Understanding and targeting oncogenic biomolecular condensates of ALK kinase

肺癌是英国第三大常见癌症，每年有近5万例新确诊病例。肺癌分为小细胞肺癌和更常见的非小细胞肺癌（80%的病例）。大约5%的患者在他们的癌细胞DNA中有一个特定的突变，导致一种叫做EML4-ALK的基因融合。EML4-ALK患者平均比大多数肺癌患者年轻得多。他们接受ALK酪氨酸激酶抑制剂治疗，这些患者的5年生存率提高到50%-60%，比标准化疗有效得多。目前仍有改进的空间，因此我们需要破译EML4-ALK蛋白促进癌细胞存活和增殖的分子机制，并确定癌症如何对ALK抑制剂治疗产生耐药性。这将使我们能够开发新的和优化的治疗方法。在这个项目中，我们将专注于EML4-ALK蛋白的分子相互作用，它们形成的细胞结构，以及它们对癌症治疗的反应。在健康细胞中，EML4调节被称为微管的细胞结构的形成。ALK蛋白通常在发育中的大脑细胞表面起作用，在那里，它对细胞外的信号分子作出反应，指示细胞增殖。在肺癌中，这些蛋白的部分融合在一起，EML4-ALK蛋白分子之间的相互作用使它们能够指导肺癌细胞在身体其他部分的控制下增殖。这些相互作用还允许EML4-ALK蛋白在细胞内形成室室，这些室室富含促进细胞增殖和存活所需的其他蛋白质。我们之前发现，一些用于癌症治疗的ALK抑制剂会破坏细胞间室的稳定性，而一种常用的ALK抑制剂则会稳定细胞间室。基于这一点和其他观察，我们认为这些隔室是由EML4-ALK蛋白分子的ALK部分之间的相互作用以及EML4部分之间的相互作用形成的。如果我们能够阻断这些相互作用，这可能会导致一种治疗EML4-ALK肺癌的新方法。我们将重点关注ALK相互作用，因为我们认为这是更可行的，因为不受控制的ALK活性是许多其他癌症的原因，而EML4功能障碍在其他癌症类型中很少发现。我们的项目将从生产破坏ALK相互作用的分子工具开始。我们已经合成了一组8个被称为纳米体的合成蛋白质分子，它们与细胞中的EML4-ALK结合。我们将通过精确描述它们如何影响ALK相互作用和活性来进一步开发这些分子工具。同时，我们将精确地确定ALK分子是如何相互作用的。这两方面的工作都将使用实验结构生物学方法和人工智能等计算方法。同时，我们将发现哪些其他蛋白质与EML4-ALK相互作用，并确定哪些蛋白质存在于细胞区室中。这些蛋白质在隔室内的组织，以及分子进出隔室的运动将使用最先进的细胞成像方法进行研究。这个项目的这一部分将告诉我们这些隔室是如何构建的，以及分子是如何从隔室传递到细胞的其他部分，并发出增殖指令的。该项目的最后一步将是利用这些新信息和我们的新分子工具来询问破坏或稳定这些隔室是否可以有效地阻断来自EML4-ALK的细胞增殖和存活信号。我们将在从肺癌患者身上培养的细胞，以及对用于癌症治疗的ALK抑制剂产生耐药性的细胞中进行测试。如果成功，我们将根据我们的发现启动一个后续项目，以改善肺癌的治疗。

Lung cancer is the 3rd most common cancer in the UK, with almost 50,000 new cases diagnosed annually. Lung cancer is classified as small cell or the more common non-small cell (80% of cases). Approximately 5% of these patients have a specific mutation in the DNA of their cancer cells, resulting in a gene fusion called EML4-ALK. EML4-ALK patients are on average much younger than most lung cancer patients. They are treated with ALK tyrosine kinase inhibitors, and 5-year survival for these patients has improved to 50%-60%, much more effective than standard chemotherapy. There is still room for improvement, and so we need to decipher the molecular mechanisms through which EML4-ALK proteins promote cancer cell survival and proliferation and to determine how the cancers become resistant to ALK inhibitor therapy. This will allow us to develop new and optimised treatments.In this project, we will focus on the molecular interactions of EML4-ALK proteins, the cellular structures they form, and how these respond to cancer therapies. In healthy cells, EML4 regulates the formation of cellular structures called microtubules. The ALK protein normally functions on the surface of cells in the developing brain where, in response to a signalling molecule from outside the cell, it instructs the cell to proliferate. In lung cancer, parts of these proteins are fused together, and interactions between EML4-ALK protein molecules allows them to instruct lung cancer cells to proliferate out of the control of the rest of the body. These interactions also allow EML4-ALK proteins to form compartments within cells that are enriched in other proteins required to promote cell proliferation and survival. We previously discovered that some ALK inhibitors used in cancer treatment destabilise the compartments, whereas one commonly used ALK inhibitor stabilises the compartments. Based on this and other observations, we think that the compartments are formed from interactions between the ALK parts of the EML4-ALK protein molecules, as well as interactions between the EML4 parts. If we could block these interactions, this might lead to a new way to treat EML4-ALK lung cancers. We will focus on the ALK interactions because we think this is more feasible, and because uncontrolled ALK activity is the cause of many other cancers, whereas EML4 dysfunction is rarely found in other cancer types. Our project will begin with the production of molecular tools to disrupt the ALK interactions. We have already made a set of eight synthetic protein molecules called nanobodies that bind to EML4-ALK in cells. We will develop these molecular tools further by characterising precisely how they affect ALK interactions and activity. In parallel, we will determine precisely how ALK molecules interact with each other. Both strands of work will use experimental structural biology methods and computational methods such as artificial intelligence. Also in parallel, we will find out which other proteins interact with EML4-ALK and determine which of them are present in the cellular compartments. The organisation of these proteins within the compartments, and the movement of molecules in and out of the compartments will be studied using state-of-the-art cell imaging methods. This part of the project will tell us how the compartments are built, and how molecules are passed from the compartment to the rest of the cell with the instructions to proliferate. The final step of the project will be to use this new information and our new molecular tools to ask whether disrupting or stabilising the compartments can effectively block the cell proliferation and survival signals from EML4-ALK. We will test this in cells that were cultured from lung cancer patients, and cells that have become resistant to ALK inhibitors used in cancer therapy . If successful, we will initiate a follow-up project towards improving the treatment of lung cancer based on our findings.