast light microscopy of living cells after 12 h treatment with pyridinyl imidazole p38 MAPK inhibitors. Scale bar: 45 µ m. The representative graph shows the mean number of vacuoles per cell quantified using ImageJ/Fiji (find maxima—bright spots above a certain threshold). Dying rounded cells were excluded from the analysis. Similar results were obtained in three independent experiments. ( B ) The content of vacuole-like vesicles was visualized by electron microscopy 24 h post-treatment with SB202190 (SB202). Control and SB202(1)—scale bar 5 µ m. SB202(2)—scale bar 1 µ m. Two independent experiments showed similar results. ( C ) Quantitative phase-imaging analysis of cellular dry mass in melanoma cells treated for 12 h with SB202190. The zero level of dry mass density was defined as the density of the observation medium. The areal density distribution of the dry mass was quantified in profiles (right panels) indicated by dark lines. Red arrows highlight the position of vacuoles. Two independent digital holography microscopy experiments showed similar results. Cancers 2020,12, 1516 7 of 24 Cancers 2020, 12, x 7 of 25 responsible for the active transport of membrane proteins, including receptors regulating growth signaling, and for the uptake of vital nutrients from the extracellular environment [38]. Small molecule fluorescent dyes such as acridine orange or LysoTracker Green DND-26 stain acidic compartments in living cells, including endosomes and lysosomes. Both dyes readily marked the SB202190-induced vacuoles in A375 melanoma cells, supporting the possibility of their endolysosomal origin (Figure 3A,B). Using confocal fluorescence microscopy, we observed that only a small proportion of SB202190-induced vacuoles with a diameter larger than 1 µm express early endosomal marker RAB5 (Figure 3C). In contrast, most SB202190-induced vacuoles with a diameter larger than 1 µm express late endosome marker RAB7 at their surface (Figure 3D), indicating that the majority of enlarged vacuoles originated from the late endosome. Moreover, sucrose-induced osmotic stress or the small molecule inhibitor 5-(N-Ethyl-Nisopropyl)amiloride (EIPA), which inhibits a form of endocytosis called macropinocytosis, prevented the formation of large vacuoles in SB202190-treated cells (Figure 3E). The negative impact of EIPA on RAB7-positive vacuole-like vesicles was also observed using fluorescence microscopy (Figure S5). These data further supported the endocytic origin of the enlarged vesicles. Furthermore, live-cell imaging indicated that most SB202190-induced vacuoles could not be recycled back to the plasma membrane, suggesting a block in later stages of endosomal trafficking (Video S1). Figure 3. Vacuole-like vesicles induced by pyridinyl imidazole compounds in melanoma cells have an endolysosomal origin. A375 cells were treated with SB202190 (SB202; 15 μM) or the equivalent amount of vehicle (DMSO) in control. (A) Cells were treated with SB202190 for 20 h and stained with acridine orange (5 µg/mL) for 15 min. Scale bar: 50 µm. The graph shows the relative change in the yellow stained area in response to SB202190. Similar results were obtained in two independent experiments. (B) Cells were treated with SB202190 for 20 h and stained with LysoTracker Green (50 nM) for 15 min. Scale bar: 50 µm. The graph shows the relative change of the LysoTracker Green signal in response to SB202190. Similar results were obtained in two independent experiments. (C) The confocal microscopy detection of endogenous early endosomal marker RAB5 after 24 h treatment with SB202190. Scale bar: 20 µm. The graph shows the number of RAB5-positive vacuoles per cell with a diameter larger than 1 µm. Similar results were obtained in three independent experiments. (D) Deconvolved wide-field fluorescence microscopy imaging of EGFP-tagged late endosomal/lysosomal marker RAB7A after 24 h SB202190 treatment. Scale bar: 10 µm. The diameter Figure 3. Vacuole-like vesicles induced by pyridinyl imidazole compounds in melanoma cells have an endolysosomal origin. A375 cells were treated with SB202190 (SB202; 15 µ M) or the equivalent amount of vehicle (DMSO) in control. ( A ) Cells were treated with SB202190 for 20 h and stained with acridine orange (5 µ g/mL) for 15 min. Scale bar: 50 µ m. The graph shows the relative change in the yellow stained area in response to SB202190. Similar results were obtained in two independent experiments. (B) Cells were treated with SB202190 for 20 h and stained with LysoTracker Green (50 nM) for 15 min. Scale bar: 50 µ m. The graph shows the relative change of the LysoTracker Green signal in response to SB202190. Similar results were obtained in two independent experiments. ( C ) The confocal microscopy detection of endogenous early endosomal marker RAB5 after 24 h treatment with SB202190. Scale bar: 20 µ m. The graph shows the number of RAB5-positive vacuoles per cell with a diameter larger than 1 µ m. Similar results were obtained in three independent experiments. ( D ) Deconvolved wide-field fluorescence microscopy imaging of EGFP-tagged late endosomal/lysosomal marker RAB7A after 24 h SB202190 treatment. Scale bar: 10 µ m. The diameter of RAB7-positive structures was determined, and the number of vacuoles larger than 1 µ m per cell was plotted in the graph. The experiment was performed three times with similar results. ( E ) Effect on vacuolization was visualized by bright-field images in cells treated for 20 h with SB202190 alone and in combination with sucrose (0.5 M) or EIPA (50 µ M). Scale bar: 50 µ m. The graph shows the number of vacuoles per cell, quantified using ImageJ/Fiji (find maxima—bright spots above a certain threshold). Dying rounded cells were excluded from the analysis. The experiment was repeated three times with a similar response to the addition of sucrose and EIPA. 2.3. Pyridinyl Imidazole Compounds Partly Mimic PIKfyve Inhibition in Melanoma Cells The apparent defect in the endocytic pathway promoting a marked increase in the volume of endolysosomes led us to search the literature for a similar phenotype. Inhibition of the FYVE finger-containing phosphoinositide kinase (PIKfyve) had been previously reported to induce extensive cytoplasmic vacuolization in some cell types [ 39 , 40 ]. In A375 melanoma cells, the phenotype induced by a small-molecule PIKfyve inhibitor YM201636 was strikingly similar to that induced by SB202190, including the characteristic accumulation of large vesicles (Figure 4A). As the BRAF inhibitor SB590885 bears significant structural similarity to p38 MAPK inhibitors SB202190 and SB203580, we hypothesized that it might also be capable of targeting endolysosomal trafficking. Indeed, we also observed the Cancers 2020,12, 1516 8 of 24 accumulation of large vacuole-like vesicles in SB590885-treated A375 melanoma cells (Figure 4A). Furthermore, a small molecule inhibitor of vacuolar H+ATPase bafilomycin A1 that can prevent the formation of vacuoles in response to PIKfyve inhibition [ 41 ] also blocked the vacuolization induced by pyridinyl imidazole compounds (Figure 4A). Cancers 2020, 12, x 8 of 25 of RAB7-positive structures was determined, and the number of vacuoles larger than 1 μm per cell was plotted in the graph. The experiment was performed three times with similar results. (E) Effect on vacuolization was visualized by bright-field images in cells treated for 20 h with SB202190 alone and in combination with sucrose (0.5 M) or EIPA (50 μM). Scale bar: 50 µm. The graph shows the number of vacuoles per cell, quantified using ImageJ/Fiji (find maxima—bright spots above a certain threshold). Dying rounded cells were excluded from the analysis. The experiment was repeated three times with a similar response to the addition of sucrose and EIPA. 2.3. Pyridinyl Imidazole Compounds Partly Mimic PIKfyve Inhibition in Melanoma Cells The apparent defect in the endocytic pathway promoting a marked increase in the volume of endolysosomes led us to search the literature for a similar phenotype. Inhibition of the FYVE fingercontaining phosphoinositide kinase (PIKfyve) had been previously reported to induce extensive cytoplasmic vacuolization in some cell types [39,40]. In A375 melanoma cells, the phenotype induced by a small-molecule PIKfyve inhibitor YM201636 was strikingly similar to that induced by SB202190, including the characteristic accumulation of large vesicles (Figure 4A). As the BRAF inhibitor SB590885 bears significant structural similarity to p38 MAPK inhibitors SB202190 and SB203580, we hypothesized that it might also be capable of targeting endolysosomal trafficking. Indeed, we also observed the accumulation of large vacuole-like vesicles in SB590885-treated A375 melanoma cells (Figure 4A). Furthermore, a small molecule inhibitor of vacuolar H+ ATPase bafilomycin A1 that can prevent the formation of vacuoles in response to PIKfyve inhibition [41] also blocked the vacuolization induced by pyridinyl imidazole compounds (Figure 4A). Figure 4. Phenotype induced in melanoma cells by pyridinyl imidazole compounds partly mimics PIKfyve inhibition. A375 cells were treated 24 h with pyridinyl imidazole inhibitors SB202190 (SB202; 15 µ M),SB590885(SB590; 5 µ M),andPIKfyveinhibitorYM201636(YM;1 µ M).( A )Aphase-contrastanalysis of the vacuolization induced by the inhibitors and the effect of the cotreatment with bafilomycin A (BafA; 50 nM). Scale bar: 50 µ m. Vacuoles per cell were quantified using ImageJ/Fiji (find maxima—bright spots above a certain threshold) and presented in the graph. Dying rounded cells were excluded from the analysis. Three independent experiments showed a similar effect of the addition of BafA. ( B ) Confocal microscopy analysis of fluorescent protein-labeled endosomal marker (mCherry-Endo-14). Scale bar: 20 µ m. The area of mCherry-positive vacuolar structures was quantified in each treatment and plotted in the graph. Three independent experiments showed similar results. ( C ) Confocal fluorescence microscopy detection of endogenous phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Scale bar: 10 µ m. Three images of different parts of the same specimen were acquired and analyzed using ImageJ/Fiji. Images were thresholded by the signal intensity, and the amount of PI(3,5)P2 foci per cell was determined. Similar results were obtained in two independent experiments. Cancers 2020,12, 1516 9 of 24 Next, we ectopically expressed the mCherry-tagged endosomal marker RhoB (mCherry-Endo-14) in A375 melanoma cells. Using confocal fluorescence microscopy, we found that it localized to the membranes of vesicles induced by the inhibitor of PIKfyve as well as vesicles induced by the pyridinyl imidazole compounds (Figure 4B). PIKfyve is responsible for the synthesis of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) [ 42 ], and this lipid appears to be critical for the proper maturation of endosomes [ 43 ]. To our surprise, the cellular staining patterns detected by immunofluorescence with an anti-PI(3,5)P2 antibody in YM201636and pyridinyl imidazole-treated A375 cells were very similar (Figure 4C). Collectively, these results suggested that the pyridinyl imidazole compounds and the PIKfyve inhibitor YM201636 caused a very similar defect in endocytosis, potentially by targeting similar signaling pathways. Nevertheless, whether these changes might impact on the growth and survival of cancer cells remained unclear. 2.4. Pyridinyl Imidazole Compounds Induce Changes in mTOR Subcellular Localization in A375 Melanoma Cells Bridges et al. reported that Raptor, an essential subunit of mTORC1, could interact with PI(3,5)P2, and PIKfyve was necessary for the subcellular localization and activation of mTORC1 in 3T3-L1 adipocytes [ 40 ]. The activation of mTORC1 requires the translocation of the mTOR kinase to the lysosomalsurface [ 22 ]. Theprocessinvolvesinteractions between lysosomalv-ATPaseand apentameric protein complex called Ragulator [ 44 ]. This complex possesses a guanine nucleotide exchange factor (GEF) activity towards the Rag GTPases that can recruit mTORC1 for activation at the lysosomal surface [19,45,46]. We hypothesized that changes induced in the endolysosomal compartment of melanoma cells in response to PIKfyve inhibition and pyridinyl imidazole compounds might interfere with mTOR subcellular localization. To study this possibility, we expressed in A375 cells EGFP-tagged lysosomal Ragulator complex protein p18/LAMTOR1 and analyzed its colocalization with endogenous mTOR kinase using fluorescence microscopy. Protein p18/LAMTOR1 serves as a scaffold for the assembly of the Ragulator–Rag GTPase complex and is responsible for its anchoring to the lysosomal membrane [ 47 ]. The mTOR-p18/LAMTOR1 colocalization pattern was also analyzed in cells treated with drugs targeting the mTORC1 kinase (Rapamycin), p38 MAPK (BIRB-796), and ERK MAPK signaling (U0126, Vemurafenib). Even though the A375 melanoma cells were grown in full media, the PIKfyve inhibitor, as well as the pyridinyl imidazole compounds SB202190 and SB590885, all seemed to disrupt lysosomal mTOR targeting as the mTOR staining pattern changed from dot-like structures to diffuse cytoplasmic staining (Figure 5A). Importantly, a switch to mTOR diffuse staining pattern upon the extended treatment was not observed in response to the p38 MAPK inhibitor BIRB796, the MEK kinase inhibitor U0126, or the BRAF inhibitor vemurafenib. The results indicated that the observed effect of pyridinyl imidazole compounds was likely not linked to their capacity to inhibit p38 and ERK MAPK signaling pathways (Figure 5A). Interestingly, the results of the confocal microscopy suggested that p18/LAMTOR1 might not be evenly distributed on the surface of enlarged endolysosomes induced by pyridinyl imidazoles. The Ragulator complex protein appeared to form clusters on the surface of the large vacuole like structures (Figure 5A). A detailed time-lapse microscopy analysis indicated that p18/LAMTOR1 might preferentially cluster at the interface of the large vacuoles (Video S2). The mTORC1 activity directly regulates the subcellular localization of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis [ 48 ]. Starvation, disruption of lysosomal function, and pharmacological inhibition of mTORC1 can stimulate TFEB-dependent transcription by promoting TFEB nuclear translocation [ 49 , 50 ]. Interestingly, PIKfyve inhibition was also shown to induce nuclear accumulation of TFEB [ 51 , 52 ]. In our experiments, the PIKfyve inhibitor YM201636 and pyridinyl imidazole inhibitors SB202190 and SB590885 all promoted nuclear localization of TFEB (Figure 5B), indicating that the drug-induced disruption of mTOR lysosomal tethering could inhibit mTORC1 activity in melanoma cells. Cancers 2020,12, 1516 16 of 24 4. Materials and Methods 4.1. Cell Culture and Treatments Human melanoma cell lines A375, G361, COLO-800, MEL-JUSO, SK-MEL-30, and IPC-298 were purchased from the European Collection of Cell Cultures (ECACC; Salisbury, UK). All cell lines were maintained at 37 ◦ C in a humidified atmosphere containing 5% CO2. IPC-298, MEL-JUSO, COLO-800, and A375 cells were cultured in RPMI-1640 (Sigma-Aldrich, Prague, Czech Republic). G361 cells were propagated in McCoy 0 s 5a (Thermo Fisher Scientific, Prague, Czech Republic) and SK-MEL-30 in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific). Growth media were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 IU/mL), and streptomycin (100 µ g/mL). The reporter cell line for measuring ERK pathway activity was prepared by stable transfection of A375 cells with pKrox24(MapErk)Luc plasmid construct [ 35 ]. Cells were regularly checked for mycoplasma contamination using Mycoplasma Detection Kit (Biotool, Munich, Germany) and DAPI staining followed by fluorescence microscopy. The following compounds were used for cell treatments: SB202190, SB239063, sucrose, EIPA, thapsigargin, and tunicamycin (Sigma-Aldrich); U0126 (Wako Chemicals, Neuss, Germany); SB203580, BIRB796, PD184352, YM201636, and rapamycin (Selleckchem, Munich, Germany); vemurafenib (Tinib-Tools, Olomouc, Czech Republic); SB590885 (MedChemExpress, Monmouth Junction, NJ, USA); and puromycin (Cayman Chemical, Ann Arbor, MI, USA). Stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO). Inhibitor stocks were diluted in pre-warmed cell culture medium and added to the cells. Controls received the corresponding amount of the vehicle. 4.2. Western Blotting Cells were lysed in 2 × Laemli sample buffer, and proteins in total cell lysates were separated by SDS-polyacrylamide gel electrophoresis (10 or 15% acrylamide) using mini vertical electrophoresis unit SE250 (Hoefer, Holliston, MA, USA). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Prague, Czech Republic) in the Trans-Blot SD semi-dry transfer system (Bio-Rad, Prague, Czech Republic). Membranes were blocked with 5% non-fat milk in tris-buffered saline +0.1% Tween 20 (TBST) for one hour at room temperature and incubated with primary antibodies overnight at 4 ◦ C. The next day, membranes were washed 3 × 10 min in TBST and incubated with secondary antibodies for one hour at room temperature. Proteins of interest were visualized with enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific) in the G:BOX detection system (Syngene, Cambridge, UK). The intensity of bands was quantified using ImageJ/Fiji. Original data are available in Figure S7. Primaryantibodies usedfor Westernblot: mouseantiα -tubulin (B-7; sc-5286), rabbitanti-pMEK1/2 (sc-7995), goat anti-MEK1 (C-18; sc-219), mouse anti-BRAF (F-7; sc-5284) (Santa Cruz Biotechnology), rabbit anti-p70 S6K (#2708), rabbit anti-phospho-p70 S6K (#9234), rabbit anti-phospho-S6 Ser235/236 (#4858), rabbitanti-S6(#2217), rabbitanti-phospho-ERK1/2T202/Y204 (#4370), rabbitanti-ERK1/2(#9102) (Cell Signaling Technology, Danvers, MA, USA), mouse anti-Puromycin (MABE343) (Sigma-Aldrich), and mouse anti-PCNA (PC-10), kindly provided by Dr. Boˇrivoj Vojtˇešek (Masaryk Memorial Cancer Institute, Brno, Czech Republic). Secondary antibodies conjugated to horseradish peroxidase (HRP): donkey anti-rabbit (sc-2357), anti-mouse (sc-516102), and anti-goat (sc-2020) (Santa Cruz Biotechnology, Heidelberg, Germany). All antibodies were used according to the manufacturers’ recommendations. 4.3. In Vitro Kinase Assays Melanoma cells (A375) were seeded at 10 cm plate, washed once with ice-cold phosphate-buffered saline (PBS), scraped out, and lysed as previously described [ 82 , 83 ]. The lysate was centrifugated at 13,000 × gfor 25 min at 4 ◦ C. Mouse anti-BRAF (sc-5284; Santa Cruz Biotechnology) antibody was added (1 µ g per each sample) to the lysate and incubated for 1–2 h at 4 ◦ C on a slow rotator. Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) were added and incubated for another Cancers 2020,12, 1516 17 of 24 2–3 h at 4 ◦ C on a slow rotator. Beads–immune complexes were washed as previously described [ 82 , 83 ]. The kinase assay was performed in the presence of 20 µ M ATP and 500 ng of a recombinant human inactive MEK-1 as the substrate (Life Technologies, Prague, Czech Republic). A corresponding amount of DMSO (control) or SB202190 (5 µM) was added to the reaction performed for 40 min at 30 ◦C with gentle agitation. The kinase reactions were stopped by the addition of 20 µ L 2 × SDS sample buffer, analyzed using Western blot, and density of bands was quantified via ImageJ/Fiji. 4.4. Transient Transfections and Fluorescence Microscopy The day after seeding the cells to a glass coverslip, cells were transfected using TurboFect transfection reagent (Thermo Fisher Scientific) or FuGENE HD (Promega, Madison, WI, USA) following the manufacturer’s instructions. Twenty-four hours post transfections, cells were treated for the indicated time, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X, and incubated with primary antibodies at 4 ◦ C overnight. Secondary antibodies were used at room temperature for one hour. Then, coverslips were stained with DAPI (sc-3598; Santa Cruz Biotechnology) and washed three times with PBS. Images were acquired using an inverted confocal microscope Carl Zeiss LSM 700 (Jena, Germany) with Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Zeiss) and processed in ZEISS ZEN Microscope Software or ImageJ/Fiji. Quantification was performed using at least three technical replicates to take into account the variability owing to differences in microscopy settings. All analyses were performed in at least two independent experiments. Live cell imaging: Melanoma cells (A375) were seeded on MatTek 35 mm glass-bottom dishes (MatTek Corporation, Ashland, MA, USA), and the next day transfected using FuGENE HD (Promega) according to the manufacturer’s instructions. Cells were washed four hours later and treated with the inhibitor (SB202190, 15 µ M) for 24 h. For imaging, the media was replaced with warm Live Cell Imaging buffer (Invitrogen) containing the inhibitor (SB202190, 15 µ M) and supplemented with 20 mM glucose. Live cell imaging was performed on the Deltavision OMX V4 microscope (GE Healthcare) equipped with three water-cooled PCO.edge sCMOS cameras, a solid-state light source, and laser-based autofocus. Heated stage and an objective heater (20/20 Technologies, Wilmington, NC, USA) provided environmental control conditions. Images were deconvolved using softWoRx software and processed in ImageJ/Fiji [84,85]. Dyes used for cell staining: LysoTracker Green DND-26 (Thermo Fisher Scientific), Acridine Orange hemi(zinc chloride) salt (A6014, Sigma-Aldrich). The following primary antibodies were used for immunofluorescence: rabbit anti-mTOR (#2983), rabbit anti-Rab5 (#3547; Cell Signaling Technology), mouse anti-PtdIns(3,5)P2 (Z-P035; Echelon Biosciences, Salt Lake City, UT, USA). Anti-rabbit Alexa Fluor 594/488 (A11012/A11008) and anti-mouse Alexa Fluor 488 (A11001) (Life Technologies) were used as secondary antibodies. List of plasmids: mCherry-Endo-14 (Addgene, #55040), EGFP-Rab7A (Addgene, #28407), N1-p18-EGFP (Addgene, #42334), and EGFP-N1-TFEB (Addgene, #38119). 4.5. Quantitative Phase Imaging Analysis Using Coherence-Controlled Holographic Microscopy Transmitted-light coherence-controlled holographic microscope (CCHM) was used to evaluate the distribution of the dry mass (DM) (e.g., proteins, carbohydrates, fats, amino acids, and nucleic acids) density in A375 cells and the drug-induced vacuoles. The microscope was designed and built at the Institute of Physical Engineering (IPE) and the Central European Institute of Technology (CEITEC), Brno University of Technology. The setup of the microscope and the principle behind the image reconstruction have previously been described in detail [ 86 ]. The CCHM quantitative phase images are formed by the phase shift between the object and reference wave detected using the interference of light. At every image point, the phase shift value is directly proportional to the cell dry-mass density [87]. For obtaining the images, we used 20 × /NA =0.4 objectives, a digital CCD camera Astropix 1.4, and software developed at the IPE and CEITEC. The light source was a halogen lamp spectrally restricted by an interference filter (FWHM =10 nm, maximum transmissivity at 650 nm). Cancers 2020,12, 1516 18 of 24 Melanoma cells (A375) were observed live in the imaging chamber with 5% CO2. Before imaging, the culture medium was replaced with an observation medium: Eagle’s minimal essential medium without phenol red (buffered to pH 7.4) (Sigma-Aldrich), supplemented with 10 % FBS and only one-third of the normal concentration of sodium bicarbonate. 4.6. Electron Microscopy A375 melanoma cells were seeded on 35 mm plates, grown for 24 h and harvested 12 h after treatment, washed three times in 0.1 M cacodylate buffer (pH 6.98), and fixed for 2.5 h in 3% glutaraldehyde solution (prepared in 0.1 M cacodylate buffer containing 0.2 M saccharose). Afterward, post-fixation was performed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 2.5 h at room temperature. Individual sections were stained with 2.5% uranyl acetate (6 min), lead citrate (3 min), and observed using transmission electron microscopy (Philips Morgagni, FEI Company, Eindhoven, The Netherlands). 4.7. Quantification of Intracellular Metabolites Using NMR Spectroscopy Acetonitrile extraction was employed to quench cell metabolism and to extract low molecular weight compounds from A375 melanoma cells quantitatively [ 83 , 88 ]. Following removal of acetonitrile via vacuum concentration, dried extracts were resuspended in 550 µ L of D 2 O (Sigma-Aldrich) containing 0.005% sodium 3-(trimethylsilyl)-propionate-2,2,3,3-d4 (TSP) (Sigma-Aldrich) used as both chemical shift reference and internal standard for metabolite quantification. Information on the concentration of metabolites in individual samples was derived from volumes of corresponding signals in 1D 1H NMR spectrum. The assignment of signals in the NMR spectra of individual samples to a metabolite was achieved via a comparison of a sample spectrum with spectra of pure metabolites (Sigma-Aldrich). The 1D 1H spectra were measured at 700 MHz using a Bruker Avance III NMR spectrometer (Bruker, Billerica, MA, USA) equipped with a triple resonance room temperature probe using the zgpr pulse sequence (standard Bruker pulse program library). All spectra were acquired at 20 ◦ C and processed using TopSpin 3.2 (Bruker). To make the comparison of metabolite concentration profiles among various samples possible, the signal intensities in individual samples were normalized to total protein concentration. 4.8. Flow Cytometry and MTT Proliferation Assay For cell viability assay, melanoma cells were collected 48 h post-treatment and washed once with ice-cold PBS. Cell pellets were resuspended in ice-cold PBS, and 1 µ g/mL propidium iodide (PI) (Sigma-Aldrich) was added to the suspension and cell fluorescence was measured using the Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific). As live cells exclude PI, the percentage of dead cells was calculated based on the proportion of PI-positive cells in the population. Uptake of BSA was measured in A375 melanoma cells, seeded in density 200,000 cells per well on a six-well plate. The next day, cells were treated with tested drugs for one hour, and then 10 ug/mL of DQ Red BSA (Thermo Fisher Scientific, D12051) was added to the medium for 30 min. Afterward, cells were chased for 75 min in DQ-BSA free medium (with inhibitors) and collected. Flow cytometry analysis was performed on LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) using FACS Diva (BD Biosciences) software. MTT assay was used to measure cellular metabolic activity as an indicator of cell viability and proliferation of melanoma cell lines (A375, G361, COLO-800, MEL-JUSO, SK-MEL-30, IPC-298). Cells were seeded at a density of 1–2 × 10 3 cells/well in a 96-well plate and grown overnight. After treatment with inhibitors for 48 h, cells were incubated with 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 h at 37 ◦ C. Afterward, cells were centrifugated, and the water-insoluble formazan product was dissolved in DMSO (200 µ L/well). The absorbance at 570 nm was determined using a microplate reader VersaMax (Molecular Devices, San Jose, CA, USA). Cancers 2020,12, 1516 19 of 24 4.9. Statistical Analysis The analyses were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Three or more independent experiments were performed for each data set, represented as mean +SD. Statistical analysis was done using Student’s t-test or analysis of variance (ANOVA) when multiple samples were compared. Values of * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 were considered statistically significant. 5. Conclusions We identified pyridinyl imidazole compounds SB2020190, SB203580, and SB590885 as dual inhibitors of mutant BRAF kinase and mTOR signaling in melanoma cells. The dual targeting of essential pro-growth pathways in melanoma cells indicates the potential for the development of BRAF inhibitors based on the pyridinyl imidazole core that could be less prone to the development of acquired drug resistance. Moreover, the disruption of the endolysosomal compartment by pyridinyl imidazole drugs can sensitize melanoma cells to ER stressors, further underscoring their therapeutic potential. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/6/1516/s1, Figure S1: Additional independent replicates for all Western blots presented in this study; Figure S2: Pyridinyl imidazole compounds inhibit ERK signaling in A375 melanoma cells; Figure S3: Analysis of the proliferation of BRAFand NRAS-mutated melanoma cell lines in the presence of pyridinyl imidazole compounds; Figure S4: Coherence-controlled holographic microscopy; Figure S5: Immunofluorescence microscopy; Figure S6. Nutrient uptake was negatively affected in A375 melanoma cells treated with pyridinyl imidazole compounds; Figure S7: Uncropped original Western blots; Video S1: SB202190 disrupts late endosomal trafficking in melanoma cells: Deconvolved wide-field live-cell fluorescence microscopy of A375 melanoma cells transiently transfected with a plasmid construct encoding EGFP-labeled late endosomal marker RAB7A (EGFP-Rab7A); Video S2: p18/LAMTOR1 clusters at contact sites of SB202190-induced vacuole-like vesicles: Live-cell imaging was performed using A375 cells transiently transfected with a plasmid construct encoding EGFP-labeled LAMTOR1 (N1-p18-EGFP). Author Contributions: Conceptualization, V.P. and S.U.; validation, V.P., T.R., M.K. (Michaela Krafˇc í kov á ), and S.U.; formal analysis, V.P., S.U., and K.O.S.; investigation, V.P., T.R., T.V., M.M., L.C., M.K. (Miriama Krut á ), M.S., H.H., I.S., H.U., A.K., V.R., K.O.S., and S.U.; resources, A.V., L.T., R.C., M.S., and P.V.; writing—original draft preparation, V.P. and S.U.; writing—review & editing, V.P. and S.U.; visualization, V.P., T.R., and K.O.S.; supervision, V.R., L.T., R.C., P.V., K.O.S., and S.U. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the European Regional Development Fund—Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868), the Specific University Research (MUNI/A/1087/2018), the Czech Science Foundation (18-01396S), and the Czech-Bioimaging: National Infrastructure for Biological and Medical Imaging (LM2018129). L.T. was supported by a grant from the Ministry of Health of the Czech Republic (NV19-08-00450). Access to the NMR infrastructure was supported by a grant from the MEYS (CIISB – LM2018127). Acknowledgments: We want to thank Dobromila Klemov á for electron microscopy sample preparation; Lucie Šupol í kov á for technical assistance; Boˇrivoj Vojtˇešek (Masaryk Memorial Cancer Institute, Brno, Czech Republic) for the anti-PCNA antibody; and Pavel Krejˇc í , Michael Davidson, Qing Zhong, David Sabatini, and Shawn Ferguson for providing plasmid constructs. The Flow Cytometry Core Facility at Oslo University Hospital is gratefully acknowledged for help with the DQ-BSA uptake assay. We also thank the Core Facility for Advanced Light Microscopy at Oslo University Hospital for help with live-cell imaging. Conflicts of Interest: The authors declare no conflict of interest. References 1. Miller, A.J.; Mihm, M.C., Jr. Melanoma. N. Engl. J. Med. 2006,355, 51–65. [CrossRef] [PubMed] 2. Berger, M.F.; Hodis, E.; Heffernan, T.P.; Deribe, Y.L.; Lawrence, M.S.; Protopopov, A.; Ivanova, E.; Watson, I.R.; Nickerson, E.; Ghosh, P.; et al. 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