Functional characterisation of dynamic BRAF signalling complexes and their modulation by tumour specific mutations and clinically relevant kinase inhibitors

动态 BRAF 信号复合物的功能特征及其通过肿瘤特异性突变和临床相关激酶抑制剂的调节

• 批准号: 421542753
• 负责人: Professor Dr. Tilman Brummer
• 金额: --
• 依托单位: Départment de Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/421542753?language=en
• 关键词: Functional; characterisation; dynamic; BRAF; signalling

BRAF plays a central role in the activation of RAS/ERK signalling. The activation cycle of this kinase is driven by RAS induced homo- or hetero-dimerisation and tightly controlled by protein-protein interaction events and post-translational modifications (PTMs).BRAF is often dysregulated in cancer. The most common mutation, V600E, cuts the incompletely understood BRAF activation cycle short. Thereby, this mutation generates an oncoprotein in which its kinase domain maintains an active conformation that is only transiently assumed by wildtype BRAF (BRAFWT) following RAS induced activation. This allowed the development of BRAFV600E selective inhibitors that yield impressive initial response rates in various entities. Unfortunately, therapeutic responses are short-lived due to the emergence of drug resistance. BRAF inhibitor induced paradoxical ERK pathway activation represents a common resistance mechanism. This phenomenon is caused by the unforeseen property of clinically applied BRAF selective inhibitors to promote heterodimers between drug-bound BRAF and other RAF isoforms in the presence of RAS activity. Drug-bound BRAF acts as a potent allosteric activator of the drug-free RAF protomer, thereby causing ERK re-activation and tumour growth. It is likely that the paradoxical action of BRAF inhibitors exploits processes occurring during physiological RAS/ERK pathway activation. In order to develop more effective and safer inhibitors, it will be critical to understand the spatio-temporal dynamics of quaternary BRAF signalling complexes in both physiological and pharmacological settings. Using Blue Native PAGE and SEC-PCP-SILAC based mass spectrometry (MS), we demonstrated that BRAFWT and BRAFV600E organise multi-protein complexes of distinct size and composition. We also showed that RAS induces BRAFWT containing complexes of similar size as those formed by BRAFV600E. Moreover, clinically relevant drugs affect the stability of these complexes, e.g. in settings with desired or paradoxical effects of BRAF inhibitors. Based on this and other data, we posit that the activity status of the kinase domain dictates the assembly of BRAF signalling complexes. In the proposed project, we aim to confirm this hypothesis by conducting an in-depth characterisation of the composition and PTM pattern of BRAF complexes formed under physiological conditions and in the presence of kinase inhibitors of (pre)clinical relevance. We will combine our MS protocols with novel biochemical approaches to identify short-lived dynamic interactions. We will extend our studies to complexes formed by non-V600E BRAF oncoproteins, which are increasingly detected by personalised medicine programs. These mutants are hardly defined in terms of their pathomechanism and drug sensitivity, currently precluding therapeutic recommendations. Thereby, we will gain novel mechanistic insights into the BRAF signalling and can provide at the same time critical information about their druggability.

BRAF在RAS/ERK信号的激活中起核心作用。该激酶的激活周期由RAS诱导的同源或异源二聚体驱动，并受到蛋白质-蛋白质相互作用事件和翻译后修饰(PTM)的严格控制。BRAF在癌症中经常处于失调状态。最常见的突变是V600E，它缩短了不完全理解的BRAF激活周期。因此，这种突变产生了一种癌蛋白，其激活域保持着一种活性构象，只有野生型BRAF(BRAFWT)在RAS诱导激活后才会短暂地呈现这种构象。这使得BRAFV600E选择性抑制剂得以开发，在各种实体中产生了令人印象深刻的初步应答率。不幸的是，由于耐药性的出现，治疗反应是短暂的。BRAF抑制剂诱导的反常ERK通路激活是一种常见的耐药机制。这种现象是由于临床上应用的BRAF选择性抑制剂在存在RAS活性的情况下促进药物结合的BRAF和其他RAF亚型之间的异二聚体的特性而引起的。药物结合的BRAF作为不含药物的RAF原的有效变构激活剂，从而导致ERK的重新激活和肿瘤的生长。BRAF抑制剂的这一矛盾作用很可能利用了生理性RAS/ERK通路激活过程。为了开发更有效和更安全的抑制剂，了解四元BRAF信号复合体在生理和药理环境下的时空动力学将是至关重要的。利用Blue Native PAGE和SEC-PCP-SILAC基质谱(MS)，我们证明了BRAFWT和BRAFV600E组织了不同大小和组成的多蛋白质复合体。我们还表明，RAS诱导的BRAFWT含有与BRAFV600E形成的复合物大小相似的BRAFWT。此外，临床上相关的药物会影响这些复合体的稳定性，例如，在具有BRAF抑制剂所期望的或相反的效果的环境中。基于这一点和其他数据，我们假设，激活域的活性状态决定了BRAF信号复合体的组装。在拟议的项目中，我们旨在通过对在生理条件下和在存在(临床前)相关的激酶抑制剂的情况下形成的BRAF复合体的组成和PTM模式进行深入的表征来证实这一假设。我们将把我们的MS协议与新的生化方法结合起来，以识别短暂的动态相互作用。我们将把我们的研究扩展到由非V600E BRAF癌蛋白形成的复合体，这些复合体越来越多地被个性化药物程序检测到。这些突变很难根据它们的致病机制和药物敏感性来定义，目前排除了治疗建议。因此，我们将获得对BRAF信号的新的机械学见解，同时可以提供关于它们的可药性的关键信息。