摘要目的:探讨ARAF突变在肺癌细胞RAF抑制剂耐药中的作用。方法:通过CRISPR-Cas9方法构建敲除ARAF基因的A549和NCI-H1688肺癌细胞株。通过慢病毒包装感染方法，在敲除ARAF基因的肺癌细胞中过表达野生型ARAF（ARAF WT）和突变型ARAF（ARAF V145L、S214F、S214C或D429A），对照组为未经过处理的敲除ARAF基因的A549或NCI-H1688细胞。使用不同浓度RAF抑制剂belvarafenib或AZ-628处理以上各组A549或NCI-H1688细胞。CCK-8法检测细胞活力，计算各组细胞对belvarafenib或AZ-628的半数抑制浓度（ IC50）。以ARAF WT组A549细胞 IC50（263 nmol/L）belvarafenib处理各组A549或NCI-H1688细胞，蛋白质印迹法检测ARAF及MEK/ERK/RSK信号通路中关键蛋白表达水平。选择6~8周龄雄性BALB/c裸鼠，腋窝皮下注射稳定过表达ARAF WT或突变型ARAF的敲除ARAF基因的A549或NCI-H1688细胞悬液，构建异种移植瘤模型，以每天30 mg/kg belvarafenib或AZ-628溶液连续灌胃，比较第16天各组肿瘤体积。 结果:与ARAF WT组比较，ARAF D429A组A549或NCI-H1688细胞belvarafenib或AZ-628的 IC50均低，ARAF S214F和ARAF S214C组 IC50均高，差异均有统计学意义（均 P<0.05）。与ARAF WT组比较，经belvarafenib处理的ARAF WT组、ARAF D429A组A549细胞或NCI-H1688细胞p-MEK、p-ERK、p-RSK的相对表达水平均低，差异均有统计学意义（均 P<0.05），ARAF、MEK、ERK、RSK的相对表达水平差异均无统计学意义（均 P>0.05）；与ARAF WT组比较，经belvarafenib处理的ARAF S214F组、ARAF S214C组p-MEK、p-ERK、p-RSK相对表达水平均高，差异均有统计学意义（均 P<0.05），ARAF、MEK、ERK、RSK的相对表达水平差异均无统计学意义（均 P>0.05）。ARAF WT组、ARAF D429A组A549细胞或NCI-H1688细胞移植瘤裸鼠经belvarafenib或AZ-628灌胃后，第16天肿瘤体积均低于对应的未经belvarafenib或AZ-628干预的ARAF WT组、ARAF D429A组细胞移植瘤裸鼠，且经belvarafenib或AZ-628灌胃的ARAF D429A组A549细胞或NCI-H1688细胞移植瘤裸鼠第16天肿瘤体积均低于对应的经belvarafenib或AZ-628灌胃的ARAF WT组移植瘤裸鼠，差异均有统计学意义（均 P<0.05）；ARAF S214F组、ARAF S214C组A549细胞或NCI-H1688细胞移植瘤裸鼠经belvarafenib或AZ-628灌胃后第16天肿瘤体积与对应的未经belvarafenib或AZ-628干预的ARAF S214F组、ARAF S214C组细胞移植瘤裸鼠比较，差异均无统计学意义（均 P>0.05），与belvarafenib或AZ-628灌胃的ARAF WT组移植瘤裸鼠间差异也均无统计学意义（均 P>0.05）。 结论:ARAF D429A突变增加了肺癌对RAF抑制剂的敏感性，ARAF S214F和S214C突变增加了肺癌对RAF抑制剂的耐药性。

abstractsObjective:To explore the role of ARAF mutation in RAF inhibitor resistance in lung cancer cells.Methods:The lung cancer cell lines A549 and NCI-H1688 with ARAF gene knockout were constructed using CRISPR-Cas9 method. Overexpression of wild-type ARAF (ARAF WT) and mutant ARAF (ARAF V145L, S214F, S214C, or D429A) in lung cancer cells with ARAF gene knockout was achieved through lentiviral packaging infection method, while the control group consisted of untreated A549 or NCI-H1688 cells with ARAF gene knockout. A549 or NCI-H1688 cells in each group were treated with different concentrations of RAF inhibitor belvarafenib or AZ-628. The CCK-8 method was used to detect cell viability, and the half maximal inhibitory concentration ( IC50) of cells in each group against belvarafenib or AZ-628 was calculated. A549 or NCI-H1688 cells in each group were treated with IC50 (263 nmol/L) of belvarafenib in the ARAF WT group, and the expression levels of key proteins in the ARAF and MEK/ERK/RSK signaling pathways were detected by Western blotting. The 6-8 week old male BALB/c nude mice were selected and subcutaneously injected stable overexpression of ARAF WT or mutant ARAF A549 or NCI-H1688 cell suspension with ARAF gene knockout into the axilla to construct a xenograft tumor model, and 30 mg/kg belvarafenib or AZ-628 solution was administered continuously by gavage per day; tumor volume was compared among the groups at the 16th day. Results:Compared with the ARAF WT group, the IC50 of belvarafenib or AZ-628 in A549 or NCI-H1688 cells of the ARAF D429A group was lower, while the IC50 of the ARAF S214F and ARAF S214C groups was higher, and the differences were statistically significant (all P < 0.05). Compared with the ARAF WT group, the relative expression levels of p-MEK, p-ERK and p-RSK in A549 or NCI-H1688 cells treated with belvarafenib in the ARAF WT and ARAF D429A groups were all lower, and the differences were statistically significant (all P < 0.05), but there were no statistically significant differences in the relative expression levels of ARAF, MEK, ERK, and RSK (all P > 0.05); compared with the ARAF WT group, the ARAF S214F and ARAF S214C groups treated with belvarafenib had higher relative expression levels of p-MEK, p-ERK and p-RSK, and the differences were statistically significant (all P < 0.05), but there were no statistically significant differences in the relative expression levels of ARAF, MEK, ERK, and RSK (all P > 0.05). On the 16th day after gavage with belvarafenib or AZ-628, the tumor volume of nude mice transplanted with A549 or NCI-H1688 cells in ARAF WT and ARAF D429A groups was lower than that in the corresponding ARAF WT and ARAF D429A groups without belvarafenib or AZ-628 intervention, and the tumor volume of nude mice transplanted with A549 or NCI-H1688 cells in ARAF D429A group was lower than that in the corresponding ARAF WT group with belvarafenib or AZ-628 gavage, and the differences were statistically significant (all P < 0.05). On the 16th day after gavage with belvarafenib or AZ-628, the tumor volume of nude mice transplanted with A549 or NCI-H1688 cells in ARAF S214F and ARAF S214C groups was not significantly different from that in ARAF S214F and ARAF S214C groups without belvarafenib or AZ-628 intervention (all P > 0.05), and compared with nude mice transplanted with A549 or NCI-H1688 cells in ARAF WT group with belvarafenib or AZ-628 gavage, there was no significant difference (all P > 0.05). Conclusions:The ARAF D429A mutation increases the sensitivity of lung cancer to RAF inhibitors, while the ARAF S214F and S214C mutations increase the resistance of lung cancer to RAF inhibitors.