Abstract 2659: Erythropoietin stimulation in BRAF mutated metastatic melanoma confers a highly proliferative and invasive phenotype

Abstract Introduction: Currently, there is a lack of understanding of the mechanisms underlying the progression of primary melanoma to a metastatic phenotype. Interestingly, RNA-sequencing data from the cancer genome atlas has demonstrated that patients with cutaneous melanoma with high expression of EPOR have poor long-term survival outcomes. This finding may be related to why some patients with anemia of malignancy treated with erythropoietin (EPO) have progression of their cancer. There has been significant discussion on this topic and whether membrane-bound EPOR is functional in solid malignancies. Here we report on how modulation of EPOR affects melanoma cell proliferation and invasion in an in-vitro model of metastatic disease. Methods: A metastatic melanoma cell line, SK-MEL1, with mutant BRAFV600E was treated with varying doses of EPO and a novel EPOR inhibitor, URV3, for 24 hours. Cell proliferation was quantified by determining absolute cell number using a hemocytometer. Further, an invasion assay was utilized to assess the effect of EPOR modulation on cell invasiveness. ∼50, 000 cells were seeded into 1:5 diluted Matrigel overlaying a porous membrane and allowed to invade for 24 hours. After 24 hours, Matrigel was removed from the membrane and cells were fixed, stained, and imaged in 4 different positions. Invaded cells were quantified by averaging the cell density in each imaged field (cells/micrometer^2) and multiplying by total membrane area. All data were performed in duplicates and reported as mean fold change from control. Non-parametric ANOVA was used for statistical analysis. Results: Modulation of EPOR with EPO, 8uM URV3, 8uM URV with EPO, 32uM URV3, and 32uM URV3 with EPO yielded a mean fold change in cell number over control of 1.43 (95% CI: 1.09-1.78), 0.89 (95% CI: 0.56-1.24), 1.38 (95% CI: 0.95-1.81), 0.88 (95% CI: 0.46-1.30), and 1.59 (95% CI: 1.15-2.04), respectively (p=0.0027). Cell invasion after 24 hours of treatment with EPO, 8uM URV3, 8uM URV with EPO, 32uM URV3, and 32uM URV3 with EPO yielded a mean fold change in cell invasion over control of 1.96 (95% CI: -0.80-4.71), 0.59 (95% CI: -4.82-5.99), 0.48 (95% CI: 0.03-0.92), 0.48 (95% CI:-1.22-2.19), and 0.60 ( 95% CI: -1.22-3.96) respectively (p=0.059). Discussion: We demonstrate that direct EPOR stimulation causes increased cell proliferation and invasion. EPOR blockade with URV3 impedes cell growth; however, it is unable to overcome the proliferative effect of additional EPO stimulation. Interestingly, URV3 demonstrates a strong inhibitory effect on melanoma cell invasion despite EPO stimulation. These results illustrate the functional capabilities of the cell-surface bound EPOR receptor in metastatic melanoma and warrant further investigation of downstream signaling that contributes to this invasive and proliferative phenotype. Citation Format: Jayesh Menon, Raza Ahmed, Muneeb Aslam, Rakesh Singh, Philip Friedlander, Omar Aljitawi. Erythropoietin stimulation in BRAF mutated metastatic melanoma confers a highly proliferative and invasive phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2659.