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

BRAF在RAS/ERK信号传导的激活中起核心作用。该激酶的活化周期由RAS诱导的同源或异源二聚化驱动，并由蛋白质-蛋白质相互作用事件和翻译后修饰（PTM）严格控制。最常见的突变V600 E缩短了不完全理解的BRAF激活周期。因此，这种突变产生一种癌蛋白，其中其激酶结构域保持活性构象，该活性构象仅在RAS诱导激活后由野生型BRAF（BRAFWT）短暂呈现。这使得BRAFV 600 E选择性抑制剂的开发在各种实体中产生令人印象深刻的初始响应率。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ERK通路的反常激活代表了一种常见的耐药机制。这种现象是由临床应用的BRAF选择性抑制剂在RAS活性存在下促进药物结合的BRAF和其他RAF同种型之间的异二聚体的不可预见的性质引起的。药物结合的BRAF充当无药物RAF原聚体的有效变构激活剂，从而引起ERK再激活和肿瘤生长。BRAF抑制剂的矛盾作用可能利用了生理性RAS/ERK通路激活期间发生的过程。为了开发更有效和更安全的抑制剂，了解四元BRAF信号复合物在生理和药理学环境中的时空动力学将是至关重要的。使用蓝色非变性PAGE和基于SEC-PCP-SILAC的质谱（MS），我们证明了BRAFWT和BRAFV 600 E组织不同大小和组成的多蛋白复合物。我们还表明，RAS诱导BRAFWT含有类似大小的复合物形成的BRAFV 600 E。此外，临床相关药物影响这些复合物的稳定性，例如在具有BRAF抑制剂的期望或矛盾作用的环境中。基于此和其他数据，我们认为激酶结构域的活性状态决定了BRAF信号复合物的组装。在拟议的项目中，我们的目标是通过对在生理条件下和存在临床（前）相关激酶抑制剂的情况下形成的BRAF复合物的组成和PTM模式进行深入表征来证实这一假设。我们将联合收割机我们的MS协议与新的生化方法，以确定短暂的动态相互作用。我们将把我们的研究扩展到由非V600 E BRAF癌蛋白形成的复合物，这些蛋白越来越多地被个性化医学项目检测到。这些突变体很难定义其病理机制和药物敏感性，目前排除治疗建议。因此，我们将获得对BRAF信号传导的新的机制见解，并可以同时提供关于其可药用性的关键信息。

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.