a Representative 16-week-old nf1a^+/−;nf1b^−/−;ptena^+/−;ptenb^−/−;p53^M214K/M214K zebrafish with one spontaneous melanoma (indicated by arrow). b Hematoxylin and eosin (H&E) staining of the melanoma tumor shown in panel a (×5 magnification, scale bar = 200 μm). c Melanoma tumor cells from the black box in (b), magnified ×100. d Melanoma tumor cells that have invaded into the dorsal muscle from the white box in (b), magnified ×100. e Cumulative frequency of spontaneous melanomas arising in zebrafish with the indicated genotypes (generated by the inbreeding of the nf1a^+/−;nf1b^−/−;ptena^+/−;ptenb^−/−;p53^M214K/M214K line, p < 0.0001, log-rank test). f Immunohistochemical analysis of melanoma tumor sections using antibodies to detect phosphorylated ERK1/2 (pERK), phosphorylated AKT (pAKT), phosphorylated S6 (pS6), proliferating cell nuclear antigen (PCNA) and cleaved caspase 3 (CC3) (×63 magnification, scale bar = 20 μm). The percentage of PCNA+ cells was determined by manually counting positive and negative melanoma cells in one representative high-power field (150–200 cells per field) within three independent tumor samples. g Pigmented nf1/pten-mutant melanoma cells were transplanted intraperitoneally into adult rag2^−/− Casper zebrafish. The implanted melanoma cells (left panel, arrow) grew rapidly into secondary tumors (within 2 weeks; right panel).

a Schematic of the melanoma tumor transplantation assay. b, c Transplanted nf1/pten-mutant melanoma tumor cells were monitored daily in 3-week-old rag2^−/− recipient zebrafish treated with DMSO (CTR; n = 12), 80 nM trametinib (n = 11), 2 μM buparlisib (n = 11), or the combination of 80 nM trametinib and 2 μM buparlisib (n = 12) for 6 days. Kaplan–Meier curves for progression-free survival (PFS, b) and overall survival (OS, c) are shown. Statistical analyses were performed by log-rank test, comparing drug-treated with DMSO-treated zebrafish. d, e Transplanted nf1/pten-mutant melanoma tumor cells were monitored daily in 3-week-old rag2^−/− recipient zebrafish treated with DMSO (CTR; n = 12, same values as in b, c), 20 μM sirolimus (n = 12), 20 μM everolimus (n = 11) or 40 μM temsirolimus (n = 11) for 6 days. Kaplan–Meier curves are shown, with statistical analyses performed as in b, c. For all experiments involving drug treatments, drugs were replenished every 2 days during the 6-day course of treatment (black arrows).

Three-week-old rag2^−/− zebrafish transplanted with pigmented nf1/pten-mutant melanoma cells were treated for 6 days with DMSO, 80 nM trametinib, 2 μM buparlisib, the combination of 80 nM trametinib and 2 μM buparlisib, or 20 μM sirolimus. a, c, e, g, and i Representative zebrafish at the end of the 6-day drug treatment. b, d, f, h, and j Representative zebrafish at 4 days following the end of drug treatment. k Quantification of melanotic nf1/pten-mutant tumor-cell area at the end of the 6-day course of drug treatment (left), and 4 days later (right). ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed, unpaired t-test. Scale bar = 1 mm.

a Representative tissue sections from a transplanted nf1/pten-mutant melanoma tumor after 2 days of treatment with DMSO (CTR), 80 nM trametinib, 2 μM buparlisib, the combination of 80 nM trametinib and 2 μM buparlisib, or 20 μM sirolimus. Sections were immunostained using antibodies to detect pERK, pAKT, pS6, PCNA, and cleaved caspase-3 (CC3). pERK-, pAKT- and pS6-positive tumor areas, as well as PCNA-positive nuclei, are quantified post-treatment in (b–e). “T + B” refers to trametinib plus buparlisib. ns p > 0.05, *p < 0.05, **p < 0.01 by Mann–Whitney test. Scale bar = 20 μm.

a Representative tissue sections from a transplanted nf1/pten-mutant melanoma tumor at 4 days after a 6-day drug treatment with DMSO (CTR), 80 nM trametinib, 2 μM buparlisib, the combination of 80 nM trametinib and 2 μM buparlisib, or 20 μM sirolimus. Sections were immunostained using antibodies to detect pERK, pAKT, pS6, PCNA, and CC3. pERK-, pAKT- and pS6-positive tumor areas, as well as PCNA-positive nuclei, are quantified in (b–e). “T + B” refers to trametinib plus buparlisib. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.0001 by Mann–Whitney test. Scale bar = 20 μm.

a Representative sagittal tissue sections from a transplanted nf1/pten-mutant melanoma tumor treated for 2 days with the indicated drugs. Sections were immunostained with antibodies to detect LC3A/B. Left panels: E = eye, B = brain, G = gut, K = kidney, L = liver, S = swim bladder, T = tumor. Right panels: ×63 magnification of tumor cells from the small black boxes in left panels. b–g Transplanted nf1/pten-mutant melanoma tumor cells were monitored daily in 3-week-old rag2^−/− recipient zebrafish treated with DMSO (CTR), venetoclax, S63845, sirolimus, or the drug combinations (n = 11 or 12 for each curve; doses as indicated). Kaplan–Meier curves for PFS (b, d, and f) and OS (c, e, and g) were compared using a log-rank test. Drugs were refreshed every 2 days during the 6-day course of treatment, as indicated by black arrows. h Representative tissue sections from transplanted nf1/pten-mutant melanoma tumors treated for 2 days with DMSO (CTR), 7.5 μM venetoclax and 2.5 μM S63845, 10 μM sirolimus, and the three-drug combination. Sections were immunostained using antibodies to detect PCNA and CC3 and quantified in (i). ns p > 0.05, ***p < 0.0001 by Mann–Whitney test. Scale bars = 20 μm.

a Western blots for NF1 and PTEN in a panel of human melanoma cell lines. HEK293 and Jurkart cells were included as positive and negative controls. The levels of total ERK1/2 expression serve as the loading control. b Relative cell viability of WM-3246 cells (Cell Titer Glo assay) upon treatment with sirolimus, venetoclax, or S63845 for 6 days. Mean ± s.d. values. c Relative cell viability of WM-3246 cells (Cell Titer Glo assay) upon treatment with the combination of sirolimus, venetoclax, and S63845 for 6 days. Mean ± s.d. values. d WM-3246 cell growth kinetics after treatment with the combination of sirolimus, venetoclax, and S63845 (for doses see panel c). Mean ± s.d. values. e Synergistic effects of venetoclax and S63845 on suppression of sirolimus-sensitized WM-3246 cells were analyzed by isobologram analysis. f Western blots for BCL2, BCLXL, and MCL1 in WM-3246 cells treated with venetoclax or S63845 for 24 h. g Western blots for cleaved caspase-3 in WM-3246 cells treated with the combination of sirolimus, venetoclax, and S63845.