Mechanistic insights into lysosomal nutrient efflux in cancer

摘要 许多RAS转化的侵袭性癌细胞能够逃脱细胞毒化疗，并在 接近饥饿的条件。使它们很难被杀死的一种适应是它们清除细胞外的能力 蛋白质，并利用自噬回收细胞成分，然后两者都在溶酶体中消化，以 回收游离氨基酸。清除和内化的过程被称为巨噬细胞吞噬。 当面临营养匮乏的条件时，癌细胞会获得突变以上调它的表达。为了抗击这样的癌症， 研究人员目前的目标是巨噬细胞吞噬机制；然而，因为这个过程不是很 经过很好的研究，可能涉及数百种具有多余功能的蛋白质，这种疗法可能会被证明 挑战在不涉及多种药物的情况下执行。我们提出了一种更好的目标定位方法 通过专注于下游过程释放消化的营养物质来清除癌症的营养 溶酶体转化为胞浆。萨巴蒂尼实验室表明，溶酶体中消化的氨基酸的释放是 由mTORC1途径，特别是SLC38A9编排。这种溶酶体膜蛋白 通过直接结合精氨酸，感觉溶酶体中消化的氨基酸水平的上升。我们的实验室发现 这种感觉与转运蛋白功能的激活相结合，并导致必要的非极性物质的外流 从溶酶体到胞浆的氨基酸，如亮氨酸。重要的是，RAS转化的胰腺癌 在没有SLC38A9的情况下，以细胞外蛋白为食的细胞无法有效地形成肿瘤。 这些结果提出了一种新的治疗思路，即针对由 致癌RAS信号转导。在这个五年计划中，我们将阐明释放的分子机制。 通过SLC38A9将氨基酸从溶酶体消化到胞浆中，因此提供了一种合理的方法 药物发现。与此同时，我们将筛选与SLC38A9特异结合的小分子，以及 将它们开发成调节其运输活动的化学探针。SLC38A9的外排功能受损 导致巨噬细胞增多症衍生的氨基酸被困在溶酶体中，我们的预期是 这种治疗将损害RAS突变和其他沉迷于蛋白质清除的肿瘤的生长，而 不考虑缺乏这一要求的正常细胞。在指导阶段的前两年，我将 在怀特黑德研究所，我将从专家那里学习细胞信号和代谢组学方法 田野。我还将冒险进入一个对我来说全新的研究领域，化学生物学，与专家合作 在布罗德学院。在完成我的K99培训后，我的愿望是领导一个 结合细胞信号、结构生物学和化学生物学研究膜转运蛋白及其作用 在癌症新陈代谢中。在了解基础生物学的同时，我希望我的实验室开发出具体的 小分子调节剂调节这些蛋白质的运输活性，促进进一步的研究 领域，从长远来看--新药。

ABSTRACT Many RAS-transformed aggressive cancer cells are able to escape cytotoxic chemotherapy and survive in near-starvation conditions. One adaptation making them hard to kill is their ability to scavenge extracellular proteins and recycle the cellular components using autophagy, both of which are then digested in lysosomes to recover free amino acids. The process of scavenging and internalization is known as macropinocytosis, and cancer cells aquire mutations to upregulate it when faced with nutrient-poor conditions. To fight such cancers, researchers are currently targeting the macropinocytosis machinery; however, because this process is not very well studied, and likely involves hundreds of proteins with redundant functions, such therapy might prove challenging to exectute without involving multiple drugs. We propose a better way of targeting nutrient-scavenging cancers by focusing on a downstream process of releasing digested nutrients from lysosomes to cytosol. The Sabatini Lab showed that the release of digested amino acids from lysosomes is orchestrated by the mTORC1 pathway, and specifically by SLC38A9. This lysosomal membrane protein senses the rising levels of digested amino acids in lysosomes by directly binding arginine. Our lab found that this sensing is coupled to activation of the transporter function, and results in the efflux of essential non-polar amino-acids, such as leucine, from lysosomes to cytosol. Importantly, RAS-transformed pancreatic cancer cells that feed on extracellular protein were unable to efficiently form tumors in the absence of SLC38A9. These results present a novel therapeutic idea of targeting a metabolic vulnerability in cancers transformed by oncogenic RAS signaling. In this five-year project we will elucidate the molecular mechanism of releasing digested amino acids from lysosomes to cytosol via SLC38A9, and therefore provide a rational approach to drug discovery. In parallel to that, we will screen for small molecules that specifically bind to SLC38A9, and develop them into chemical probes that modulate its transport activity. Impaired efflux function of SLC38A9 will lead to entrapment of macropinocytosis-derived amino-acids within the lysosomes, and our expectation is that this treatment will impair the growth of RAS-mutant and other tumors addicted to protein scavenging, while sparing normal cells that lack this requirement. Over the first two years of the mentored phase, I will be based at the Whitehead Institute, where I will learn cell signaling and metabolomics approaches from the experts in the field. I will also venture into a completely new research area to me, chemical biology, working with experts at the Broad Institute. After the completion of my K99 training, my aspiration is to lead a laboratory that combines cell signaling, structural biology, and chemical biology to study membrane transporters and their role in cancer metabolism. In parallel to understanding basic biology, I want my lab to develop specific small-molecule modulators that adjust transport activities of those proteins, facilitating further research in the field, and in long term – new medicines.