Inhibition of MERTK Promotes Suppression of Tumor Growth in BRAF Mutant and BRAF Wild-Type Melanoma

Abstract

Molecularly-targeted agents have improved outcomes for a subset of patients with BRAF-mutated melanoma, but treatment of resistant and BRAF wild-type tumors remains a challenge. The MERTK receptor tyrosine kinase is aberrantly expressed in melanoma and can contribute to oncogenic phenotypes. Here we report the effect of treatment with a MERTK-selective small molecule inhibitor, UNC2025, in preclinical models of melanoma. In melanoma cell lines, treatment with UNC2025 potently inhibited phosphorylation of MERTK and downstream signaling, induced cell death, and decreased colony formation. In patient-derived melanoma xenograft models, treatment with UNC2025 blocked or significantly reduced tumor growth. Importantly, UNC2025 had similar biochemical and functional effects in both BRAF-mutated and BRAF wild-type models and irrespective of NRAS mutational status, implicating MERTK inhibition as a potential therapeutic strategy in tumors that are not amenable to BRAF-targeting and for which there are limited treatment options. In BRAF-mutated cell lines, combined treatment with UNC2025 and the BRAF inhibitor vemurafenib provided effective inhibition of oncogenic signaling through ERK, AKT, and STAT6, increased induction of cell death, and decreased colony-forming potential. Similarly, in NRAS-mutated cell lines, addition of UNC2025 to cobimetinib therapy increased cell death and decreased colony-forming potential. In a BRAF-mutated patient-derived xenograft, treatment with combined UNC2025 and vemurafenib was well-tolerated and significantly decreased tumor growth compared with vemurafenib alone. These data support the use of UNC2025 for treatment of melanoma, irrespective of BRAF or NRAS mutational status, and suggest a role for MERTK and targeted combination therapy in BRAF and NRAS-mutated melanoma.

Figure 1:. UNC2025 inhibits MERTK activation and downstream signaling in melanoma cell lines.

A) Cultures of the indicated cell lines were treated with UNC2025 for 90 minutes, and then with pervanadate phosphatase inhibitor for 3 minutes. MERTK was immunoprecipitated from cell lysates, and phosphorylated and total MERTK proteins were detected by immunoblot. B) Serum starved cultures of the indicated cell lines were treated with UNC2025 for 90 min and then stimulated with 200 nM GAS6 ligand or an equivalent volume of vehicle for an additional 10 min. Cell lysates were prepared and the indicated phosphorylated (denoted by p-) and total proteins were detected by immunoblot. ACTIN is shown as a loading control. C) The indicated cell lines were treated with UNC2025 for 72 h. Cell lysates were prepared and SURVIVIN was detected by immunoblot. Data are representative of at least 3 independent experiments.

Figure 2:. UNC2025 reduces colony-forming potential in BRAF^mt and NRAS^mt melanoma cell lines.

Melanoma cell lines were cultured at low density and treated with UNC2025 for 10 days, then colonies were stained with crystal violet and enumerated. A) Images from a representative experiment. B) Colony number in BRAF^mt G361, A101D, and SK-MEL-5 cell lines relative to cultures treated with vehicle (0 = DMSO), n = 4–7 independent experiments. C) Relative colony number in NRAS^mt cell lines HMCB, SK-MEL-2, and SK-MEL-119, n = 3–5 independent experiments. Mean values and standard errors are shown. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, one-way ANOVA.

Figure 3:. UNC2025 inhibits oncogenic signaling and reduces colony-forming potential in BRAF^wt/NRAS^wt melanoma cell lines.

A-B) MeWo BRAF^wt/NRAS^wt cell cultures were treated with the indicated concentrations of UNC2025 for 90 minutes (A) or 72 hours (B). Cell lysates were prepared and the indicated phosphorylated (denoted by p-) and total proteins were detected by immunoblot. C) BRAF^wt/NRAS^wt cell lines MeWo, MB2141, MB2204, and MB2724 were cultured in triplicate at low density and treated with the indicated concentrations of UNC2025 for 10 days. Colonies were stained with crystal violet and enumerated. Colony numbers relative to vehicle-treated (DMSO) cultures are shown. n=3–4 independent experiments, *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA.

Figure 4:. Combined treatment with UNC2025 and a BRAF or MEK inhibitor enhances functional anti-tumor effects in BRAF^mt and NRAS^mt melanoma cells.

A) Serum starved BRAF^mt G361 cell cultures were treated with 300 nM UNC2025 and/or 675 nM vemurafenib or DMSO vehicle for 90 min and then stimulated with 200 nM GAS6 ligand or an equivalent volume of vehicle for an additional 10 min. Cell lysates were prepared and the indicated phosphorylated (denoted by p-) and total proteins were detected by immunoblot. B) G361 and A101D cell lines were cultured at low density and treated with 1μM vemurafenib alone or in combination with the indicated concentrations of UNC2025, or with vehicle (DMSO) only for 10 days. Colonies were stained with crystal violet and enumerated. Colony numbers relative to vehicle treated cultures are shown. C) G361 and A101D cell cultures were treated with 1μM vemurafenib alone or in combination with the indicated concentrations of UNC2025 or with vehicle (DMSO) only for 48 h and then stained with PO-PRO^™−1 iodide (PO-PRO) and 7-AAD. Apoptotic (PO-PRO+, 7-AAD negative) and dead (7-AAD+) cells were detected by flow cytometry. Dead and dying cells were enumerated via flow cytometric analysis based on PO-PRO™-1 and 7-AAD uptake. D&E) HMCB cell cultures were treated with the indicated concentrations of cobimetinib (cobi), UNC2025, the combination, or vehicle and colony formation (D) and induction of cell death (E) were assessed as above. n=3–4 independent experiments, *p < 0.05, **p < 0.01, ***p<0.001, one-way ANOVA.

Figure 5:. Treatment with UNC2025 inhibits tumor growth in a BRAF^mt patient-derived melanoma xenograft model alone and in combination with vemurafenib.

A xenograft-passaged BRAF^mt melanoma patient sample (MB2117) was transplanted into nude mice, and mice with established tumors were treated with 40 or 50 mg/kg UNC2025 twice daily (BID), 40 mg/kg vemurafenib once daily, or equivalent doses of UNC2025 and vemurafenib in combination, or vehicle. Tumor volumes were measures at intervals. A) n=6, * p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA B) n=10–13, ** p <0.01 versus vehicle, *** p < 0.001 versus vehicle, # p <0.05 versus vemurafenib monotherapy, two-way ANOVA. Asterisks denote comparison between vehicle and combination treatment. Data shown are representative of two independent experiments.