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

项目摘要。肺癌是全球癌症死亡的主要原因，其中非小细胞肺癌 癌症（NSCLC）是肺癌的主要组织学亚型，肺腺癌是主要亚型 非小细胞肺癌。 ALK 基因重排（例如 EML4-ALK 融合）是 NSCLC 和当前 ALK 中经过验证的靶标 激酶抑制剂产生令人印象深刻的反应。尽管临床取得了进展，耐药性仍然是一个问题 这限制了患者的生存。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、多学科的合作项目，希望能够提高 NSCLC 的生存率 我们通过研究 ALK 融合发现了一种新的致癌信号传导机制，从而对患者进行治疗 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 （冷凝物）作为癌症中致癌激酶信号传导的独特机制。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癌蛋白激酶融合的范例 例如 RET 融合，从头形成它们自己的相分离的基于蛋白质的亚细胞区室，缺乏 脂质膜并利用高阶蛋白质组装作为致癌的区分原理 输出。这些无膜细胞质蛋白颗粒包含一种致癌信号传导模式，即 与天然受体酪氨酸激酶 (RTK) 信号传导和其他 RTK 的致癌、突变形式不同 例如 EGFR，它使用经典的基于脂质膜的信号传导。致病性生物分子凝聚物 由 ALK（和其他 RTK）融合癌蛋白形成，局部集中 RAS 激活复合物 GRB2/SOS1 并以不依赖脂膜的方式激活 RAS。 RTK 蛋白颗粒的形成对于 细胞中致癌的 RAS/MAPK 信号输出。我们鉴定了一组蛋白质颗粒信号传导成分 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 融合癌蛋白之外，还需要衔接蛋白 GRB2 和 SHC。我们的研究结果表明 无膜、高阶细胞质蛋白组装作为独特的亚细胞组织平台 癌症中的致癌 RTK 和 RAS 信号传导。我们利用创新提出了 2 个互补的具体目标 探索凝聚生物学的方法，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、致癌信号传导和肿瘤生长所需的蛋白质。拟议的 研究将为 RTK 聚变凝结物生物学的机制理解奠定坚实的基础 未来设计基于机制的治疗策略，以干扰 ALK 蛋白颗粒组装 se 并补充了传统的 ALK 靶向临床药物，即 ALK 激酶抑制剂。这个项目 将深入了解这种独特形式的致癌信号传导，重点关注 ALK，具有更广泛的影响 了解凝析油和 RTK 融合生物学以及差异化治疗策略的设计。

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.