Regulation of Ras-Dependent Signal Transduction Pathways

Ras 依赖性信号转导途径的调节

• 批准号: 10262070
• 负责人: Deborah Morrison
• 金额: $ 108.04万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10262070
• 关键词: Affinity; BRAF gene; Basic Science; Binding; Binding Proteins; Biochemical; Biological Assay; Bioluminescence; Cell Survival; Cell membrane; Cells; Cellular Stress; Chimeric Proteins; Clinical Research; Collaborations; Collection; Complementarity Determining Regions; Development; Dimerization; Disease; Disease Progression; Division of Cancer Epidemiology and Genetics; Down-Regulation; Drug resistance; Effectiveness; Embryo; Energy Transfer; Environment; Enzymes; Epitopes; Event; Exhibits; Extramural Activities; Family; Family member; Feedback; Germ-Line Mutation; Goals; Growth; Growth and Development function; Guanosine Triphosphate Phosphohydrolases; Health Priorities; Human; KRAS2 gene; Laboratories; Lead; Link; MAP Kinase Gene; MEKs; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methodology; Molecular; Molecular Target; Monitor; Mutation; N-terminal; NCI Center for Cancer Research; Natural Products; Neoplasm Metastasis; Pathway interactions; Pharmacotherapy; Phosphorylation; Post-Translational Protein Processing; Protein Kinase; Proteins; RAS genes; Ras Signaling Pathway; Ras/Raf; Regulation; Research; Research Personnel; Role; Route; Severities; Signal Transduction; Signal Transduction Pathway; Somatic Mutation; Structure; System; Techniques; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Work; Zebrafish; cancer cell; cancer therapy; developmental disease; dimer; gain of function; high resolution imaging; high throughput screening; human disease; inhibitor/antagonist; kinase inhibitor; member; mutant; novel therapeutics; patient advocacy group; preference; programs; raf Kinases; screening; targeted treatment; tool; transmission process; tumor progression; tumorigenesis

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 Ras signaling can promote human disease states, with somatic mutations in the RAS genes (HRAS, KRAS and NRAS) being prominent drivers of tumorigenesis and Ras germline mutations contributing to a group of related developmental disorders known collectively as the RASopathies. Elucidating the molecular mechanisms that regulate RAS pathway signaling and identifying strategies to disrupt signal transmission in human disease states is a major health priority and 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 live 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 still occur, events that can strongly influence protein binding as well as signal progression. During this review period, we have utilized the BRET assay to investigate the RAS/RAF interaction and our studies have 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, whereas BRAF demonstrates a unique selectivity for KRAS. By generating different RAS and RAF chimeric proteins, our work revealed that the KRAS selectivity of BRAF is determined by the acidic N-terminal segment of BRAF, which interacts with polybasic residues in the hypervariable region of KRAS, thus identifying a new RAS-binding epitope that uniquely contributes to the KRAS/BRAF interaction. 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. Thus, these findings have revealed a previously unappreciated role for CRAF in potentiating BRAF function. Working in collaboration with the NCI-Molecular Targets Program and utilizing the NCI's large and diverse collection of natural product extracts, our lab has gone on to utilize the BRET assay in a high-throughput screen to identify 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. Importantly, the screen has also resulted in the identification of numerous promising compounds that inhibit RAS/RAF binding (Kim et al., 2020) and may lead to the development of new therapeutic agents to counteract aberrant RAS signaling in human disease states. 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 two studies evaluating a number of the most prevalent RASopathy-associated CRAF and BRAF mutants (Kota et al., 2019). 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.

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 信号传导失调可以促进人类疾病状态，RAS 基因（HRAS、KRAS 和 NRAS）的体细胞突变是肿瘤发生的主要驱动因素，而 Ras 种系突变则导致一组相关的发育障碍。统称为 RASopathies。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信号传递的策略是一项重要的健康优先事项，也是我们实验室近 30 年来努力的重点。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，并充当三层 ERK/MAPK 级联（由 RAF、MEK 和 ERK 蛋白激酶组成）中的起始酶。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。在本次审查期间，我们利用 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药物的反应方式。疗法（Terrell 等人，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，而 BRAF 对 KRAS 表现出独特的选择性。通过生成不同的RAS和RAF嵌合蛋白，我们的工作揭示了BRAF的KRAS选择性是由BRAF的酸性N端片段决定的，该片段与KRAS高变区的多元残基相互作用，从而鉴定出新的RAS结合表位这对 KRAS/BRAF 相互作用有独特的贡献。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。因此，这些发现揭示了 CRAF 在增强 BRAF 功能中的先前未被认识到的作用。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，继续在高通量筛选中利用 BRET 测定来识别可以调节 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。重要的是，该筛选还鉴定出了许多有前景的抑制 RAS/RAF 结合的化合物（Kim 等人，2020），并可能导致新治疗药物的开发，以抵消人类疾病状态下异常的 RAS 信号传导。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两项研究，评估了一些最流行的 RAS 病相关 CRAF 和 BRAF 突变体（Kota 等人，2019）。通过这项努力并与 LCDS 斑马鱼设施合作，我们建立了一种使用斑马鱼胚胎的筛选方法，可以监测 RAS 病相关突变体的功能获得活动。该测定将用于分析通过 RASopathy Initiative 鉴定的任何以前未表征的 RASopathy 突变体。此外，该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。