Regulation of Ras-Dependent Signal Transduction Pathways

RAS通路是细胞信号转导的重要途径，其功能是传递控制细胞存活、增殖和分化的重要信号。与其在细胞信号传导中的核心作用一致，RAS通路的失调可以促进人类疾病状态，包括癌症和RASopathy发育障碍。阐明调节RAS信号通路的分子机制和确定在人类疾病状态中破坏RAS信号通路的策略是我们实验室近30年来的工作重点。我们的研究主要集中在RAF蛋白激酶（ARAF， BRAF和CRAF）上。RAF激酶家族成员是活化RAS的直接效应器，在由RAF、MEK和ERK蛋白激酶组成的三层ERK/MAPK级联反应中起起始酶的作用。我们对该领域工作的主要贡献是我们对调节RAF功能的关键蛋白质相互作用和磷酸化事件的识别和表征。我们小组的早期研究首次发现了破坏RAS结合的RAF激酶突变，为研究人员提供了研究RAS/RAF相互作用功能意义的关键工具。此外，我们在分析RAF磷酸化的工作中发现了抑制反馈磷酸化回路，该回路可以影响某些癌症治疗的有效性，并且对于正常生长条件下和细胞应激期间RAS信号的下调至关重要。我们的研究证明了RAF二聚体的作用，也对癌症治疗具有重要意义，揭示了促进RAF二聚体形成的继发性突变或抑制剂治疗如何改变疾病进展。此外，这些研究提供了抑制RAF二聚化具有治疗潜力的“原理证明”。意识到在活细胞条件下研究信号事件的重要性，我们的团队最近开发了生物发光共振能量转移（BRET）方法来分析活细胞中RAF调节相互作用（RAS/RAF结合和RAF二聚化）。BRET系统的优势在于，它允许在质膜环境的背景下，在翻译后修饰和脂质加工仍然发生的条件下，监测关键的信号相互作用，这些事件可以强烈影响蛋白质结合和信号进展。使用BRET分析来研究RAS/RAF相互作用，我们的研究揭示了高度保守的RAS和RAF家族成员之间不同的结合偏好，这些偏好直接影响癌症进展，并可以改变癌细胞对靶向治疗的反应（Terrell等人，2019）。更具体地说，我们发现突变体KRAS， ras介导的肿瘤发生的主要贡献者，与所有RAF成员高亲和力结合。相比之下，突变体HRAS和NRAS表现出对CRAF的优先结合，其中BRAF对KRAS具有独特的选择性。此外，通过耗尽研究，我们发现CRAF对于突变型HRAS驱动的信号传导至关重要，促进稳定BRAF/CRAF二聚体形成的事件，如某些BRAF突变或RAF抑制剂治疗，可以使突变型HRAS以更高的亲和力与BRAF结合，促进肿瘤发生。在此回顾期间，我们的实验室继续在合作研究中使用BRET RAS/RAF相互作用测定，以确定KRAS或NRAS的特定突变如何影响这些RAS蛋白与各种RAF成员相互作用的能力（Johnson等人，2022年和Murphy等人，2022年）。此外，我们的实验室与NCI分子靶标项目合作，利用NCI大量多样的天然产物提取物，利用BRET分析进行高通量药物筛选，以确定可以调节RAS/RAF相互作用的化合物。BRET检测已被证明是一种非常敏感的检测激酶抑制剂和其他药物疗法的方法，这些药物疗法具有增强RAS/RAF结合的有害作用，从而促进耐药性和/或继发性肿瘤形成（Durrant等，2021）。此外，筛选发现了许多能够抑制RAS/RAF结合的化合物（Kim et al.， 2020和Senadeera et al., 2022），其中一些可能具有治疗潜力。在回顾期间，我们的实验室还完成了与张平博士在nci -结构生物学项目中的重要合作项目。该项目确定了从哺乳动物细胞中分离的全长BRAF复合物的三种高分辨率冷冻电镜结构：自抑制的单体BRAF:14-3-3 32； MEK和BRAF:14-3-3 -32复合物，以及RAF抑制剂结合的二聚体braf2:14-3-3 -32复合物。值得注意的是，BRAF的RAS结合域（RBD）在我们的两个BRAF单体结构中都得到了很好的解析，首次揭示了这一关键结构域在全长、自抑制BRAF单体中的位置和方向（Martinez-Fiesco et al., 2022）。最后，在审查期间，我们的实验室还参与了NCI-CCR推进RASopathy疗法（ART）计划的启动。这项倡议包括临床和基础研究两部分，并将汇集癌症研究中心（CCR）、癌症流行病学和遗传学部门（DCEG）的研究人员、患者倡导团体和研究这些发育障碍的校外专家。我们小组最近完成了一个项目，评估了一些最普遍的rasopathy相关的CRAF和BRAF突变体。通过这一努力以及与LCDS斑马鱼设施的合作，我们已经建立了一种使用斑马鱼胚胎的筛选试验，可以监测rasopathy相关突变体的功能获得活性。该试验将用于分析通过RASopathy Initiative确定的任何先前未表征的RASopathy突变体。此外，该分析有望提供有关突变严重程度以及各种药物治疗有效性的有价值的信息。

The RAS pathway is an important route of cellular signal transduction, functioning to relay vital signals that control cell survival, proliferation, and differentiation. Consistent with its central role in cell signaling, dysregulation of the RAS pathway can promote human disease states including cancer and the RASopathy developmental disorders. Elucidating the molecular mechanisms that regulate RAS pathway signaling and identifying strategies to disrupt RAS signaling in human disease states has been the focus of our laboratory's efforts for almost 30 years. Much of our research has centered on the RAF protein kinases (ARAF, BRAF and CRAF). Members of the RAF kinase family are direct effectors of activated RAS and function as the initiating enzymes in the three-tiered ERK/MAPK cascade, comprised of the RAF, MEK and ERK protein kinases. A primary contribution of our work to the field has been our identification and characterization of key protein interactions and phosphorylation events that modulate RAF function. Early studies from our group were the first to identify a mutation in the RAF kinases that disrupts RAS binding, providing researchers with a key tool to investigate the functional significance of the RAS/RAF interaction. In addition, our work analyzing RAF phosphorylation led to the discovery of inhibitory feedback phosphorylation loops that can impact the effectiveness of certain cancer therapies and are critical for the downregulation of RAS signaling under normal growth conditions and during cellular stress. Our studies demonstrating the role of RAF dimerization have also had important implications for cancer treatment, revealing how disease progression can be altered by secondary mutations or inhibitor treatments that promote RAF dimer formation. Moreover, these studies provided the "proof-of-principle" that inhibiting RAF dimerization has therapeutic potential. Realizing the importance of studying signaling events under live cell conditions, our group has recently developed bioluminescence resonance energy transfer (BRET) methodologies for analyzing RAF regulatory interactions (RAS/RAF binding and RAF dimerization) in living cells. The advantage of the BRET system is that it allows for crucial signaling interactions to be monitored in the context of the plasma membrane environment and under conditions where post-translational modifications and lipid processing still occur, events that can strongly influence protein binding as well as signal progression. Using the BRET assay to investigate the RAS/RAF interaction, our studies revealed distinct binding preferences between the highly conserved RAS and RAF family members that directly impact cancer progression and can alter how a cancer cell responds to targeted therapies (Terrell et al., 2019). More specifically, we found that mutant KRAS, the major contributor to RAS-mediated tumorigenesis, binds with high affinity to all RAF members. In contrast, mutant HRAS and NRAS exhibit preferential binding to CRAF, with BRAF demonstrating a unique selectivity for KRAS. Moreover, through depletion studies, we found that CRAF is critical for mutant HRAS-driven signaling and that events promoting stable BRAF/CRAF dimer formation, such as certain BRAF mutations or RAF inhibitor treatments, can allow mutant HRAS to engage BRAF with increased affinity to promote tumorigenesis. During this review period, our lab has continued to use the BRET RAS/RAF interaction assay in collaborative studies to determine how specific mutations in KRAS or NRAS impact the ability of these RAS proteins to interact with the various RAF members (Johnson et al, 2022 and Murphy et al, 2022). In addition, working in collaboration with the NCI-Molecular Targets Program and utilizing the NCI's large and diverse collection of natural product extracts, our lab utilized the BRET assay to conduct a high-throughput drug screen for identifying compounds that can modulate the RAS/RAF interaction. The BRET assay has proven to be a very sensitive way of detecting kinase inhibitors and other drug therapies that have the deleterious effect of augmenting RAS/RAF binding, which in turn can promote drug resistance and/or secondary tumor formation (Durrant et al. 2021). In addition, the screen identified numerous compounds that were able to inhibit RAS/RAF binding (Kim et al., 2020 and Senadeera et al., 2022), some of which may have therapeutic potential. In this review period, our lab also completed an important collaborative project with Dr. Ping Zhang in the NCI-Structural Biology Program. This project resulted in the determination of three high-resolution cryo-electron microscopy structures of full-length BRAF complexes that were isolated from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3- 32 complexes, and a RAF inhibitor-bound, dimeric BRAF2:14-3-32 complex. Notably, the RAS binding domain (RBD) of BRAF was well-resolved in both of our monomeric BRAF structures, revealing for the first time the position and orientation of this critical domain in the context of the full-length, autoinhibited BRAF monomer (Martinez-Fiesco et al., 2022). Finally, during the review period, our lab was also engaged in the kick-off of the NCI-CCR Initiative on Advancing RASopathy Therapies (ART). This initiative has both clinical and basic research components and will bring together investigators in the Center for Cancer Research (CCR), Division of Cancer Epidemiology and Genetics (DCEG), patient advocacy groups, and extramural experts working on these developmental disorders. Our group has recently completed a project evaluating a number of the most prevalent RASopathy-associated CRAF and BRAF mutants. Through this effort and in collaboration with the LCDS Zebrafish Facility, we have established a screening assay using zebrafish embryos that can monitor the gain-of-function activities of RASopathy-associated mutants. This assay will be employed to analyze any previously uncharacterized RASopathy mutants that are identified through the RASopathy Initiative. Moreover, this assay is expected to provide valuable information regarding the severity of the mutation as well as the effectiveness of various drug treatments.