The role of Mevalonate pathway inhibition, p53 and STAT3 in PD-L1 modulation in melanoma

Melanoma is one of the most aggressive cancers characterized by distinct molecular subtypes. While melanoma treatment strategies have been revolutionized by BRAF inhibitors and immune checkpoint inhibitors (ICIs) targeting Programmed death ligand 1 (PD-L1), NRAS-mutant melanoma lacks targeted therapies and resistance to ICIs remains common. The mevalonate pathway produces cholesterol and isoprenoids essential for protein prenylation and may play an important role in PD-L1 expression. This thesis investigated whether mevalonate pathway inhibitors could reduce PD-L1 in melanoma and examined the roles of p53 and STAT3 in this regulation. Three melanoma cell lines: A375 (BRAF V600E, p53-wt), SkMel28 (BRAF V600E, p53-GOF), SkMel2 (NRAS Q61R, p53-LOF) were treated with IFNγ (10 ng/ml, 24h) followed by respective statins, FTIs, GGTIs and squalene synthase inhibitors (24h). The protein levels of p53, STAT3 and PD-L1 were measured using SDS-PAGE and Western blot. IFNγ robustly induced PD-L1 expression across the cell lines, while mevalonate pathway inhibitors showed limited effects with a trend towards reduction of PD-L1 observed for atorvastatin at a concentration of 25 µM (p=0.081) in SkMel2 and a trend to increase observed for GGTI-2133 on basal conditions (p=0.0985) in SkMel28. Additionally, we observed a mutation-dependent phosphorylation response of p53 to IFNγ stimulation. Cells with strong phosphorylation induction of p53 demonstrated consistent PD-L1 expression, highlighting the importance of its dynamic response to IFNγ for stable PD-L1 expression. STAT3 responses, on the other hand, were divergent – decreased in A375, increased in SkMel2 and undetectable in SkMel28. These observations demonstrate that PD-L1 regulation patterns in melanoma are genotype-dependent with p53 and STAT3 playing context-dependent roles. The novel observation of dynamic p53 phosphorylation response represents a new insight into PD-L1 dynamics. Together with NRAS dependency on prenylation, our observations provide an important insight for future combination therapy strategies.