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Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids.
Journal of Biological Chemistry ( IF 4.0 ) Pub Date : 2020-04-03 , DOI: 10.1074/jbc.ra119.011025
Svenja Wiechmann 1, 2 , Pierre Maisonneuve 3 , Britta M Grebbin 4, 5, 6 , Meike Hoffmeister 1, 7 , Manuel Kaulich 1, 8 , Hans Clevers 9, 10, 11 , Krishnaraj Rajalingam 12 , Igor Kurinov 13 , Henner F Farin 4, 5, 6 , Frank Sicheri 3 , Andreas Ernst 2, 14
Affiliation  

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity to the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity to the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

中文翻译:

活化的Ras GTPases的构象特异性抑制剂显示,患者衍生的类癌类器官对Ras的依赖性有限。

小的GTPases H,K和NRAS是适当调节细胞增殖和生长必不可少的分子开关。编码该蛋白家族成员的基因中的几种突变与癌症有关,并导致由下游效应蛋白的募集失控引起的信号传导过程异常激活。在这项研究中,我们设计了C-Raf原癌基因Ras结合域(RBD),Ser / Thr激酶(CRAF)的变体。这些变体以活性构象与Ras的效应子结合位点高亲和力结合。结构表征揭示了与WT RBD相比,新近鉴定出的RBD突变如何协同作用,从而增强了对Ras中效应子结合位点的亲和力。工程改造的RBD变体紧密模仿天然存在的Ras效应子的相互作用模式,并充当显性-负亲和力试剂,可阻断Ras信号转导。癌细胞实验表明,这些RBD变体的表达抑制Ras信号传导,减少细胞生长并诱导细胞凋亡。使用这些优化的RBD变体,我们根据患者对Ras抑制的反应,将具有已知Ras突变状态的患者来源的结直肠癌类器官分层。这些结果表明,Ras突变的存在不足以预测对Ras抑制的敏感性,这表明并非所有这些肿瘤都需要Ras信号进行增殖。总之,通过设计CRAF-RBD的Ras / Raf接口,
更新日期:2020-04-03
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