Abstract Induction of ferroptosis is an emerging strategy to suppress melanoma progression. Strategies to enhance the sensitivity to ferroptosis induction would be a major advance in melanoma therapy. Here, we used a drug synergy screen that combined a ferroptosis inducer, RSL3, with 240 anti-tumor drugs from the FDA-approved drug library and identified lorlatinib to synergize with RSL3 in melanoma cells. We further demonstrated that lorlatinib sensitized melanoma to ferroptosis through inhibiting PI3K/AKT/mTOR signaling axis and its downstream SCD expression. Moreover, we found that lorlatinib's target IGF1R, but not ALK or ROS1, was the major mediator of lorlatinib-mediated sensitivity to ferroptosis through targeting PI3K/AKT/mTOR signaling axis. Finally, lorlatinib treatment sensitized melanoma to GPX4 inhibition in preclinical animal models, and melanoma patients with low GPX4 and IGF1R expression in their tumors survived for longer period. Altogether, lorlatinib sensitizes melanoma to ferroptosis by targeting IGF1R-mediated PI3K/AKT/mTOR signaling axis, suggesting that combination with lorlatinib could greatly expand the utility of GPX4 inhibition to melanoma patients with IGF1R-proficient expression. Keywords: Melanoma, Ferroptosis, Lorlatinib, Synergy, IGF1R 1. Introduction Ferroptosis is an iron-dependent and non-apoptotic form of programmed cell death characterized by lethal accumulation of lipid peroxides [[37]1,[38]2]. It has been well documented that therapy-resistant tumor cells, particularly those of the mesenchymal-like state and prone to metastasis, are highly susceptible to ferroptosis [[39]3,[40]4]. These works highlighted induction of ferroptosis by the inhibition of glutathione peroxidase 4 (GPX4) as a promising strategy for cancer treatment [[41]2,[42]5]. However, the sensitivity of ferroptosis varies greatly among cancer cells, and melanoma is relatively insensitive to erastin-induced ferroptosis, compared with other tumors, especially diffuse large B cell lymphoma and renal cell carcinomas [[43]2,[44]6]. Therefore, there is mounting interest in explore the mechanisms that underpin the sensitivity of melanoma cells to ferroptosis. Several excellent studies have clarified multiple, complex and inter-related signaling pathway to regulate the susceptibility of ferroptosis to melanoma. For example, some microRNAs such as miR-9 and miR-137 affect the sensitivity of ferroptosis by regulating glutamine catabolism [[45]7,[46]8]. Moreover, some proteins regulated by ferroptosis induction, including aldo-keto-reductase-1C (AKR1C) 1/2/3, neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) and calcium/calmodulin dependent protein kinase 2 (CAMKK2), render melanoma cells not responsive any longer to ferroptosis through degrading the 12/15-LOX-generated lipid peroxides [[47]9], down-regulating voltage dependent anion channel 2/3 (VADC2/3) expression [[48]10], or activating the AMP-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway [[49]11], respectively. Additionally, sterol regulatory element binding transcription factor 2 (SREBP2) or oleic acid protect circulating melanoma cells or melanoma cells in lymph from ferroptosis by inducing transferrin expression or reducing the amount/density of polyunsaturated fatty acids (PUFA) available for oxidation in membranes [[50]12,[51]13]. However, drugs targeting these ferroptosis suppressors are far from being used in the clinic, highlighting the significance of screening the FDA-approved drug library to synergize with GPX4 inhibition in melanoma cells. Lorlatinib is an FDA-approved, third-generation, ATP-competitive small-molecule tyrosine kinase inhibitor for the treatment of non-small cell lung cancer caused by an abnormal anaplastic lymphoma kinase (ALK) gene [[52]14]. However, the role of lorlatinib in melanoma and whether it is involved in the regulation of ferroptosis sensitivity are completely unknown. Here, we identified lorlatinib to synergize with GPX4 inhibition in melanoma cells from 240 FDA-approved anti-tumor drugs. Mechanistically, lorlatinib sensitizes melanoma to ferroptosis through targeting insulin like growth factor 1 receptor (IGF1R)-mediated PI3K/AKT/mTOR signaling axis and its downstream stearoyl-CoA desaturase 1 (SCD) expression. Consistently, melanoma patients with low GPX4 and IGF1R expression in their tumors survive for longer period. Thus, lorlatinib-mediated IGF1R inhibition plays a critical role in promoting melanoma ferroptosis, indicating that combination with lorlatinib could greatly expand the utility of GPX4 inhibition to melanoma patients with IGF1R-proficient expression. 2. Results 2.1. Identification of lorlatinib to synergize with GPX4 inhibition in melanoma The sensitivity of ferroptosis varies greatly among cancer cells ([53]Fig. S1A). To uncover clinically applicable drugs that synergize with GPX4 inhibition in melanoma, we performed a screening of 240 anti-tumor drugs identified from the FDA-approved drug library combined with ferroptosis inducer - RSL3 using in-vitro drug combination assay ([54]Fig. 1A). The coefficient of drug interaction (CDI) was applied to evaluate the effect of combined medication [[55]15]. Lorlatinib was identified as one of the most potential drugs in both A375 and SK-MEL-28 cells, in addition to several drugs including regorafenib and its monohydrate [[56]16], temsirolimus [[57]17], sorafenib and sorafenib tosylate [[58]16], which have been reported to synergize with ferroptosis inducers in cancer cells ([59]Fig. 1B–C). Consistent with previous findings, our data also demonstrated that sorafenib synergized with RSL3 in melanoma ([60]Figs. S1B–C), thus supporting the validity of our screens. To further clarify whether lorlatinib synergizes with RSL3, we conducted a series of 6 × 6 screening experiments in both A375 and SK-MEL-28 cells, indicating a strong synergy ([61]Fig. 1D–E). RSL3 functions primarily through binding and inactivation of peroxidase activity of GPX4 [[62]2]. To further support our findings, we constructed GPX4 knockout melanoma cell lines ([63]Fig. 1F), and found that GPX4 deficient melanoma cells was vulnerable to lorlatinib in a dose-dependent manner ([64]Fig. 1G), suggesting that lorlatinib sensitizes melanoma to GPX4 inhibition. To visualize the morphological features after drugs treatment, we performed live cell time-lapse imaging. We observed that lorlatinib had minimal toxic effects on melanoma cells. However, after 6 h, melanoma cells cotreated with lorlatinib and RSL3 experienced more cell death which shared morphological characteristics of necrosis, including cell rounding, swelling and plasma membrane rupture ([65]Fig. 1H). Fig. 1. [66]Fig. 1 [67]Open in a new tab Identification of lorlatinib to synergize with GXP4 inhibition in melanoma. (A) Schematic of the identification of clinically applicable drug from the FDA-approved drug library that sensitize melanoma to RSL3. (B-C) Summary scatter plot of CDI in A375 (B) and SK-MEL-28 (C) cells indicating lorlatinib as one of the most potential drugs that synergize with RSL3. Indicated was the drugs that have been reported to synergize with RSL3. (D-E) Percentage of inhibition rate was presented in a series of 6 × 6 screening experiments in A375 (D) and SK-MEL-28 (E) cells. Synergy was evaluated by Chou-Talalay combination index (CI) for lorlatinib and RSL3 across the indicated cell lines. The x axis of CI plots represents fraction affected. (F) GPX4 protein levels were quantified by western blotting in control (sgCtrl) and GPX4 deficient (sgGPX4) cells. (G) Cell viability of GPX4 deficient cells treated with different concentrations of lorlatinib for 12 h. (H) Cell morphological features at different time point after the indicated treatment. Lorlatinib, 5 μM; RSL3, 2.5 μM. Images were taken at 200X magnification. P values were calculated using two-way ANOVA analysis. ***, P < 0.001. 2.2. Combination of lorlatinib and GPX4 inhibition drives melanoma ferroptosis To further determine the type of cell death driven by lorlatinib and RSL3, we cotreated the cells with various cell death inhibitors. We found that the toxic effect of the combination therapy could be completely negated by the anti-oxidant N-acetyl-cysteine (NAC) and the iron chelator deferoxamine (DFO), but not by inhibitors of apoptosis (Z-VAD-FMK), necroptosis (Nec-1s), or autophagy (CQ) ([68]Fig. 2A–B), suggesting that combination of lorlatinib and RSL3 drives melanoma ferroptosis. To further support the notion, we used four different approaches to assess whether ferroptosis was induced by lorlatinib and RSL3 co-treatment. First, more ferroptosis inhibitors including ferrostatin-1, liproxstatin-1 and DFO could reverse the cell death in GPX4-deficient melanoma cells cotreated with lorlatinib ([69]Fig. 2C–D). Second, mRNA levels of CHAC1 and PTGS2, markers for assessment of ferroptosis, were notably increased after lorlatinib and RSL3 co-treatment ([70]Fig. 2E–F). Third, malondialdehyde (MDA), aldehyde secondary products of lipid peroxidation, and lipid peroxidation, the hallmark of ferroptosis, were markedly increased in combination group, compared with other groups ([71]Fig. 1G–H). Fourth, transmission electron microscopy analysis displayed a striking ferroptosis-associated morphologic change in melanoma cells with combination therapy, characterized by shrunken mitochondria with increased membrane density and reduced numbers of mitochondrial cristae ([72]Fig. 1I). Collectively, these findings suggest that co-treatment of lorlatinib and RSL3 leads to melanoma ferroptosis. Fig. 2. [73]Fig. 2 [74]Open in a new tab Combination of lorlatinib and GPX4 inhibition drives melanoma ferroptosis. (A-B) Indicated melanoma cells were treated with lorlatinib (5 μM), RSL3 (2.5 μM), or a combination of both drugs with or without cell death inhibitors (ZVAD-FMK, 5 μM; Necrostatin-1s, 10 μM; CQ, 10 μM; NAC, 1 mM; DFO, 100 μM) for 6h, and cell viability was assessed. (C) GPX4 deficient cells treated with different concentrations of lorlatinib for 12 h in the absence or presence of Fer-1 (2 μM), Lip-1 (10 μM) or DFO (100 μM). (D) Cell death of GPX4 deficient cells induced by the indicated treatment were shown by microscope and quantified by PI-staining coupled with flow cytometry. Lorlatinib, 5 μM; RSL3, 2.5 μM. (E-F) Real-time PCR analysis of CHAC1 (E) and PTGS2 (F) expression in A375 and SK-MEL-28 cells after the indicated treatment for 6 h. Lorlatinib, 5 μM; RSL3, 2.5 μM. (G) Analysis of MDA in A375 and SK-MEL-28 cells after the indicated treatment for 6 h. Lorlatinib, 5 μM; RSL3, 2.5 μM. (H) Lipid ROS were quantified with BODIPY-C11 using flow cytometry. Cells were treated as indicated for 6 h. Lorlatinib, 5 μM; RSL3, 2.5 μM. (I) Transmission electron microscopy images of A375 cells after the indicated treatment for 6 h. Lorlatinib, 5 μM; RSL3, 2.5 μM. Scale bar, upper, 2 μm; lower, 500 nm. One-way ANOVA analysis was performed in B, D, E, F, G. ***, P < 0.001. 2.3. Lorlatinib sensitizes melanoma to ferroptosis through SCD To illuminate the underlying mechanism by which lorlatinib enhance RSL3-mediated ferroptosis, we pretreated melanoma cells with lorlatinib overnight and then replaced with new medium only containing RSL3, finding that the effect of promoting ferroptosis still existed ([75]Fig. S1D). This result suggested that pretreatment with lorlatinib puts melanoma cells in a state that is sensitized to ferroptosis. Several pathways have been reported to be associated with the sensitivity of ferroptosis [[76]3,[77]4]. We firstly check the effect of lorlatinib on the SLC7A11/GPX4 axis. As expected, the levels of GSH were dramatically reduced under imidazole ketone erastin treatment and GPX4 activity was significantly abrogated by RSL3 treatment [[78]1,[79]2]. However, lorlatinib treatment did not affect the levels of GSH and GPX4 activity ([80]Fig. 3A–B). Moreover, lorlatinib treatment failed to affect the intracellular levels of iron ([81]Fig. 3C) and the mRNA expression of genes for iron metabolism ([82]Figs. S2A–B), as well as coenzyme Q10 ([83]Fig. 3D), a main downstream of the newly discovered ferroptosis suppressor - FSP1 [[84]18,[85]19]. Furthermore, we generated DHODH and GCH1 deficient melanoma cells ([86]Figs. S2C–D), and found that lorlatinib could still sensitize these cells to ferroptosis ([87]Figs. S2E–F), indicating that lorlatinib-mediated sensitivity to ferroptosis is independent on the expression of DHODH or GCH1, another two newly discovered ferroptosis suppressors [[88]20,[89]21]. Consistent with these findings, lorlatinib treatment had no obvious effects on the protein expression of key regulators in GPX4/GSH axis, iron metabolism, or these ferroptosis suppressors ([90]Fig. 3E). Fig. 3. [91]Fig. 3 [92]Open in a new tab Lorlatinib sensitizes melanoma to ferroptosis through SCD. (A) The relative levels of GSH were assayed in A375 and SK-MEL-28 cells treated with DMSO, lorlatinib (5 μM), or IKE (2.5 μM) for 12 h. (B) Relative glutathione peroxidase activity were quantified in A375 and SK-MEL-28 cells with the indicated treatment for 6 h. Lorlatinib, 5 μM; RSL3, 2.5 μM. (C) Relative Fe^2+ levels in A375 and SK-MEL-28 cells following treatment with DMSO or lorlatinib (5 μM) for 12 h. (D) CoQ10 levels at different time point in A375 cells after treatment with 5 μM lorlatinib, or in A375 cells treated with different concentrations of lorlatinib for 12 h. (E) Western blotting analysis of proteins at different time point in A375 cells after treatment with lorlatinib (5 μM), or proteins in A375 cells treated with different concentrations of lorlatinib for 12 h. (F) Fold change of lipid species in A375 cells treated with 5 μM lorlatinib for 12 h in negative and positive ionization modes. (G) SCD protein levels were quantified by western blotting in control (sgCtrl) and SCD deficient (sgSCD) cells. (H-I) Dose response of RSL3-induced death of sgCtrl and sgSCD cells in the presence of DMSO or lorlatinib (5 μM) for 6 h. (J) SCD protein levels were quantified by western blotting in cells with control (vector) or SCD overexpression (SCD ov). (K) Viability of the indicated cells with control or SCD overexpression after treatment with RSL3 (2.5 μM), lorlatinib (2.5 μM), or RSL3 + lorlatinib. P values were determined using one-way ANOVA analysis in A, B, D. Two-tailed unpaired Student's t-test was performed in C. Two-way ANOVA analysis was performed in K. **, P < 0.01; ***, P < 0.001; ns, no significance. Lipid metabolic processes has been reported to impinge on cells susceptibility toward ferroptosis [[93]22]. We further evaluated the protein expression of genes regulating lipid metabolism, finding that SCD, but not ACSL4 or LPCAT3, was down-regulated by lorlatinib in a time- and dose-dependent manner ([94]Fig. 3E). SCD converts saturated fatty acids to monounsaturated fatty acids (MUFA) and promotes ferroptosis resistance, providing a mechanistic explanation to our observation [[95]23]. In line with this finding, lipidomics showed that lorlatinib could decrease the abundance of some lipids, especially some MUFA - phospholipids (MUFA-PLs) which tend to confer resistance to ferroptosis, while polyunsaturated fatty acyl-PLs (PUFA-PLs) were comparable with or without lorlatinib treatment ([96]Fig. 3F, [97]Fig. S3). Furthermore, we constructed SCD deficient cells ([98]Fig. 3G), and observed that SCD knockout had a strong sensitization effect on RSL3-induced ferroptosis in the absence of lorlatinib, but almost no sensitization effect in the presence of lorlatinib ([99]Fig. 3H–I). Conversely, SCD overexpression rendered melanoma cells resistant to the combination of lorlatinib with RSL3 ([100]Fig. 3J–K). These data indicated that lorlatinib sensitized melanoma cells to RSL3-mediated ferroptosis through inhibiting the expression of SCD. 2.4. Lorlatinib inhibits the expression of SCD via PI3K/AKT/mTOR signaling To elaborate how lorlatinib regulates the expression of SCD, we performed KEGG enrichment analysis based on RNA-seq data ([101]Fig. S4A). Strikingly, as one of the pathways that are affected most by lorlatinib, PI3K/AKT signaling pathway was suggested to play a significant role ([102]Fig. 4A). Gene Set Enrichment Analysis (GSEA) further demonstrated that lorlatinib could significantly inhibit PI3K/AKT/mTOR pathways ([103]Fig. 4B, [104]Fig. S4B). Previous studies have proved that inhibition of PI3K/AKT/mTOR signaling potentiates the cancer therapeutic effect of ferroptosis inducer via SREBP1/SCD-mediated lipogenesis [[105]17]. SREBP1 is a transcription factor and particularly related to fatty acid metabolism [[106]24]. We wondered whether lorlatinib regulated the expression of SCD through a similar mechanism. Interestingly, gene signatures associated with SREBP1 activity and fatty acid metabolism were markedly abrogated by the treatment of lorlatinib ([107]Fig. 4C). In agreement with these findings, lorlatinib treatment caused a decrease of p-PI3K, p-AKT, p-mTOR, p-p70s6, and the level of the mature form of SREBP1 (SREBP1m), which could translocate into the nucleus to regulate its downstream transcriptional targets including SCD, FASN, ACLY and ACACA ([108]Fig. 4D, [109]Figs. S4C–F). Moreover, inhibition of PI3K/AKT/mTOR pathway with PI3K inhibitor (GDC-0941), AKT inhibitor (MK-2206), and mTOR inhibitor (CCI-779) strongly sensitized melanoma to RSL3-induced ferroptosis, but did not further enhance RSL3-induced ferroptosis in the presence of lorlatinib ([110]Fig. 4E–G, [111]Figs. S5A–C). Additionally, a marked degree of sensitization to RSL3 was observed after knockout of SREBP1 in control cells, but not in cells treated with lorlatinib ([112]Fig. 4H–J). Conversely, SREBP1 overexpression protected melanoma cells from ferroptosis induced by the combination of RSL3 with lorlatinib ([113]Fig. 4K-L). These findings suggested that lorlatinib inhibits the expression of SCD via PI3K/AKT/mTOR/SREBP1 signaling. Fig. 4. [114]Fig. 4 [115]Open in a new tab Lorlatinib inhibits the expression of SCD via PI3K/AKT/mTOR signaling. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes between DMSO and lorlatinib treated A375 cells. (B) GSEA showing that PI3K-Akt and MTOR signaling pathways were down regulated in lorlatinib treatment group. (C) GSEA showing that FATTY_ACID_METABOLISM and SREBP1 signaling were down regulated in lorlatinib treatment group. (D) Western blotting analysis of the indicated proteins in A375 cells treated with DMSO or lorlatinib (5 μM) for 12 h. (E) Dose response of RSL3-induced death of DMSO or PI3Ki (GDC-0941) treated- A375 cells in the absence or presence of lorlatinib for 6 h. (F) Dose response of RSL3-induced death of DMSO or AKTi (MK-2206) treated- A375 cells in the absence or presence of lorlatinib for 6 h. (G) Dose response of RSL3-induced death of DMSO or mTORi (CCI-779) treated- A375 cells in the absence or presence of lorlatinib for 6 h. (H) SREBP1 protein levels were quantified by western blotting in control (sgCtrl) and SREBP1 deficient (sgSREBF1) cells. (I-J) Dose response of RSL3-induced death of sgCtrl and sgSREBF1 A375 (I) or SK-MEL-28 (J) cells in the presence of DMSO or lorlatinib (5 μM) for 6 h. (K) SREBP1 protein levels were quantified by western blotting in cells with control (vector) or SREBF1 overexpression (SREBF1 ov). (L) Viability of the indicated cells with control or SREBF1 overexpression after treatment with RSL3 (2.5 μM), lorlatinib (2.5 μM), or RSL3 + lorlatinib. P values were calculated using two-way ANOVA analysis in L. **, P < 0.01; ***, P < 0.001. 2.5. ALK and ROS1 are not the major mediators of lorlatinib-mediated sensitivity to ferroptosis Lorlatinib is well known as an FDA-approved, third-generation, ATP-competitive small-molecule tyrosine kinase inhibitor of ALK/ROS1 [[116]25]. We next sought to determine whether lorlatinib-mediated sensitivity to ferroptosis is dependent on the expression of ALK and ROS1. However, ALK or ROS1 knockdown by shRNA failed to sensitize melanoma cells to RSL3-induced ferroptosis ([117]Figs. S6A–D). Consistently, ALK or ROS1 knockdown by siRNA or knockout by sgRNA still could not sensitize melanoma cells to RSL3-induced ferroptosis ([118]Figs. S6E–I). To further rule out the possibility of ALK and ROS1 as the major mediator of lorlatinib-mediated sensitivity to ferroptosis, we performed a 6 × 6 screening experiments. We observed that ALK inhibitor (alectinib or alectinib hydrochloride) or inhibitors targeting both ALK and ROS1 (entrectinib or brigatinib) failed to synergize with RSL3 in melanoma cells ([119]Figs. S6J–K). Furthermore, ferroptosis induction could still be potently sensitized by lorlatinib even after ALK or ROS1 knockdown. ALK or ROS1 overexpression also failed to reverse the inhibitory effect caused by lorlatinib and RSL3 treatment ([120]Fig. S6L-M). These results demonstrated that lorlatinib-mediated sensitivity to ferroptosis is independent of the expression ALK and ROS1. 2.6. Lorlatinib regulates melanoma susceptibility to ferroptosis and PI3K/AKT/mTOR pathway through IGF1R Lorlatinib is also reported to potently inhibit other tyrosine kinases, including LTK, FER, FES, PTK2B, TNK2, PTK2, NTRK1/2/3, FRK, EGFR, IGF1R, TSSK2, EPHA1, JAK2 and INSR [[121]14]. By analyzing the associations between GPX4 inhibitors and the expression of lorlatinib targets using the Cancer Therapeutics Response Portal, we found that IGF1R is one of these kinases positively correlated with resistance to ferroptosis inducers including RSL3, erastin, ML162, and ML120. As expected, FSP1 and SLC7A11 were positively while ACSL4 were negatively associated with the logIC50 of these ferroptosis inducers ([122]Fig. 5A, [123]Figs. S7A–C). To further evaluate the association between lorlatinib targets and the sensitivity of ferroptosis, we generated these kinases knockout cells using CRISPR/Cas9 technology, respectively ([124]Fig. S7D), finding that IGF1R knockout sensitized melanoma to ferroptosis mostly, but failed to further enhance ferroptosis in the presence of lorlatinib in SK-MEL-28 cells ([125]Fig. 5B–C). This result was further validated in A375 cells ([126]Fig. 5D–E). Consistently, knockdown of IGF1R using shRNA also sensitized melanoma cells to ferroptosis ([127]Fig. 5F–H). Pharmacologically, IGF1R selective inhibitor linsitinib sensitized the effect of RSL3 on the induction of ferroptosis ([128]Fig. S7E), but failed to further enhance RSL3-induced ferroptosis in the presence of lorlatinib ([129]Fig. 5I). Collectively, these finding indicated that IGF1R is the major mediator of lorlatinib-mediated sensitivity to ferroptosis. Fig. 5. [130]Fig. 5 [131]Open in a new tab Lorlatinib regulates melanoma susceptibility to ferroptosis and PI3K/AKT/mTOR pathway through IGF1R. (A) The correlation between logIC50 of RSL3 and gene expression of lorlatinib targets. Red dots, significant positive correlation; blue dots, significant negative correlation; black dots, no significance. (B) Dose response of RSL3-induced death of SK-MEL-28 cells transfected with control sgRNA or sgRNA of lorlatinib targets. SK-MEL-28 cells was sensitive to RSL3 the most when IGF1R was deficient. (C) Dose response of RSL3-induced death of sgCtrl and sgIGF1R SK-MEL-28 cells in the presence of lorlatinib for 6 h. (D) IGF1R protein levels were quantified by western blotting in control (sgCtrl) and IGF1R deficient (sgIGF1R) cells. (E) Dose response of RSL3-induced death of sgCtrl and sgIGF1R A375 cells in the presence of DMSO or lorlatinib (5 μM) for 6 h. (F) IGF1R knock down efficiency in A375 and SK-MEIL-28 cells assessed by real-time PCR (F) and western blotting (G). (H) Dose response of RSL3-induced death of shCtrl and shIGF1R cells. (I) Dose response of RSL3-induced death of DMSO or IGF1R inhibitor (linsitinib) treated- A375 cells in the absence or presence of lorlatinib for 6 h. (J) Gene set variation analysis (GSVA) of TCGA-SKCM segregated by IGF1R expression. High IGF1R group demonstrated significant activation of PI3K/AKT/mTOR related pathways. (K) Western blotting analysis of the indicated proteins in sgCtrl and sgIGF1R A375 cells. (L) Dose response of RSL3-induced death of shIGF1R A375 (upper) and SK-MEL-28 (lower) cells overexpressed vector, SCD or SREBF1. P values were calculated using two-tailed unpaired Student's t-test in F, J. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to