J Cancer 2015; 6(12):1320-1330. doi:10.7150/jca.11126

Research Paper

PLX4032 Mediated Melanoma Associated Antigen Potentiation in Patient Derived Primary Melanoma Cells

Andrea L. George1, Robert Suriano2, Shilpi Rajoria1, Maria C. Osso4, Neha Tuli1, Elyse Hanly1, Jan Geliebter1, Angelo N. Arnold5, Marc Wallack1,3, Raj K. Tiwari1✉

1. New York Medical College, Department of Microbiology and Immunology, Valhalla, New York, 10595;
2. College of Mount Saint Vincent, Division of Natural Sciences, Bronx, New York, 10471;
3. Metropolitan Hospital Center GNS, Department of Surgery, New York, New York, 10029;
4. McDaniel College, Westminster, Maryland 21157;
5. Westchester Medical Center, Transplant Immunogenetics Laboratory, Valhalla, New York, 10595.

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George AL, Suriano R, Rajoria S, Osso MC, Tuli N, Hanly E, Geliebter J, Arnold AN, Wallack M, Tiwari RK. PLX4032 Mediated Melanoma Associated Antigen Potentiation in Patient Derived Primary Melanoma Cells. J Cancer 2015; 6(12):1320-1330. doi:10.7150/jca.11126. Available from http://www.jcancer.org/v06p1320.htm

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Over expression of various immunogenic melanoma associated antigens (MAAs) has been exploited in the development of immunotherapeutic melanoma vaccines. Expression of MAAs such as MART-1 and gp100 is modulated by the MAPK signaling pathway, which is often deregulated in melanoma. The protein BRAF, a member of the MAPK pathway, is mutated in over 60% of melanomas providing an opportunity for the identification and approval by the FDA of a small molecule MAPK signaling inhibitor PLX4032 that functions to inactivate mutant BRAFV600E.

To this end, we characterized five patient derived primary melanoma cell lines with respect to treatment with PLX4032. Cells were treated with 5μM PLX4032 and harvested. Western blotting analysis, RT-PCR and in vitro transwell migration and invasion assays were utilized to determine treatment effects. PLX4032 treatment modulated phosphorylation of signaling proteins belonging to the MAPK pathway including BRAF, MEK, and ERK and abrogated cell phenotypic characteristics such as migration and invasion. Most significantly, PLX4032 led to an up regulation of many MAA proteins in three of the four BRAF mutated cell lines, as determined at the protein and RNA level. Interestingly, MAGE-A1 protein and mRNA levels were reduced upon PLX4032 treatment in two of the primary lines.

Taken together, our findings suggest that the BRAFV600E inhibitor PLX4032 has therapeutic potential over and above its known target and in combination with specific melanoma targeting vaccine strategies may have further clinical utility.

Keywords: BRAF Kinases, MAPK, Melanoma, Melanoma Associated Antigens, PLX4032, Vaccine.