PHI-501, a novel pan-RAF inhibitor, enhances immunotherapy efficacy by converting cold tumors into hot tumors

Background: Immune checkpoint blockade (ICB) has significantly improved survival outcomes in patients with melanoma; however, a substantial proportion of patients remain non-responsive. Therefore, converting “cold” tumors into “hot” tumors represents a critical therapeutic strategy to enhance responsiveness to ICB therapy. PHI-501, a novel pan-RAF inhibitor, is proposed to enhance immunotherapy responsiveness by promoting immune activation and converting “cold” tumors into “hot,” immune-inflamed tumors. In this study, we sought to characterize the immunomodulatory mechanisms by which PHI-501 primes antitumor immunity in melanoma. Methods: RNA sequencing was performed on SK-MEL-3 (BRAF V600E ) melanoma cells treated with PHI-501 (10 μM, 48 h), followed by gene set enrichment analysis (GSEA) to identify immune-related transcriptomic alterations. To validate immune activation, SK-MEL-3 melanoma cells were treated with PHI-501 and analyzed for JAK/STAT signaling and type I interferon (IFN) response using quantitative PCR (qPCR). Additional experiments with IFN-gamma (24 h) co-treatment were performed to assess MHC class I induction. Western blotting was performed on cell lysates and culture supernatants to measure intracellular immune markers and the extracellular release of HMGB1 in SK-MEL-3 and SK-MEL-29 (BRAF V600E ). Results : GSEA of RNA-seq data from PHI-501-treated SK-MEL-3 cells revealed enrichment of the type I interferon response (NES = 1.17, p = 0.14) and the tissue-specific immune response (NES = 1.64, p = 0.01). PHI-501 treatment downregulated the expression of key MAPK pathway regulators such as DUSP6, SPRY4, ETV4, and ETV5, while upregulating canonical interferon-stimulated genes (ISGs) including IFIT1, IFIT2, IFIT3, and IRF1 (p<0.05). Consistent with the transcriptomic results, qPCR analysis demonstrated robust induction of immune-related genes. In SK-MEL-3, STAT2, IFNA1, and IRF1 transcripts were upregulated approximately 12-, 9-, and 7-fold, respectively. Furthermore, IFN-gamma co-treatment synergistically enhanced MHC class I expression, increasing levels by more than 7-fold in SK-MEL-3 compared with controls. Additionally, extracellular HMGB1 levels increased in culture supernatants of SK-MEL-3 and SK-MEL-29 cells, accompanied by elevated IRF1 and NLRP3 protein expression in cell lysates. Conclusion : Taken together, these findings demonstrate that PHI - 501 activates immune-related signaling pathways, including the type I IFN and downstream JAK/STAT cascades. Overall, these results suggest that PHI-501 may serve as a promising therapeutic agent capable of enhancing ICB responsiveness when used in combination with immunotherapy.