High-throughput drug screening in advanced pre-clinical 3D melanoma models identifies potential first-line therapies for NRAS-mutated melanoma

Background: Despite significant advances in targeted (BRAFi + MEKi) and immune (anti-PD1/PD-L1, anti-CTLA4, and anti-LAG3) therapies, treatment options for NRASmut melanoma remain limited. Currently, NRASmut patients rely on immune checkpoint inhibitors, classical chemotherapy, and off-label MEK inhibitors, with over 50% experiencing rapid disease progression. One of the key challenges in developing effective targeted therapies is the lack of preclinical models that accurately recapitulate the tumor microenvironment (TME) and the intrinsic resistance of melanoma cells bearing NRAS mutations. Methods: To address this, we performed high-throughput screening (HTS) of over 1,300 compounds in 3D NRASmut melanoma spheroids. A multi-step analysis was performed to identify hits, which were further tested by performing drug-response curve (DRC) analysis. Most promising compounds were further validated using mono- and co-culture 3D in vitro models that mimic three main metastatic sites in melanoma, such as skin/dermal, lung, and liver, utilizing spheroid and hydrogel systems. Ultimately, validation was conducted using zebrafish xenograft models to enable a more refined and accurate assessment of drug response. Results: High-throughput drug screening of NRASmut melanoma spheroids identified 17 candidate compounds, which were subsequently validated through DRC analyses. Among the most promising drugs, Daunorubicin HCl (DH) and Pyrvinium Pamoate (PP) were selected for further investigation, demonstrating potent anti-melanoma activity in advanced 3D co-culture systems and zebrafish xenograft models. Notably, PP demonstrated higher cytotoxicity compared to Trametinib, the off-label MEK inhibitor, with an inhibitory effect on AKT and invasive behavior in the patient-derived metastatic melanoma cell lines. Additionally, combinatorial treatment with Trametinib resulted in additive effects on cell proliferation and viability. Importantly, both compounds showed similar efficacy in NRASmut and BRAFwt/NRASwt melanoma cell lines that were resistant to Trametinib (MEK inhibitor). Conclusions: Using advanced 3D melanoma models that incorporate key TME elements and zebrafish xenograft models, this study highlights the potential of Daunorubicin HCl and Pyrvinium Pamoate as novel first-line therapies for NRASmut melanoma, with a noteworthy effect also on MEKi-resistant cells. These findings support drug repurposing strategies and underscore the importance of physiologically relevant preclinical models in identifying effective therapies.