BRAF抑制剂广泛用于治疗带有BRAF V600E突变的黑色素瘤。然而，耐药性的发展损害了它们的治疗效果。在 BRAF 抑制剂耐药性黑色素瘤中发现了多种基因组和转录组改变，迫切需要收敛的可药物靶点来逆转具有不同耐药机制的耐药性肿瘤。进行 CRISPR-Cas9 筛选以鉴定新的靶基因，其抑制选择性靶向 A375VR（一种BRAF V600E 突变细胞系对维莫非尼具有获得性耐药性。进行了各种体外和体内测定，包括细胞竞争测定、水溶性四唑（WST）测定、活死测定和异种移植测定，以确认协同细胞死亡。液相色谱-质谱分析定量分析维莫非尼耐药黑色素瘤中的多胺生物合成和蛋白质组变化。通过 O-炔丙基嘌呤霉素标记测定、mitotracker、mitoSOX 标记和海马测定来测定 EIF5A hypusination 依赖性蛋白翻译以及线粒体生物合成和活性的后续变化。使用生物信息学分析来确定多胺生物合成与黑色素瘤患者队列中 BRAF 抑制剂耐药性和不良预后的关系。我们阐明了多胺生物合成及其在促进 BRAF 抑制剂耐药性中的调节机制。利用 CRISPR-Cas9 筛选，我们确定了 AMD1（S-腺苷甲硫氨酸脱羧酶 1）（一种多胺生物合成的关键酶）作为药物靶标，其抑制作用可降低维莫非尼耐药性。代谢组学和蛋白质组学分析表明，在威罗非尼耐药的癌症中，多胺生物合成上调，导致 EIF5A 抑制、线粒体蛋白翻译和氧化磷酸化增强。我们还发现，维莫非尼耐药癌症中持续的 c-Myc 水平是导致多胺生物合成升高的原因。抑制多胺生物合成或 c-Myc 可逆转体外细胞系模型和体内异种移植模型中的威罗非尼耐药性。在黑色素瘤队列中，多胺生物合成特征与 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，突出了我们研究结果的临床相关性。我们的研究结果描绘了涉及多胺-EIF5A 抑制-线粒体呼吸途径赋予 BRAF 抑制剂耐药性的分子机制在黑色素瘤中。这些靶点将作为有效的治疗靶点，可以最大限度地提高现有 BRAF 抑制剂的治疗效果。© 2024。作者。

BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms.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.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.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.© 2024. The Author(s).