A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer.
A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer.
复制标题
高内涵克隆生存药物筛选确定 mek 抑制剂是 KRAS 突变非小细胞肺癌的有效放射增敏剂。
DOI：
10.1097/jto.0000000000000199
复制
发表时间：
2014-07
期刊：
影响因子：
--
通讯作者：
Heymach JV
中科院分区：
文献类型：
--
作者：
Lin SH;Zhang J;Giri U;Stephan C;Sobieski M;Zhong L;Mason KA;Molkentine J;Thames HD;Yoo SS;Heymach JV
传统的克隆存活率和高通量比色分析不足以作为药物筛选来识别新型辐射增敏剂。我们开发了一种方法，我们称之为高含量克隆生存测定（HCSA），这将允许筛选药物库，以确定候选辐射增敏剂。在96孔板中使用HCSA进行药物筛选。在药物处理、照射和孵育后，将菌落用结晶紫染色并在INCell 6000®（GE Health）上成像。使用INCell Developer图像分析软件对达到50个或更多个细胞的集落进行计数。在KRAS突变肺癌细胞系H460和定制临床收集（Custom Clinical Collection）（146种化合物）上进行原理验证筛选。同一类别的多种药物被发现是辐射增敏剂，效力水平似乎反映了这些药物的临床相关性。例如，几种PARP抑制剂在HCSA筛选中被鉴定为良好的辐射增敏剂。然而，也有一些PARP抑制剂未被发现具有致敏性，这些抑制剂要么没有进入临床开发，要么在BSI-201的情况下，甚至被证明不是PARP抑制剂。我们发现，激活的突变KRAS（PI 3 K，AKT，mTOR和MEK 1/2）下游通路的抑制剂使H460细胞对辐射敏感。此外，有效的MEK 1/2抑制剂trmenitib选择性地增强了KRAS突变型而非野生型肺癌细胞中的辐射效应。使用HCSA方法筛选新型辐射增敏剂是可行的。这是一项使能技术，将有助于加速发现用于临床测试的新型放射增敏剂。
Traditional clonogenic survival and high throughput colorimetric assays are inadequate as drug screens to identify novel radiation sensitizers. We developed a method which we call the High Content Clonogenic Survival Assay (HCSA) that will allow screening of drug libraries to identify candidate radiation sensitizers. Drug screen using HCSA was done in 96 well plates. After drug treatment, irradiation, and incubation, colonies were stained with crystal violet and imaged on the INCell 6000® (GE Health). Colonies achieving 50 or more cells were enumerated using the INCell Developer image analysis software. A proof-of-principle screen was done on the KRAS mutant lung cancer cell line H460 and a Custom Clinical Collection (146 compounds). Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several PARP inhibitors were identified as good radiation sensitizers in the HCSA screen. However there were also a few PARP inhibitors not found to be sensitizing that have either not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor. We discovered that inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation. Furthermore, the potent MEK1/2 inhibitor tramenitib selectively enhanced radiation effects in KRAS mutant but not wild type lung cancer cells. Drug screening for novel radiation sensitizers is feasible using the HCSA approach. This is an enabling technology that will help accelerate the discovery of novel radiosensitizers for clinical testing.