A functional genomics approach to determine the mechanism of cellular response to new anti-cancer drugs

 描述(由申请人提供)：理论基础和背景：细胞毒性和靶向药物是癌症治疗的关键组成部分。对于一些癌症，如睾丸癌和某些血癌，化疗可以治愈大多数患者。尽管取得了这些成功，但内在和获得性耐药仍然是一个主要的临床问题。由于基因组学革命，我们对癌症中常见的基因组变化的理解现在相当广泛。观察性癌症基因组学为初治和复发肿瘤开辟了一片潜在的治疗靶点。现在的当务之急是确定药物目标的优先顺序，并绘制和阻止抗药性的路线。基因组学识别癌症中的驱动因素突变，使我们能够创造新的靶向药物，尽管单一药物靶向治疗往往最终失败，因为肿瘤细胞的子集逃逸。此外，还有许多对癌细胞存活和药物反应至关重要的基因从未发生突变。在癌症治疗中理解这种“非癌基因成瘾”是至关重要的。我们需要更好的方法来从功能上描绘癌细胞的反应和抗癌药物的耐药性，以便开发出阻止癌细胞逃避治疗的合理方法。在魏斯曼实验室，我创造了新的功能基因组学工具，以探索癌症治疗中的药物反应。利用核酸酶失活形式的CRISPR效应蛋白Cas9(DCas9)，我们可以针对特定的DNA序列靶向转录激活因子(CRISPRa)或抑制因子(CRISPRi)结构域，从而强大而特异地控制人类基因组编码的单个转录本的表达。我们已经建立了基因组规模的CRISPRi/a文库，并表明当CRISPRi/a结合在一起时，CRISPRi/a屏幕可以更完整地显示控制细胞生长、分化和对毒素的反应的过程。目的：我建议使用我们的CRISPRi/a功能基因组平台来研究癌症治疗中的药物反应。为了完善我们的功能基因组工具包，我还提议开发一个CRISPRi/a遗传互作(GI)图谱平台。GI图谱是识别合成-致死基因对的强大方法，这是癌症治疗中理想的药物靶点。我建议进行一段时间的指导培训，以应用我们新的CRISPR功能基因组学平台来确定细胞对新类别抗癌药物的反应机制，并学习如何构建和分析GI图谱。我的主要导师乔纳森·韦斯曼是基因相互作用图谱方面的世界专家，他的实验室是我学习成功实现这一目标所需的技术和分析技能的完美场所。我选择使用CRISPRi/a来表征HSP70抑制剂和K-RAS(G12C)抑制剂，这是我的共同导师Jason Gestwicki和Kevin Shokat创造的两类令人兴奋的抗癌药物。我在加州大学旧金山分校的一个社区工作，在研究蛋白质伴侣和RAS方面有着深厚而成功的历史。我的两位同事都是化学家，他们都是抗癌药物设计的原则，他们对我进行了一段时间的培训，这将使我能够独特地使用我们的CRISPR功能基因组学平台作为研究抗癌药物的工具。这项工作将为利用CRISPRi/a研究癌基因(RAS)和非癌基因(蛋白伴侣)成瘾在癌症中的作用提供通用模板。为了实现这些目标，我提出了以下具体目标：(1)确定细胞对HSP70伴侣抑制剂的反应机制(2)确定细胞对K-RAS(G12C)抑制剂的反应机制(3)实现CRISPRi/a遗传相互作用(GI)定位平台。为了追求这些目标，我将获得知识基础、分析技能和独特的视角，开始我作为一名癌症生物学家的独立研究生涯。

 DESCRIPTION (provided by applicant): Rationale and Background: Cytotoxic and targeted agents are a critical component of cancer therapy. For some cancers, such as testicular cancer and certain blood cancers, chemotherapy cures most patients. Despite these select successes, intrinsic and acquired drug resistance remains a major clinical problem. Due to the genomics revolution, our understanding of the common genomic alterations in cancer is now quite extensive. Observational cancer genomics has opened up a world of potential therapeutic targets in naïve and relapsed tumors. There is now an imperative to prioritize drug targets and to map and block routes of drug resistance. Genomics identifies driver mutations in cancer enabling us to create new-targeted drugs, although single agent targeted therapies frequently eventually fail because a subset of tumor cells escape. Additionally, there are many genes critical for cancer cell survival and drug response that are never mutated. Understanding such "non-oncogene addictions" in cancer therapy is critical. We need better ways of functionally mapping out cancer cell response and resistance to anti-cancer drugs in order to develop rational ways of blocking cancer cell escape from therapy. In the Weissman lab, I have created new functional genomics tools to explore drug response in cancer therapy. Using a nuclease-inactivate form of the CRISPR effector protein Cas9 (dCas9), we can target transcription activator (CRISPRa) or repressor (CRISPRi) domains to specific DNA sequences to robustly and specifically control expression of individual transcripts encoded by the human genome. We have generated genome-scale CRISPRi/a libraries and shown that when combined CRISPRi/a screens yield a more complete view of processes controlling cell growth, differentiation, and response to toxins. Objective: I am proposing to use our CRISPRi/a functional genomic platform to study drug response in cancer therapy. To complete our functional genomic toolset, I am also proposing to develop a CRISPRi/a genetic-interaction (GI) mapping platform. GI maps are a powerful method of identifying synthetic-lethal gene pairs, which are ideal drug targets in cancer therapy. I propose a period of mentored training to apply our new CRISPR functional genomics platform to determine mechanisms of cellular response to new classes of anti-cancer drugs and crucially to learn how to construct and analyze a GI map. My primary mentor, Jonathan Weissman, is a world expert in genetic- interaction mapping and his lab is the perfect place for me to learn the technical and analytical skills I need to be successful in this aim. I have chosen to use CRISPRi/a to characterize HSP70 inhibitors and K-Ras(G12C) inhibitors, two new exciting classes of anti-cancer drugs that were created by my co-mentors Jason Gestwicki and Kevan Shokat. I am embedded in a community at UCSF with a deep and successful history of studying protein chaperones and Ras. A period of training by my co-mentors who are both chemists in principles of anti-cancer drug design will uniquely enable me to use our CRISPR functional genomics platform as a tool for studying anti-cancer drugs. This work will serve as a general template for using CRISPRi/a to study oncogene (Ras) and non- oncogene (protein chaperones) addictions in cancer. To achieve these goals, I propose the following specific aims: (1) Determine the mechanism of cellular responses to HSP70 chaperone inhibitors (2) Determine the mechanism of cellular response to K-Ras(G12C) inhibitors (3) Implement a CRISPRi/a genetic-interaction (GI) mapping platform. In pursuit of these aims, I will acquire the knowledge base, analytical skills and unique perspective to launch my independent research career as a cancer biologist.