High-Throughput Drug Discovery for NRAS-Mutant Melanoma: Leveraging Advanced Preclinical Co-Culture Models

Significant therapeutic advancements have been achieved in the treatment of advanced BRAFmut melanoma through the approval of combined targeted therapies (BRAFi+MEKi) and immunotherapies (anti-PD1/PD-L1 and anti-CTLA4). However, patients with NRASmut and wild-type (WT) melanoma continue to face limited therapeutic options, relying on immune checkpoint inhibitors and off-label treatments such as MEKi, with over 50% experiencing disease progression due to primary and acquired resistance. A critical challenge in developing effective therapies lies in
the limitations of conventional pre-clinical models, which fail to adequately replicate the physiological complexity of the tumor microenvironment (TME). This doctoral thesis addresses these gaps through the development of advanced multicomponent 3D melanoma models and their application in the evaluation of compound efficacy identified by 3D high-throughput drug screening. Advanced in vitro 3D melanoma models were established, incorporating co-cultures of melanoma cells with endothelial cells and fibroblasts, simulating the metastatic niches of skin, lung, and liver. 3D models were also integrated into a 3D high-throughput drug screening platform, enabling the evaluation of over 1300 compounds in NRASmut and WT melanoma spheroids. Promising hits were identified and two compounds, Daunorubicin HCl and Pyrvinium pamoate, underwent detailed validation. Both compounds demonstrated potent anti-melanoma activity in advanced 3D coculture
models and zebrafish in vivo models, with Pyrvinium pamoate notably having cytotoxic
effects in NRASmut melanoma cells. Comparative studies revealed Pyrvinium pamoate’s superior efficacy over Trametinib, the standard MEKi used off-label or for disease control in clinics for NRASmut melanoma, particularly in 3D models. Furthermore, combinatory treatments with Trametinib showed additive effects on cell proliferation and viability, while both compounds exhibited substantial efficacy in MEKi-resistant NRASmut and WT melanoma cell lines. In conclusion, this thesis presents a robust pipeline for pre-clinical melanoma drug evaluation
through physiologically relevant 3D models, addressing critical challenges in the development of effective first-line therapies for NRASmut and WT melanoma. These findings highlight a potential interest in Pyrvinium pamoate as a first-line therapeutic candidate for the treatment of NRASmut melanoma patients.