PO.IM01.04 · 免疫学
PHI-501，一种新型泛RAF抑制剂，通过将冷肿瘤转化为热肿瘤增强免疫治疗疗效
PHI-501, a novel pan-RAF inhibitor, enhances immunotherapy efficacy by converting cold tumors into hot tumors
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：免疫检查点阻断（ICB）显著改善了黑色素瘤患者的生存结局；然而，相当比例的患者仍无应答。因此，将“冷”肿瘤转化为“热”肿瘤代表了增强对ICB治疗反应性的一项关键治疗策略。PHI-501是一种新型泛RAF抑制剂，被提出通过促进免疫活化并将“冷”肿瘤转化为“热”的免疫炎性肿瘤来增强免疫治疗反应性。在本研究中，我们旨在表征PHI-501在黑色素瘤中启动抗肿瘤免疫的免疫调节机制。
方法：对用PHI-501（10 μM，48小时）处理的SK-MEL-3（BRAF V600E）黑色素瘤细胞进行RNA测序，随后进行基因集富集分析（GSEA）以鉴定免疫相关的转录组改变。为验证免疫活化，用PHI-501处理SK-MEL-3黑色素瘤细胞，并使用定量PCR（qPCR）分析JAK/STAT信号传导和I型干扰素（IFN）应答。进行了额外的IFN-gamma（24小时）共处理实验以评估MHC I类诱导。对细胞裂解物和培养上清液进行蛋白质印迹，以测量SK-MEL-3和SK-MEL-29（BRAF V600E）中的细胞内免疫标志物和HMGB1的细胞外释放。
结果：对PHI-501处理的SK-MEL-3细胞的RNA-seq数据进行GSEA显示，I型干扰素应答（NES=1.17，p=0.14）和组织特异性免疫应答（NES=1.64，p=0.01）富集。PHI-501处理下调了关键MAPK通路调节因子（如DUSP6、SPRY4、ETV4和ETV5）的表达，同时上调了经典干扰素刺激基因（ISGs），包括IFIT1、IFIT2、IFIT3和IRF1（p<0.05）。与转录组结果一致，qPCR分析显示免疫相关基因的强劲诱导。在SK-MEL-3中，STAT2、IFNA1和IRF1转录本分别上调约12倍、9倍和7倍。此外，IFN-gamma共处理协同增强MHC I类表达，使SK-MEL-3中的水平较对照增加超过7倍。此外，SK-MEL-3和SK-MEL-29细胞培养上清液中的细胞外HMGB1水平升高，同时细胞裂解物中IRF1和NLRP3蛋白表达升高。
结论：综上所述，这些发现表明PHI-501激活免疫相关信号通路，包括I型IFN和下游JAK/STAT级联反应。总体而言，这些结果提示PHI-501可能作为一种有前景的治疗剂，在与免疫治疗联合使用时能够增强ICB反应性。
查看英文原文 English abstract
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.
利益披露 Disclosure
S. Kim, None..
S. Kim, None..
K. Nam, None..
J. Yoon, None..
S. Shin, None.