Fig 2: CD36+ MAPKi-tolerant melanoma cells have retained peroxisome levels and UGCG.(A) Heatmap showing relative expression of indicated melanoma cell-state-specific markers, a panel of peroxisomal genes, and PPARGC1A in 4 drug-tolerant melanoma populations in the MEL006 PDX model during early dabrafenib+trametinib treatment (day 4). (B) Expression of CD36 and a peroxisomal gene signature, UGCG, GBA, and PPARGC1A (HTSeq-FPKM) in the GDC TCGA Melanoma data set (SKCM, n = 472). Spearman’s rank-order. (C and D) Violin plots of scRNA-seq data highlighting the distribution of (C) a gene signature indicating cancer cell metabolic activity and (D) indicated peroxisomal genes and UGCG in CD36– (<2.2) versus CD36+ (≥2.2) cells, before or after dabrafenib+trametinib treatment for 28 days (MRD). (E) Schematic of experimental design for panels F and G. (F) Relative expression of AGPS (left) and UGCG (right) in CD36– and CD36+ cell populations isolated from A375M melanoma xenografts following vehicle (n = 4) or PLX4720+cobi (n = 7) treatment for 8 days. (G) Fold change in AGPS (left) and UGCG (right) transcripts in CD36+ cells relative to CD36– cells isolated from A375M melanoma xenografts following PLX4720+cobi treatment for 8 days. RPLP0 was used as a reference gene. Cells from a total n = 7 tumors were pooled before sorting. Two-sided unpaired t test. (H) Schematic of experimental design for I. (I) Relative expression of AGPS (left) and UGCG (right) in CD36– versus CD36+ A375M cells following treatment with DMSO, vemu (2.5 μM), or vemu (2.5 μM) combined with cobi (100 nM) for 96 hours. Significance assessed with 2-way ANOVA (C, D, F, and I). Data in F–I represent mean ± SD.

Fig 3: Increased peroxisomal and UGCG activity commonly occurs in melanomas that rapidly acquire resistance to MAPK-targeted therapy.(A) Normalized gene expression of CD36 (left), relative expression of a peroxisomal gene signature (right), and (B) expression of AGPS, SCP2, PEX1, and UGCG in a cohort of melanoma samples (n = 17 out of a total of 22 patients, Kwong 2015 data set) collected before, while on, or relapsed on MAPK-targeted therapy showing an overall trend of CD36 induction upon MAPKi. Data represent mean + SEM. One-way ANOVA. (C and D) Normalized abundance of indicated lipid species grouped by carbon chain length detected in (C) MEL006 PDX tumor samples collected before (Pre, n = 6) or relapsed (Res, n = 5) on dabrafenib+trametinib treatment, or (D) 451Lu parental (Par) cells versus vemu-resistant 451Lu-R cells (n = 3). Two-way ANOVA. PE-O, 1-alkyl,2-acylphosphatidylethanolamine; PC-O, 1-alkyl,2-acylphosphatidylcholine. (E) Fold change in AGPS and UGCG mRNA levels in a panel of MAPKi-resistant melanoma cells relative to their corresponding parental cells, normalized to ACTB as a reference gene (n = 4). Data in C–E represent mean ± SD. (F) Western blot analysis of the indicated proteins in a panel of parental versus MAPKi-resistant melanoma cells. (G) Waterfall plots showing the best overall response and (H) Kaplan-Meier curves showing PFS of melanoma patients treated with MAPK-targeted therapy. Risk score ranging between 0 and 3 was calculated based on expression of CD36, AGPS, and UGCG before and after treatment (see Supplemental Table 5 for detailed information). Patients were subsequently grouped into high-risk (risk score ≥2) versus low-risk (risk score ≤1). Values in G represent percentage response of individual patient from baseline. Significance assessed by 2-sided unpaired t test (E and G) or log-rank test (H).

Fig 4: NNC 55-0396 and PPMP potentiate melanoma response to combined BRAF/MEK inhibition.(A) Schematic of experimental design for B–E. (B) Tumor growth curve and (C) waterfall plots showing the STR of A375M-derived melanomas to PLX4720+cobi treatment. Tumors were allowed to grow to a volume of 750 mm3 before indicated treatments started. (D) Percentage of CD36+ cells and (E) relative AGPS expression among total (CD45–) tumor cells, isolated from A375M-derived melanomas following indicated treatment for 10 days. (F) Kaplan-Meier curves showing OS of mice bearing 1205Lu-VCDR–derived melanomas treated with NNC+PPMP or vehicle. All mice were kept on PLX4720 chow and simultaneously treated with cobi. NNC+PPMP treatment started when PLX4720/cobi dual-resistant tumors first reached a volume of 250 mm3. Detailed experimental design and individual tumor growth curves are shown in Supplemental Figure 9, E and F, respectively. In B–F, the number of biological replicates (mice) is indicated in each graph. Data in B, D, and E represent mean ± SEM. Values in C represent percentage volume change of each tumor from baseline. Significance assessed by 2-way ANOVA (B), 2-sided unpaired t test (C–E), or log-rank test (F).

Fig 5: Peroxisomes and UGCG are required for survival of CD36+ MAPKi-tolerant melanoma cells.(A) Schematic of experimental design for (B–E). A375M cells were treated with vemu (2.5 μM) for 48 hours before CD36 staining and subsequent sorting. (B) Fold change in the indicated mRNAs in vemu-exposed CD36+ A375M cells relative to CD36– A375M cells, normalized to RPLP0 as a reference gene (n = 4). The SCP2 primers amplify the N-terminus of the transcript initiated from the proximal promoter, encoding the peroxisome-specific protein SCPx. (C) Number of ABCD3 puncta and (D) relative UGCG expression in vemu-exposed CD36+ versus CD36– A375M cells. Representative immunofluorescent staining for (C) CD36 (red), ABCD3 (green), and DAPI (blue) or (D) CD36 (red), UGCG (green), and DAPI (blue) are presented. Red arrows highlight cells stained positive for CD36. (E) Percentage apoptosis (left) and representative images (right) of vemu-exposed CD36+ versus CD36– A375M cells following PEX3 knockdown and/or PPMP treatment (n = 5). Scale bars: 10 μm (C), 20 μm (D), and 50 μm (E). (F–H) Percentage of CD36+ populations in (F) A375M cells following PEX3 knockdown and the indicated treatment (n = 3), (G) A375M-Ctrl versus PEX3-KO AG3 cells upon indicated treatment (n = 3), or (H) A375M-Ctrl versus PEX3-KO AG3 cells following UGCG knockdown and subsequently treated with vemu (n = 4). Significance assessed by 2-sided unpaired t test (B–D) or 2-way ANOVA (E–H). Data represent mean ± SD.