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. BRAF_V600E mutation is the most common melanoma mutation¹. BRAF is a proto-oncogene that promotes cell growth, proliferation, and survival^1,2. 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 survival^3,4. Combined drug therapy has shown promising results in the treatment of BRAF_V600E 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 progression^7,8.

1. Paolo A Ascierto, John M Kirkwood, Jean-Jacques Grob, et al. J Transl Med. 2012; 10: 85. Published online 2012 Jul 9. doi: 10.1186/1479-5876-10-85. “The role of BRAF V600 mutation in melanoma.”
2. Hawryluk, E., Tsao H. “Melanoma: Clinical Features and Genomic Insights.” Cold Spring Harb Prospect Med. 2014 Sep; 4(9):a015388. Doi:10.1101/cshperspect.a015388.
3. Griffin Merope, Scotto Daniele, Josephs Debra H., et al. “BRAF inhibitors: resisitance and the promise of combination treatments for melanoma.” Oncotarget.2017. https://doi.org/10.18632/oncotarget.19836
4. Niessner H, Schmitz J, Tabatabai G, Schmid AM, et al. “PI3K Pathway Inhibition Achieves Potent Antitumor Activity in Melanoma Brain Metastases In Vitro and In Vivo”. Clin Cancer Res. 2016; 22:5818–28. https://doi.org/10.1158/1078-0432.CCR-16-0064.
5. Chante Karimkhani, Robert P. Dellavalle, Luc E. Coffeng, et al. JAMA Dermatol. 2017 May; 153(5): 406–412. Published online 2017 Mar 1. doi: 10.1001/jamadermatol.2016.5538. “Global Skin Disease Morbidity and Mortality An Update From the Global Burden of Disease Study 2013.”
6. Daniele Perna, Florian A. Karreth, Alistair G. Rust, et al. “BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model.” January 26, 2015 https://doi-org.srv-proxy1.library.tamu.edu/10.1073/pnas.1418163112
7. Xian Yang Chan, Alamdeep Singh, Narin Osman, Terrence J. Piva Int J Mol Sci. 2017 Jul; 18(7): 1527. Published online 2017 Jul 14. doi: 10.3390/ijms18071527. “Role Played by Signalling Pathways in Overcoming BRAF Inhibitor Resistance in Melanoma.”
8. Georgina V. Long, M.D., Ph.D., Daniil Stroyakovskiy, M.D., Helen Gogas, et al. “Combined BRAF and MEK Inhibition versus BRAF Inhibition Alone in Melanoma.” N Engl J Med 2014. DOI: 10.1056/NEJMoa1406037