The Effects of Combined Drug Therapy in the Treatment of BRAF Mutated Melanoma

Mojahed Mohammad K Shalabi

 Introduction: Skin cancer is the most common form of cancer in the world, with melanoma being the deadliest form of skin cancers contributing to approximately 75% of skin cancer deaths1. The prognosis of melanoma is determined by the stage of the disease, with a 5-year survival rate for Stage IV melanoma being around 25%. As a result, early detection of melanoma is key to drastically improving prognosis for melanoma patients. BRAFV600E mutation is the most common melanoma mutation1. BRAF is a proto-oncogene that promotes cell growth, proliferation, and survival1,2. BRAF is the main target for drug therapy in metastatic melanoma; however, cancerous cells quickly gain resistance to BRAF inhibitors by upregulating other intracellular growth pathways such as the Phosphatidylinositol-3-kinase/Protein Kinase B (PI3/AKT) pathway to maintain cell survival3,4. Combined drug therapy has shown promising results in the treatment of BRAFV600E mutated melanoma5. Methods: Splinkerette PCRs were performed to determine the BRAF inhibitor resistant cell lines with ERAS expression6. Western blot analysis was used to show that ERAS induces phosphorylation of AKT in the presence or absence of both a BRAF inhibitor, PLX4720, and an AKT inhibitor, MK2206. Western Blot analyses was also performed to determine the expression of Bcl-2 agonist of cell death (BAD), myeloid cell leukemia 1 (MCL1), and other apoptotic proteins in the cell lines in the presence or absence of PLX4720 and MK22066. Control and ERAS cells were stained with Annexin-V and analyzed by flow cytometry to record apoptotic responses of both cell lines in the presence of a PLX4720 alone, MK2206 alone, or PLX+MK6. Results: From the western blot analyses, ERAS expressed cell lines showed phosphorylation of AKT which in turns hyperphosphorylated BAD and decreased apoptosis in melanoma cells. This is resolved by a concomitant treatment with PLX4720 and MK2206, which blocks phosphorylation of BAD (p<0.05)6. From the flow cytometry, combinatorial BRAF and AKT inhibition showed enhanced apoptotic response in ERAS expressing cells and control cell lines (p<0.01)6. Conclusion: The BRAF gene plays an important role in the progression of melanoma by constitutively activating growth pathways and disrupting cellular mechanisms such as apoptosis. Resistance to treatment is very prevalent in melanoma treated with BRAF inhibitors alone7. Combined drug therapy that targets BRAF and a downstream target, or a protein in another growth signaling pathway such as AKT has shown to increase death of melanoma cells, increase delay of resistance, and significantly decrease disease progression7,8.

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