Sensitivity of Oncogenic KRAS-Expressing Cells to CDK9 Inhibition.
Sensitivity of Oncogenic KRAS-Expressing Cells to CDK9 Inhibition.
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DOI：
10.1177/24725552211008853
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发表时间：
2021-08
期刊：
影响因子：
--
通讯作者：
McCormick F
中科院分区：
文献类型：
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作者：
Lai LP;Brel V;Sharma K;Frappier J;Le-Henanf N;Vivet B;Muzet N;Schell E;Morales R;Rooney E;Basse N;Yi M;Lacroix F;Holderfield M;Englaro W;Marcireau C;Debussche L;Nissley DV;McCormick F
已知 KRAS 蛋白的致癌形式是胰腺癌、结直肠癌和肺癌的驱动因素。本研究的目的是确定抑制致癌 KRAS 信号传导的化学先导物。我们首先开发了小鼠胚胎成纤维细胞 (MEF) 细胞系的同基因组，其携带野生型 RAS、致癌 KRAS 和致癌 BRAF。我们通过在基于三磷酸腺苷 (ATP) 的细胞活力测定中筛选 1402 个注释抑制剂的工具化合物库来验证这些细胞系。随后，该 MEF panel 用于使用专有化合物库在细胞活力测定中进行高通量表型筛选。所有 126 种对突变型 KRAS 表现出选择性活性的化合物均根据其在二次测定中的活性进行了选择和优先排序。最后，选择了五个化学簇。它们对 Colo320 结直肠癌细胞系中的 SW620 和 LS513 具有特异性活性。此外，根据体外酶活性测定，它们对 BRAFV600E、MEK1、细胞外信号调节激酶 2 (ERK2)、磷酸肌醇 3-激酶 α (PI3Kα)、AKT1 或哺乳动物雷帕霉素靶点 (mTOR) 没有影响。生物物理测定表明这些化合物不直接与 KRAS 结合。我们进一步确定了作用机制，并表明其中三种具有 CDK9 抑制活性。总之，我们开发并验证了同基因 MEF 组合，该组合已成功用于识别 RAS 致癌或野生型等位基因特异性漏洞。此外，我们还确定了表达 KRAS 的致癌细胞对 CDK9 抑制剂的敏感性，这为未来使用 CDK9 抑制剂治疗 KRAS 驱动的癌症的研究提供了依据。
Oncogenic forms of KRAS proteins are known to be drivers of pancreatic, colorectal, and lung cancers. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type RAS, oncogenic KRAS, and oncogenic BRAF. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in an adenosine triphosphate (ATP)-based cell viability assay. Subsequently, this MEF panel was used to conduct a high-throughput phenotypic screen in a cell viability assay with a proprietary compound library. All 126 compounds that exhibited a selective activity against mutant KRAS were selected and prioritized based on their activities in secondary assays. Finally, five chemical clusters were chosen. They had specific activity against SW620 and LS513 over Colo320 colorectal cancer cell lines. In addition, they had no effects on BRAFV600E, MEK1, extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3-kinase alpha (PI3Kα), AKT1, or mammalian target of rapamycin (mTOR) as tested in in vitro enzymatic activity assays. Biophysical assays demonstrated that these compounds did not bind directly to KRAS. We further identified the mechanism of action and showed that three of them have CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that was used successfully to identify RAS oncogenic or wild-type allele-specific vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibitors, which warrants future studies of treating KRAS-driven cancers with CDK9 inhibitors.
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