Relation between TP53 GOF mutation and Osimertinib treatment in EGFR mutant lung cancer

Background: Although TP53 gain-of-function (GOF) mutations promote cancer survival, its effect on EGFR-TKI efficacy remains unclear. We established EGFR-mutant lung cancer cell lines expressing various TP53 genotypes using CRISPR-Cas9 technology and found that TP53-GOF mutant cells develop an early resistance to EGFR-TKI osimertinib.The goal of this study is to elucidate the mechanisms underlying resistance to osimertinib treatment in TP53 GOF mutations through comprehensive gene analysis using RNA-seq with next-generation sequencing (NGS). Methods: Total RNA was isolated from PC-9 cells overexpressing TP53 R248Q mutation (PC9/p53R248Q: TP53 GOF mutation) and PC-9 cells overexpressing empty vector plasmid (PC9/p53EV: TP53 null) treated with DMSO or osimertinib for 24hours, using the RNeasy Micro Kit , in accordance with the manufacturer’s instructions. RNA samples were quantified by NanoDrop-2000 spectrophotometer, and the quality was confirmed with a 2200 TapeStation. rRNA was removed using MGI Easy rRNA Depletion Kit according to manufacturer's instructions followed by library construction using MGIEasy RNA Directional Library Prep Set (MGI). MGI DNBseq-G400 FAST was used to perform the amplicons deep sequencing following the standard operation protocol. The sequence format was 150bp pair read for all samples. All sequencing reads were trimmed low-quality bases and adapters with Trimmomatic (v.0.38) , and RNA sequencing reads were mapped to hg38 using HISAT2 software . Raw counts for each gene were estimated in each sample using RSEM version 1.3.0 and Bowtie 2. Calculation of the log fold-change (log FC) and p-value were performed using edgeR. Results: We explored the functions of specifically upregulated genes in TP53 GOF mutation after osimertinib treatment by KEGG pathway-enrichment analysis and found that the cytokine-cytokine receptor interaction was the most significantly altered pathway. Hallmark pathway analysis identified the TNF-α/NF-κB pathway was significantly enriched. Furthermore, TRRUST analysis showed enhanced activity of transcription factors especially RELA (p65) and NF-kB1. Conclusions: TP53 GOF mutaion induces osimertinib resistance by activating TNF-α/NF-κB pathway. mRNA profiles of PC9/p53R248Q cells and PC9/p53EV cells treated by osimertinib or DMSO (control) for 24 hours.

背景：尽管TP53功能获得型（gain-of-function, GOF）突变可促进肿瘤细胞存活，但其对表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）疗效的影响仍尚不明确。本研究采用CRISPR-Cas9技术构建了携带不同TP53基因型的表皮生长因子受体（EGFR）突变型肺癌细胞系，发现携带TP53功能获得型突变的细胞会对EGFR-TKI奥希替尼（osimertinib）产生早期耐药。本研究旨在通过下一代测序（next-generation sequencing, NGS）技术开展RNA测序（RNA-seq）的全基因分析，阐明TP53功能获得型突变介导奥希替尼耐药的具体机制。方法：采用RNeasy Micro试剂盒，严格遵循厂商说明书，从经二甲基亚砜（dimethyl sulfoxide, DMSO）或奥希替尼处理24小时的两组PC-9细胞中提取总RNA：一组为过表达TP53 R248Q突变（PC9/p53R248Q：TP53功能获得型突变）的细胞，另一组为转染空载体质粒（PC9/p53EV：TP53缺失型）的细胞。采用NanoDrop-2000分光光度计对RNA样品进行定量，并通过2200 TapeStation检测RNA质量。依照厂商说明书使用MGI Easy rRNA Depletion试剂盒去除核糖体RNA（rRNA），随后使用MGIEasy RNA Directional Library Prep Set（MGI）构建链特异性RNA文库。按照标准操作流程，采用MGI DNBseq-G400 FAST平台完成扩增子深度测序，所有样本均采用150bp双端测序读段模式。使用Trimmomatic（v.0.38）对测序读段进行低质量碱基及接头序列修剪，借助HISAT2软件将RNA测序读段比对至hg38参考基因组。采用RSEM v1.3.0与Bowtie 2软件对每个样本的各基因原始计数进行估算，通过edgeR软件计算对数倍变化（log fold-change, log FC）与P值。结果：本研究通过京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析，对奥希替尼处理后TP53功能获得型突变细胞中特异性上调的基因进行功能注释，发现细胞因子-细胞因子受体相互作用通路是改变最为显著的通路。特征通路（Hallmark pathway）分析显示，肿瘤坏死因子-α/核因子-κB（TNF-α/NF-κB）通路显著富集。此外，TRRUST分析结果表明，转录因子活性显著增强，尤其是RELA（p65）与NF-κB1。结论：TP53功能获得型突变通过激活TNF-α/NF-κB通路介导奥希替尼耐药。本数据集包含经奥希替尼或DMSO（对照）处理24小时的PC9/p53R248Q细胞与PC9/p53EV细胞的mRNA表达谱。