本研究は、転移・再発性ホルモン受容体陽性・HER2陰性乳癌の治療において中心的役割を果たすサイクリン依存性キナーゼ(CDK)4/6阻害剤の耐性獲得メカニズムを解明する目的で施行された。乳癌細胞株であるMCF7を対象に、CRISPR/Cas9ゲノム編集を用いたwhole-genome knockout screenを行った。具体的には、Cas9を安定発現したMCF7に対してレンチウイルスを媒介としてwhole-genome libraryを感染させ、1細胞あたり1つのsingle guide RNA(sgRNA)が挿入され、対応する遺伝子がknockoutされるようにpooled screenを行った。Cas9-MCF7にwhole-genome libraryを感染させ、hygromycinで感染細胞を選択した後に、CDK4/6阻害剤であるpalbociclibを添加し、約2週間の培養を行った。培養後に生存した最終産物の細胞からgenomic DNAを抽出した。それぞれのsgRNAに付随するバーコードをPCRにて増幅させ、NGSシーケンサーで解析を行い、耐性遺伝子の候補を解析した。細胞周期に関わるRb遺伝子など、すでに既報で確認された耐性遺伝子の発現を確認した。現在、詳細なsgRNAの発現量の解析を施行中であり、どの遺伝子がknockoutされることでpalbociclib投与下の細胞増殖につながったかを解析中である。この解析にて耐性遺伝子の候補が選定出来た際には、それらの候補遺伝子に対応するsgRNAのカスタムライブラリーを作成し、secondary screenを行うことが可能となる。そのように同定された耐性遺伝子をCRISPR/Cas9のゲノム編集技術でノックアウトし、in vitroおよびin vivoで実際に耐性を獲得するか確認を行う。
This study was conducted to elucidate the mechanisms of resistance acquisition to cyclin-dependent kinase (CDK) 4/6 inhibitors, which play a central role in the treatment of patients with metastatic or recurrent hormone receptor-positive, HER2-negative breast cancer. A whole-genome knockout screen using CRISPR/Cas9 genome editing was performed on MCF7, a breast cancer cell line. Specifically, the whole-genome library was infected via lentivirus into MCF7 cells which stably express Cas9, so that one single guide RNA (sgRNA) would be inserted per cell, and the corresponding gene would be knocked out. After infection of Cas9-MCF7 with the whole-genome library and selection of infected cells with hygromycin, palbociclib, a CDK4/6 inhibitor, was added and the cells were cultured for about 2 weeks. Genomic DNA was extracted from the end product cells that survived after culture. Barcodes associated with each sgRNA were amplified by PCR and analyzed by NGS sequencing to identify candidate resistance genes. The expression of resistance genes already confirmed in previous reports, such as the Rb gene involved in the cell cycle, was confirmed. Detailed analysis of sgRNA expression levels is currently underway to determine which genes were knocked out, leading to cell proliferation under palbociclib treatment. When candidate genes for resistance are selected through this analysis, a custom library of sgRNAs corresponding to these candidate genes will be created and a secondary screen can be performed. The identified resistance genes will be knocked out using CRISPR/Cas9 genome editing technology, and whether they actually acquire resistance in vitro and in vivo will be confirmed.
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