METHODS: CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts.
RESULTS: We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings.
CONCLUSIONS: Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.
方法:进行CRISPR-Cas9筛选以鉴定新的靶基因,其抑制选择性靶向A375VR,对vemurafenib具有获得性抗性的BRAFV600E突变细胞系。各种体外和体内试验,包括细胞竞争试验,水溶性四唑(WST)测定,进行活死试验和异种移植试验以确认协同细胞死亡.液相色谱-质谱分析定量多胺生物合成和维罗非尼耐药黑色素瘤蛋白质组的变化。EIF5A催眠依赖的蛋白质翻译和随后的线粒体生物发生和活性的变化通过O-炔丙基-嘌呤霉素标记测定,mitotracker,mitoSOX标记和海马测定。生物信息学分析用于确定多胺生物合成与BRAF抑制剂抗性和黑色素瘤患者队列中不良预后的关联。
结果:我们阐明了多胺生物合成及其调节机制在促进BRAF抑制剂抗性中的作用。利用CRISPR-Cas9屏幕,我们鉴定了AMD1(S-腺苷甲硫氨酸脱羧酶1),多胺生物合成的关键酶,作为一种药物靶标,其抑制作用降低了维罗非尼的耐药性。代谢组学和蛋白质组学分析显示,维罗非尼耐药癌症中多胺生物合成上调,导致增强的EIF5A催眠,线粒体蛋白的翻译和氧化磷酸化。我们还确定维罗非尼耐药癌症中持续的c-Myc水平是多胺生物合成升高的原因。在体外细胞系模型和在异种移植模型中体内抑制多胺生物合成或c-Myc逆转维罗非尼抗性。多胺生物合成特征与黑色素瘤患者BRAF/MAPK抑制剂治疗后的不良预后和较短的无进展生存期相关,强调我们的研究结果的临床相关性。
结论:我们的发现描述了在黑色素瘤中涉及多胺-EIF5A催眠-线粒体呼吸途径赋予BRAF抑制剂抗性的分子机制。这些靶标将作为有效的治疗靶标,其可以最大化现有BRAF抑制剂的治疗功效。