Regulation of Ras-Dependent Signal Transduction Pathways

Ras 依赖性信号转导途径的调节

基本信息

批准号：
10926001
负责人：
Deborah Morrison
金额：
$ 134.23万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10926001
关键词：
Affinity BRAF gene Basic Science Binding Binding Proteins Biochemical Biological Assay Bioluminescence Cell Survival Cell membrane Cells Cellular Stress Clinical Research Collaborations Collection Complex Cryoelectron Microscopy Dimerization Disease Disease Progression Division of Cancer Epidemiology and Genetics Down-Regulation Drug resistance Effectiveness Embryo Energy Transfer Environment Enzymes Event Exhibits Extramural Activities Family Family member Feedback Goals Growth Growth and Development function Guanosine Triphosphate Phosphohydrolases Human KRAS2 gene Laboratories Length Link Lipids MAP Kinase Gene MEKs Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Mediating Methodology Molecular Molecular Target Monitor Mutation NCI Center for Cancer Research Natural Products Neoplasm Metastasis Pathway interactions Pharmacotherapy Phosphorylation Positioning Attribute Post-Translational Protein Processing Proliferating Protein Kinase Proteins RAS genes RAS inhibition Ras Signaling Pathway Ras/Raf Regulation Research Research Personnel Resolution Role Route Severities Signal Transduction Signal Transduction Pathway Structure System Techniques Therapeutic Therapeutic Intervention Time Work Zebrafish cancer cell cancer therapy developmental disease dimer gain of function high resolution imaging high-throughput drug screening human disease inhibitor kinase inhibitor member monomer mutant patient advocacy group preference programs raf Kinases ras Proteins structural biology targeted treatment tool tumor progression tumorigenesis

项目摘要

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 (Spencer-Smith et al., 2023). Through this effort and in collaboration with the LCDS Zebrafish Facility, we have established a panel of assays using zebrafish embryos that can monitor the gain-of-function activities of RASopathy-associated mutants. These assays will be employed to analyze any previously uncharacterized RASopathy mutants that are identified through the RASopathy Initiative. Moreover, these assays are expected to provide valuable information regarding the severity of the mutation as well as the effectiveness of various drug treatments.

RAS通路是细胞信号转导的重要途径，负责传递控制细胞存活、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相一致，RAS 通路的失调可以促进人类疾病状态，包括癌症和 RAS 病发育障碍。近 30 年来，阐明调节 RAS 通路信号传导的分子机制并确定破坏人类疾病状态下 RAS 信号传导的策略一直是我们实验室的工作重点。我们的大部分研究都集中在 RAF 蛋白激酶（ARAF、BRAF 和 CRAF）上。 RAF 激酶家族的成员是激活 RAS 的直接效应子，并作为三层 ERK/MAPK 级联（由 RAF、MEK 和 ERK 蛋白激酶组成）中的起始酶发挥作用。我们对该领域工作的主要贡献是对调节 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 RAF 激酶中破坏 RAS 结合的突变，为研究人员提供了研究 RAS/RAF 相互作用的功能意义的关键工具。此外，我们分析 RAF 磷酸化的工作发现了抑制性反馈磷酸化环，它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 信号传导的下调至关重要。我们的研究证明 RAF 二聚化的作用也对癌症治疗具有重要意义，揭示了二次突变或促进 RAF 二聚体形成的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”。认识到研究活细胞条件下信号传导事件的重要性，我们的小组最近开发了生物发光共振能量转移 (BRET) 方法，用于分析活细胞中 RAF 调节相互作用（RAS/RAF 结合和 RAF 二聚化）。 BRET 系统的优势在于，它可以在质膜环境中以及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传导相互作用，这些事件可以强烈影响蛋白质结合以及信号进展。使用 BRET 测定来研究 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，2019））。更具体地说，我们发现突变的 KRAS（RAS 介导的肿瘤发生的主要贡献者）以高亲和力与所有 RAF 成员结合。相比之下，突变的 HRAS 和 NRAS 表现出与 CRAF 的优先结合，其中 BRAF 对 KRAS 表现出独特的选择性。此外，通过耗竭研究，我们发现 CRAF 对于突变型 HRAS 驱动的信号传导至关重要，并且促进稳定 BRAF/CRAF 二聚体形成的事件（例如某些 BRAF 突变或 RAF 抑制剂治疗）可以允许突变型 HRAS 以增加的亲和力与 BRAF 结合。促进肿瘤发生。在此审查期间，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 中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 et al, 2022和墨菲等人，2022）。此外，我们的实验室与 NCI 分子靶标计划合作，利用 NCI 大量且多样化的天然产物提取物集合，利用 BRET 测定进行高通量药物筛选，以识别可以调节 RAS/RAF 的化合物相互作用。 BRET 检测已被证明是一种非常灵敏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，进而促进耐药性和/或继发性肿瘤形成 (Durrant et al. 2021 ）。此外，筛选还发现了许多能够抑制 RAS/RAF 结合的化合物（Kim 等人，2020 和 Senadeera 等人，2022），其中一些可能具有治疗潜力。在此回顾期间，我们实验室还与NCI结构生物学项目张平博士完成了一项重要的合作项目。该项目确定了从哺乳动物细胞中分离出的全长 BRAF 复合物的三种高分辨率冷冻电子显微镜结构：自抑制单体 BRAF:14-3-32:MEK 和 BRAF:14-3-32 复合物，以及 RAF 抑制剂结合的二聚体 BRAF2:14-3-32 复合物。值得注意的是，BRAF 的 RAS 结合域 (RBD) 在我们的两个单体 BRAF 结构中得到了很好的解析，首次揭示了该关键域在全长、自抑制 BRAF 单体背景下的位置和方向（Martinez -Fiesco 等人，2022）。最后，在审查期间，我们的实验室还参与了 NCI-CCR 推进 RAS 病治疗 (ART) 倡议的启动。该计划包含临床和基础研究两个部分，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、患者倡导团体以及研究这些发育障碍的校外专家。我们的小组最近完成了一个项目，评估了一些最流行的 RAS 病相关 CRAF 和 BRAF 突变体（Spencer-Smith 等人，2023）。通过这项努力并与 LCDS 斑马鱼设施合作，我们建立了一组使用斑马鱼胚胎的检测方法，可以监测 RAS 病相关突变体的功能获得活动。这些检测将用于分析通过 RASopathy Initiative 鉴定的任何以前未表征的 RASopathy 突变体。此外，这些测定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。

项目成果

期刊论文数量（21）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Targeting the Raf kinases in human cancer: the Raf dimer dilemma.

靶向人类癌症中的 Raf 激酶：Raf 二聚体困境。

DOI：
发表时间：
2018-01
期刊：
影响因子：
8.8
作者：
Durrant, David E;Morrison, Deborah K
通讯作者：
Morrison, Deborah K

LAT-independent Erk activation via Bam32-PLC-γ1-Pak1 complexes: GTPase-independent Pak1 activation.

通过 Bam32-PLC-γ1-Pak1 复合物激活 LAT 独立的 Erk：GTPase 独立的 Pak1 激活。

DOI：
发表时间：
2012-10-26
期刊：
影响因子：
16
作者：
Rouquette;Sommers, Connie L;Kortum, Robert L;Morrison, Deborah K;Samelson, Lawrence E
通讯作者：
Samelson, Lawrence E

KSR2 is a calcineurin substrate that promotes ERK cascade activation in response to calcium signals.

KSR2 是一种钙调神经磷酸酶底物，可响应钙信号促进 ERK 级联激活。

DOI：
10.1016/j.molcel.2009.06.001
发表时间：
2009-06-26
期刊：
Molecular cell
影响因子：
16
作者：
Dougherty, Michele K.;Ritt, Daniel A.;Zhou, Ming;Specht, Suzanne I.;Monson, Daniel M.;Veenstra, Timothy D.;Morrison, Deborah K.
通讯作者：
Morrison, Deborah K.

Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling.

反馈磷酸化和 Raf 异二聚化对正常和突变 B-Raf 信号传导的影响。