The differential regulation of poly(ADP-ribose) in cancer and its role in protein

描述(申请人提供)：Krukenberg博士的背景是化学和生物化学，她在加州大学旧金山分校获得了化学和化学生物学博士学位。在David Agard博士的指导下，她致力于分子伴侣Hsp90的结构和功能表征。作为哈佛医学院的博士后，她在蒂莫西·米奇森博士的指导下进行细胞生物学方面的培训。克鲁肯伯格博士的最终目标是领导一个学术研究小组，专注于了解蛋白质在信号通路中的功能和调节，以及它们对癌症的影响。NIH独立之路奖将在基于细胞的分析方面提供必要的培训，包括显微镜、基因组学、蛋白质组学和生物信息学，以及癌症药理学和基础科学在翻译研究中的应用。为了实现她的目标，克鲁肯伯格博士正在研究聚(ADP-核糖)聚合酶(PAP)及其与癌症的相关性。PARPS催化多聚(ADP-核糖)加成到受体蛋白上，参与DNA损伤反应、炎症和转录等多种过程。PARP还调节少数具有不同生物功能的蛋白质的稳定性。多种PARP抑制剂目前正在进行临床试验，要么与糖尿病肾病损伤剂联合使用，要么作为DNA损伤修复缺陷癌症(BRCA1/2缺陷癌症)的单一药物。最近的研究表明，PARP抑制剂可能在治疗一些不依赖DNA损伤的癌症方面有用。了解PARP和pADPr的更广泛作用可能提供新的治疗靶点，并扩大PARP抑制剂的临床应用。使用克鲁肯伯格博士开发的一种测定pADPr水平的方法，她发现不同乳腺癌细胞的pADPr水平差异很大。初步数据表明，这是某些细胞中PARP1过度激活的结果。本研究的目的是1)研究PARP1过度激活的机制和功能后果，包括它在药物敏感性中的作用；2)探讨pADPr修饰在调节蛋白质稳定性中的作用。将阐明pADPr调控的癌症信号通路，并研究pADPr水平与化疗反应的关系。在指导阶段(K99)，将完成PARP1调控的机制研究和不同细胞系中pADPr修饰靶点的初步鉴定。还将完成对其稳定性受pADPr修饰影响的蛋白质的调查，并探索pADPr水平与药物敏感性之间的关系。如果数据显示pADPr水平具有作为生物标志物的潜力，随着更多临床合作的启动，这一令人兴奋的方向将在独立阶段得到进一步发展。同样在独立阶段期间， 将调查pADPr修饰在选定途径上的生物学后果。在理解pADPr的生物学功能及其在癌症中的作用方面有望取得重大进展。作为联合导师，朱迪思·斯蒂恩博士将提供质谱学和蛋白质组学方面的专业知识和仪器。作为一名撰稿人，西里尔·贝内斯博士将提供用于分析的细胞系，以及它们的药物敏感性和基因数据。贝内斯博士还将提供癌症药理学方面的关键专业知识。拟议的实验将进一步确定PARP的生物学作用和调控。这项研究还将确定pADPr水平在预测药物敏感性方面的潜力，并确定在临床上使用PARP抑制剂的可能新策略。

DESCRIPTION (provided by applicant): Dr. Krukenberg's background is in chemistry and biochemistry, and she completed her Ph.D. in Chemistry and Chemical Biology at the University of San Francisco, California. Under the supervision of Dr. David Agard, she worked on the structural and functional characterization of the molecular chaperone Hsp90. As a postdoctoral fellow at Harvard Medical School, she is training in cell biology under the guidance of Dr. Timothy Mitchison. Dr. Krukenberg's ultimate goal is to lead an academic research group focused on understanding the function and regulation of proteins in signaling pathways and their impacts on cancer. The NIH Pathway to Independence Award would provide necessary training in cell-based assays including microscopy, genomics, proteomics, and bioinformatics as well as in cancer pharmacology and the application of basic science to translational research. In pursuit of her goals, Dr. Krukenberg is investigating poly(ADP-ribose) polymerases (PARPs) and their relevance to cancer. PARPs catalyze the addition of poly(ADP-ribose) onto acceptor proteins involved in a variety of processes including the DNA damage response, inflammation, and transcription. PARPs also regulate the stability of a handful of proteins with diverse biological functions. Multiple PARP inhibitors are currently in clinical trials, either in combination with DN damaging agents or as single agents in cancers deficient in DNA damage repair (BRCA1/2 deficient cancers). Recent studies suggest that PARP inhibitors may have utility in treating some cancers independent of DNA damage. Understanding the broader roles of PARPs and pADPr may provide new therapeutic targets and expand the clinical uses of PARP inhibitors. Using an assay, which Dr. Krukenberg developed, for quantitating pADPr levels, she found that pADPr levels vary widely between breast cancer cells. Preliminary data suggests that this results from PARP1 hyperactivation in some cells. The aims of this proposal are to 1) investigate the mechanism and functional consequences of PARP1 hyperactivation, including its role in drug sensitivity and to 2) explore the role of pADPr modification in regulating protein stability. Cancer signaling pathways regulated by pADPr will be elucidated, and the relationship between pADPr levels and chemotherapeutic responsiveness will be investigated. During the mentored phase (K99), mechanistic studies of PARP1 regulation and initial identification of pADPr modified targets in different cell lines will be completed. A survey of proteins whose stability is influenced by pADPr modification and an exploration of the relationship between pADPr levels and drug sensitivity will also be completed. If the data suggest pADPr levels have potential as a biomarker this exciting direction will be further developed in the independent phase with the initiation of additional clinical collaborations. Also during the independent phase, the biological consequences of pADPr modification on selected pathways will be investigated. Significant progress in understanding the biological function of pADPr and its role in cancer is anticipated. As a co-mentor, Dr. Judith Steen will provide mass spectrometry and proteomics expertise and instrumentation. As a contributor, Dr. Cyril Benes will provide cell lines for analysis along with data on their drug sensitivity and genotype. Dr. Benes will also provide critical expertise in cancer pharmacology. The proposed experiments will further define the biological roles and regulation of PARPs. This study will also determine the potential of pADPr levels in predicting drug sensitivity and identify possible new strategies for the use of PARP inhibitors in the clinic.