Dissecting the role and mechanism of EML4-ALK condensates in oncogenic signaling and tumor growth

项目摘要。肺癌是世界范围内癌症死亡的主要原因，非小细胞肺癌。 肺癌(NSCLC)是肺癌的主要组织亚型，肺腺癌是肺癌的主要亚型 非小细胞肺癌。ALK基因重排(例如，EML4-ALK融合)是非小细胞肺癌和当前ALK的有效靶点 激酶抑制剂会产生令人印象深刻的反应。尽管取得了这一临床进展，但耐药性仍然是一个问题 这限制了患者的存活率。改进的治疗策略对于确定改善临床结果至关重要。 我们提出了一个创新的、多学科的、协作的项目，希望能改善非小细胞肺癌的生存 通过定义我们通过研究ALK融合而发现的致癌信号的新机制来治疗患者 癌蛋白。我们的目标是利用我们对无膜细胞质蛋白颗粒的发现 (凝结物)作为癌症中致癌激酶信号的一种不同机制。我们的数据表明一个新兴的 某些ALK融合癌蛋白以及其他临床相关的癌蛋白激酶融合的范例 例如RET融合，从头形成它们自己相分离的基于蛋白质的亚细胞室 并利用高阶蛋白质组装作为致癌的区分原则 输出。这些无膜的细胞质蛋白颗粒包括一种致癌信号模式，即 不同于天然受体酪氨酸激酶(RTK)信号和其他RTK的致癌、突变形式 如EGFR，使用经典的基于脂膜的信号转导。致病生物分子凝聚体 由ALK(和其他RTK)融合的癌蛋白形成的局部浓缩RAS激活复合体Grb2/SOS1 并以不依赖于脂膜的方式激活RAS。RTK蛋白颗粒的形成对 致癌的RAS/MAPK信号在细胞内输出。我们确定了一组蛋白质颗粒信号组件和 建立了定义ALK蛋白颗粒形成的结构规则。例如，蛋白质颗粒的形成 除了ALK融合癌蛋白外，还需要连接蛋白Grb2和SHc。我们的发现揭示了 作为组织的独特亚细胞平台的无膜、高阶细胞质蛋白组装 癌基因RTK和RAS信号与癌症我们提出了两个互补的具体目标，使用创新 探索凝析油生物学以了解相分离在ALK融合致癌中的作用 发信号。我们进一步定义了ALK融合蛋白颗粒的蛋白质结构，并确定了关键的相互作用 ALK融合蛋白颗粒形成、致癌信号和肿瘤生长所需的蛋白质。建议数 研究将建立对RTK融合凝析油生物学的机理理解，为 干扰碱性磷酸酶蛋白颗粒组装的机制治疗策略的未来设计 Se和它补充了传统的ALK靶向临床药物，这些药物是ALK激酶抑制剂。这个项目 将提供对这种不同形式的致癌信号的洞察，重点是ALK，具有更广泛的影响 以了解凝析油和RTK融合生物学并设计差异化治疗策略。

PROJECT ABSTRACT. Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) the predominant histologic subtype of lung cancer and lung adenocarcinoma the major subset of NSCLC. ALK gene rearrangements (e.g., EML4-ALK fusions) are validated targets in NSCLC and current ALK kinase inhibitors yield impressive responses. Despite this clinical progress drug resistance remains a problem that limits patient survival. Improved therapeutic strategies are critical to identify to improve clinical outcomes. We propose an innovative, multidisciplinary, and collaborative project to hopefully improve the survival of NSCLC patients by defining a new mechanism of oncogenic signaling that we uncovered by studying ALK fusion oncoproteins. We aim to capitalize on our discovery of membraneless cytoplasmic protein granules (condensates) as a distinct mechanism of oncogenic kinase signaling in cancer. Our data suggest an emerging paradigm in which certain ALK fusion oncoproteins, as well as other clinically-relevant oncoprotein kinase fusions such as RET fusions, form de novo their own phase separated protein-based subcellular compartment devoid of lipid membranes and utilize higher-order protein assembly as distinguishing principles underlying oncogenic output. These membraneless cytoplasmic protein granules comprise a mode of oncogenic signaling that is different from that of native receptor tyrosine kinase (RTK) signaling and oncogenic, mutant forms of other RTKs such as EGFR, which use classical lipid membrane-based signaling. The pathogenic biomolecular condensates formed by ALK (and other RTK) fusion oncoproteins locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in cells. We identified a set of protein granule signaling components and established structural rules that define ALK protein granule formation. For instance, protein granule formation requires the adaptor proteins GRB2 and SHC, in addition to the ALK fusion oncoprotein. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling in cancer. We propose 2 complementary Specific Aims using innovative methodologies to probe condensate biology to understand the role of phase separation in ALK fusion oncogenic signaling. We further define the protein architecture of ALK fusion protein granules and identify the key interacting proteins required for ALK fusion protein granule formation, oncogenic signaling and tumor growth. The proposed studies will establish a mechanistic understanding of RTK fusion condensate biology to lay a firm foundation for the future design of mechanism-based therapeutic strategies to interfere with ALK protein granule assembly per se and that complement conventional ALK-targeted clinical agents, which are ALK kinase inhibitors. This project will provide insight into this distinct form of oncogenic signaling with a focus on ALK, with broader implications for the understanding of condensate and RTK fusion biology and the design of differentiated treatment strategies.