Cross-Species Comparison of the Pan-RAF Inhibitor LY3009120's Anti-Tumor Effects in Equine, Canine, and Human Malignant Melanoma Cell Lines

Abstract

Malignant melanomas (MMs) are the abnormal proliferation of melanocytes and are one of the lethal skin cancers in humans, equines, and canines. Accordingly, MMs in companion animals can serve as naturally occurring animal models, completing conventional cancer models. The common constitutive activation of the MAPK and PI3K pathways in MMs has been described in all three species. Targeting the related pathways is considered a potential option in comparative oncologic approaches. Herein, we present a cross-species comparative analysis exposing a set of ten melanoma cell lines (one human, three equine, and six canine) derived from primary tumors or metastasis to a pan-RAF and RAF dimer inhibitor (LY3009120). Cellular response (proliferation, biomass, metabolism, early and late apoptosis/necrosis, and morphology) and the presence of pathogenic single-nucleotide variants (SNVs) within the mutational hotspot genes BRAF exon 11 and 15, NRAS exon 2 and 3, KRAS exon 2, and KIT exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the KRAS p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry NRAS p.G13R. Except for EMM metastasis cells eRGO6 (wild type of the above-mentioned hotspot genes), all melanoma cell lines exhibited a decrease in dose dependence after 48 and 72 h of exposure to LY3009120, independent of the mutation hotspot landscape. Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in all melanoma cell lines except for eRGO6. The anti-tumor effects of LY3009120 were observed in nine melanoma cell lines, indicating the potential feasibility of experimental trials with LY3009120. The present study reveals that the irradiation-resistant canine metastasis cells (cRGO1.2) harboring the NRAS p.G13R mutation are significantly LY3009120-sensitive, while the equine metastases-derived eRGO6 cells show significant resistance to LY3009120, which make them both valuable tools for studying resistance mechanisms in comparative oncology.

Keywords: DNA sequencing; LY3009120; Pan-RAF inhibitor; canine malignant melanoma; cellular response; equine malignant melanoma; genetic evaluation.

Figure 1

Proliferation, biomass, and part of the metabolic activity changes in A375 and all equine cell lines. Subfigures (a–d) are line graphs of proliferation and biomass changes in A375 and all equine cell lines after exposure to different concentrations of LY3009120 in 48 and 72 hours. Proliferation is evaluated with trypan blue staining. Biomass is evaluated by means of crystal violet staining, and metabolic activity is evaluated with a WST-1 assay. The significance of a treatment effect compared to the DMSO control is determined by means of a one-way ANOVA and is displayed as *: p < 0.033, **: p < 0.002, and ***: p < 0.001 (n ≥ 3).

Figure 2

Proliferation, biomass, and metabolic activity changes in all canine cell lines. Proliferation is assessed by means of trypan blue staining. Biomass was evaluated with crystal violet staining, and metabolic activity was evaluated via a WST-1 assay. The significance of a treatment effect compared to the DMSO control is determined by means of a one-way ANOVA and is displayed as *: p < 0.033, **: p < 0.002, and ***: p < 0.001 (n ≥ 3).

Figure 3

Induction of early and late apoptosis/necrosis in human, equine, and canine melanoma cell lines after 48 and 72 h incubation with LY3009120 identified via Annexin V FITC and flow cytometry by means of propidium iodide (PI) double staining. The significance of a treatment effect compared to the DMSO control is determined via a one-way ANOVA and displayed as *: p < 0.033, **: p < 0.002, and ***: p < 0.001 (n ≥ 3).

Figure 4

Morphological changes in ten melanoma cell lines with the application of LY3009120 by means of Pappenheim staining. Magnification: 1000×. Ten human, canine, and equine melanoma cell lines after DMSO or LY3009120 inhibitor exposure for 48 or 72 h. Melanin is observed in the cytoplasm of MelDuWi (solid black arrow in the upper right of the 0.1 μM). These indicators include the presence of membrane blebs (black arrows), numerous nuclear vacuoles (red arrows) and plasma vacuoles (grey arrows), chromatin condensation (solid green arrow), nuclear fragmentation (karyorrhexis, solid blue arrow), cellular fragmentation (solid purple arrow), rupture of the plasma membrane (solid grey arrow), phagocytosis (solid orange arrow), and marked distortions in cellular morphology. Except for eRGO6, comparable morphological changes are observed in all the samples treated with LY3009120 at the concentration above 0.1 μM. More pronounced evidence is observed in A375, cRGO1.2, cRGO4, eRGO1, cRGO6, Mel1268, and MelDuWi.