Quantitative analysis of the evolving genotype-to-phenotype map

描述(申请人提供)：癌基因导向的癌症治疗和抗菌治疗经常受阻于快速获得耐药性，这是持久缓解的主要障碍。一个看似遥远的领域--蛋白质折叠--正在显现出有希望的洞察力。为了发挥功能，蛋白质必须采用复杂的、往往是亚稳定的构象。危险的是，许多疾病都是由单一蛋白质的折叠或错误折叠引起的。我之前的研究主要集中在Hsp90上，这是一种分子伴侣，可以折叠对致癌转化和信号转导至关重要的亚稳态蛋白。通过影响一组精英调控蛋白的折叠和功能，这种伴侣蛋白有能力影响新特征的进化。在我剩下的指导培训和最初的独立研究生涯中，我将解决以下目标：(I)确定Hsp90如何转变基因变异。HSP90强烈影响Mec1/ATR的多态效应，Mec1/ATR是癌症信号的核心角色，能够以牺牲其他人的利益为代价，对某些基因毒性应激做出反应。从生物化学和功能上，我将研究这如何影响DNA损伤反应、细胞周期和生存能力。此外，IS还将研究Hsp90如何影响Pdr8的转录网络，Pdr8是一种能够对许多药物产生耐药性的转录因子。最后，我将研究Hsp90抑制如何转化NDI1顺式调节元件的多态效应，以产生对氧化应激的强大抵抗力。(2)研究Hsp90伴随表型的同化作用。为了研究最终的突破性耐药性，我将在最初和同化后分离致病变异(当耐药性不依赖于Hsp90时)。高通量基因技术和下一代测序将提供对这一现象的机械性理解。(Iii)识别和表征允许高度突变的细胞存活、增殖和进化新特征的其他因素。癌细胞必须承受巨大的突变负荷，因此有毒的蛋白质组不稳定。我将筛选那些挽救高度突变菌株的生长但不影响未突变父母的蛋白质。(4)确定和确定促进适应新环境的其他基于蛋白质的机制的特点。利用数字核糖体图谱、酵母和细菌遗传学以及生物化学，我将确定两个普里恩，[PSI+]和[GAR+]的特征，并确定它们如何影响对不断变化的环境的适应。这些研究的结果将提供对Hsp90以及更广泛的蛋白质稳态如何控制导致耐药的信号通路以及这些机制如何促进突破性耐药的详细见解。它们还将暴露出所有癌症共同的致命弱点--对能够维持大规模突变负载的因素的依赖。

DESCRIPTION (provided by applicant): Oncogene-directed cancer treatments and antimicrobial therapies are often thwarted by rapid acquisition of drug resistance, a chief barrier to lasting remission. Promising insight is emerging from a seemingly distant field - protein folding. To function, proteins must adopt complex, often metastable conformations. Perilously, many diseases arise from folding or misfolding of a single protein. My previous studies have focused on Hsp90, a molecular chaperone that folds metastable proteins critical for oncogenic transformation and signaling. By influencing the fold and function of an elite cohort of regulatory proteins, this chaperone has the power to influence the evolution of new traits. I will address the following aims during my remaining mentored training and initial independent research career: (I) Determine how Hsp90 transforms genetic variation. Hsp90 strongly impacts the effects of polymorphisms in Mec1/ATR, a central player in cancer signaling, enabling responses to certain genotoxic stresses at the expense of others. Biochemically and functionally, I will examine how this affects the DNA damage response, cell cycle, and viability. Additionally, is will investigate how Hsp90 impacts the transcriptional network of Pdr8, a transcription factor that enables resistance to many drugs. Finally, I will examine how Hsp90 inhibition transforms the effects of polymorphisms in cis-regulatory elements of NDI1, to create strong resistance to oxidative stresses. (II) Investigate assimilation of Hsp90-contingent phenotypes. To investigate eventual breakthrough drug resistance I will isolate causative variation initially and after assimilation (when resistance is Hsp90-independent). High-throughput genetic techniques and next-generation sequencing will provide a mechanistic understanding of this phenomenon. (III) Identify and characterize additional factors that allow highly mutated cells to survive, proliferate, and evolve new traits. Cancer cells must sustain massive mutation loads and consequently toxic proteome destabilization. I will screen for proteins that rescue growth of highly mutated strains but do not affect unmutated parents. (IV) Identify and characterize additional protein-based mechanisms that facilitate adaptation to new environments. Using digital ribosome profiling, yeast and bacterial genetics, and biochemistry I will characterize two prions, [PSI+] and [GAR+], and determine how they affect adaptation to changing environments. The results of these studies will offer detailed insight into how Hsp90, and protein homeostasis more generally, controls signaling pathways that enable drug resistance and how these mechanisms contribute to breakthrough resistance. They will also expose an Achilles' heel common to all cancers - addiction to factors that enable maintenance of massive mutation loads.