Searching for the Best Drug Combination to Treat Melanoma Patients: From Lab to Bedside

The search for the optimal drug combinations for treating melanoma is a relevant debate from the clinical perspective. Since the promising results with targeted therapy for treating unresectable or metastatic melanoma patients with Ipilimumab, a monoclonal antibody against CTLA-4 in 2011,¹ several studies have focused on drug combination to enhance sustained clinical response. Achieving long-term and continued tumor response for advanced melanoma patients receiving new targeted therapies remains challenging. As recently revised by Niezgoda,² an ample list of different drugs, either alone or combined, are in clinical testing for this purpose. Some FDA (US Food and Drug Administration) and EMA (European Medicines Agency) novel agents for treating advanced melanoma include BRAF (Vemurafenib, Dabrafenib, and LGX818), MEK (Trametinib, Selumetinib, Cobimetinib, and MEK162), C-KIT (imatinib, sunitinib, nilotinib, and masatinib) inhibitors, and immunomodulating drugs (CTLA-4 monoclonal antibodies, PD-1 inhibitors, and PD-L1 inhibitors). Far less explored is the association of some of these new inhibitors with classic chemotherapeutic compounds, particularly their radiosensitizing effects for melanoma cells. In a previous volume of JDD,³ we addressed the prospective use of the PLK1 inhibitor BI 2536 as an attractive strategy to impair melanoma progression and dissemination. However, regardless of promises, the clinical use of BI 3526 has been restricted by low intratumor levels,⁴ acquired resistance,⁵ and mild antitumor activity with drug-related adverse events in clinical trials.⁶ We would like to share our approach in treating human melanoma cell lines and two primary melanoma cell cultures with a combination of the second generation PLK1 inhibitors volasertib (BI6727) and GSK461364 with classical chemotherapy drugs for treating melanoma. Additionally, the radiosensitizing effects of both PLK1 inhibitors were evaluated. The human melanoma HT144 (HTB-63TM) cell line was purchased from the American Type Culture Collection, (ATCC, Rockville, MD). The LB373-MEL cell line was kindly provided by Dr. Dimas Tadeu Covas (Regional Blood Center of Ribeirão Preto). HT-144 is a malignant human melanoma cell line derived from a subcutaneous metastatic site of a 29-year-old Caucasian male.⁷ The LB373-MEL cell line was obtained from an in-transit metastasis (N2cM0III) at the lower limb of a 32-year-old female.⁸ Additionally, two different primary melanoma cell cultures were studied: TU2000 cells were cultured from a metastatic melanoma of a 40-year-old male, and TU2284 cells derived from a locally invasive melanoma diagnosed in a 64-year-old male. Cells were cultured in HAM-F10 (Life Technologies® #11550043, Carlsbad, CA) supplemented with 10% fetal bovine serum (Life Technologies® #A12618DG), penicillin (100 U/mL; Sigma-Aldrich, P3032), and streptomycin (100 ug/mL; Sigma-Aldrich, S9137) at 37 ?C in a humidified 5% CO2 incubator. For viability experiments, cells were treated with concentrations ranging from 10 to 150 nM of BI 6727 or GSK461364 (Axon Medchem® #1473 and #1688) or combinations with Cisplatin (CDDP), Temozolomide (TMZ), and Doxorubicin (DXR) (Sigma-Aldrich, P4394, T2577, and D1515), and analyzed after 24, 48, and 72 hours thorough the XTT® assay as described before.⁹ To test the effect of PLK1 inhibition on radioresistance, clonogenic assays were performed as previously reported.¹⁰ Our results showed significantly reduced proliferation in both melanoma cell lines (P<0.05), however, compared to BI 3526, the antiproliferative effects of BI 6727 and GSK461364 were more moderate, with the maximum