The role of the first mammalian N-term. methyltransferase, NRMT, in tumorigenesis

描述（申请人提供）：在我的博士后研究期间，我成功地纯化并鉴定了第一个哺乳动物N-末端甲基转移酶NRMT。通过微阵列分析显示在人乳腺癌中具有降低的表达/拷贝数，我还说明了NRMT敲低导致细胞增殖增加和多纺锤体表型。鉴于NRMT敲低导致这两种癌症易感表型，我打算进一步研究它是如何调节的，N-末端甲基化如何影响底物功能，以及NRMT失调如何导致肿瘤发生。我的目标是在完成我的博士后工作后，在学术研究机构获得终身职位，并建立一个独立的研究计划。成功完成我的研究目标包括在小鼠乳腺移植模型中进行培训。由于我以前没有老鼠研究经验，K99提供的额外培训时间对于学习这些技能至关重要。获得K99还将允许在质谱、荧光激活细胞分选和FRET分析方面的进一步训练。我研究的长期目标是使用NRMT小鼠模型来剖析导致乳腺癌的信号通路。随着个性化药物治疗变得越来越必要，我想创建一个模型，可以轻松地同时操纵基因组合。这些模型可用于测试目前哪些药物治疗对具有特定突变组合的患者最有效，并筛选新的更有效的治疗药物。研究肿瘤发生中涉及的基因列表相当广泛，然而，它们的许多生物学功能仍然未知。一个这样的基因，Mettl 11 a（现在更名为NRMT），已被证明在乳腺癌中表达不足，但最近才被确定为第一个哺乳动物N-末端甲基转移酶。NRMT的减少也导致多纺锤体表型，并且相关的非整倍性通常被认为有助于癌症进展。由于NRMT是一种新发现的蛋白质，因此本研究的目的是了解N-末端甲基化对蛋白质和细胞功能的作用，以研究其失调如何导致肿瘤发生。前两个目标，确定N-末端甲基化是否是组成性的，并确定N-末端甲基化是否普遍改变其底物的DNA结合，旨在更好地了解N-末端甲基化的基础生物学。这些目标将涉及质谱，荧光激活细胞分选，FRET分析，和构建敲除小鼠。第三个目标是确定NRMT在发育和肿瘤发生中的作用。将建立小鼠乳腺移植模型，用于测定多纺锤体NRMT敲低表型是否导致发育缺陷和/或肿瘤发生。本提案职业发展方面的关键要素将是学习成功完成第三个目标所需的鼠标系统。目标一和目标二的实验是为手术和腺体或肿瘤收获之间的停机时间设计的。环境弗吉尼亚大学有能力完成这一建议的所有三个目标。咨询委员会成员Don Hunt博士的校内质谱设施具有区分N-末端甲基化和乙酰化所需的傅立叶变换质谱仪。UVA流式细胞术核心将提供所有荧光激活细胞分选实验所需的培训。UVA基因靶向和转基因设施将创建NRMT敲除小鼠。研究组织学核心将石蜡包埋并制作所有正常小鼠组织和肿瘤的切片。我的顾问委员会成员Amy Bouton博士将协助解释组织学。咨询委员会成员托德斯图肯伯格博士的专业知识和试剂将有助于NRMT多纺锤体表型的表征。弗吉尼亚大学W.M.凯克细胞成像中心提供最先进的成像设备，用于小鼠组织样本的免疫荧光成像和FRET分析培训。Macara实验室目前有三名专家小鼠生物学家可用于小鼠处理和手术技术的培训。此外，UVA提供了许多旨在职业发展/培训的课程，包括UVA转基因方法和应用研讨会，流式细胞术培训研讨会，FRET显微镜研讨会，以及由博士后专业发展办公室提供的每月职业发展研讨会系列。 公共卫生相关性：许多与肿瘤发生有关的基因的生物学作用仍然未知。我们需要研究它们的功能，以全面了解癌症进展，并获得潜在的药物靶点清单。一种这样的蛋白质，NRMT，新发现的哺乳动物N-末端甲基转移酶，在乳腺癌中表达不足，其敲低导致有丝分裂缺陷，通常被认为有助于癌症进展。该提案的目标是了解NRMT是如何调节的，以及其失调如何导致肿瘤发生。 个别评审员的书面评论和标准评分在下面的“评论”部分以基本上未经编辑的形式提供。请注意，这些评论和标准评分是在会议之前准备的，在审查会议上进行任何讨论之后可能没有进行修订。上述“讨论摘要和摘要”总结了委员会的最终意见。

DESCRIPTION (provided by applicant): During my postdoctoral fellowship, I successfully purified and identified the first mammalian N-terminal methyltransferase, NRMT. Shown by microarray analysis to have reduced expression/copy number in human breast cancers, I have also illustrated NRMT knockdown results in increased cell proliferation and a multi- spindle phenotype. Given that NRMT knockdown results in these two cancer prone phenotypes, I intend to further study how it is regulated, how N-terminal methylation affects substrate function, and how NRMT misregulation leads to tumorigenesis. My goal after completion of my postdoctoral work is to obtain a tenure- track position at an academic research institution and establish an independent research program. Successful completion of my research goals includes training in a mouse mammary transplant model. As I have no previous mouse research experience, the additional training time provided by a K99 will be imperative for learning these skills. Obtaining a K99, will also allow for further training in mass spectrometry, fluorescent- activating cell sorting, and FRET analysis. The long-term goal of my research is to use the NRMT mouse model to dissect the signaling pathways that lead to breast cancer. As personalized medicine treatments become increasingly necessary, I want to create a model where combinations of genes can be easily manipulated simultaneously. These models can be used to test which current drug treatments are most effective for patients with specific combinations of mutations and to screen for new and more effective therapeutic agents. Research The list of genes involved in tumorigenesis is quite extensive, however, many of their biological functions remain unknown. One such gene, Mettl11a (now renamed NRMT), has been shown to be under-expressed in breast cancers, but has only recently been identified as the first mammalian N-terminal methyltransferase. Reduction of NRMT also results in a multi-spindle phenotype, and the associated aneuploidy is often considered to contribute to cancer progression. As NRMT is a newly discovered protein, the goals of this proposal are to understand the role of N-terminal methylation on protein and cellular function, in order to study how its misregulation leads to tumorigenesis. The first two aims, determining whether N-terminal methylation is constitutive and determining whether N-terminal methylation universally alters the DNA binding of its substrates, are designed to better understand the basic biology of N-terminal methylation. These aims will involve mass spectrometry, fluorescent-activating cell sorting, FRET analysis, and construction of a knockout mouse. The objective of the third aim is to determine the role of NRMT in development and tumorigenesis. A mouse mammary transplant model will be established for assaying if the multi-spindle NRMT knockdown phenotype leads to developmental defects and/or tumorigenesis. The key element of the career development aspect of this proposal will be learning the mouse system needed for successful completion of the third aim. The experiments of aims one and two have been designed for the downtime between surgeries and gland or tumor harvesting. Environment The University of Virginia is well equipped for completion of all three aims of this proposal. The on-campus mass spectrometry facility, of advisory committee member Dr. Don Hunt, has a Fourier transform mass spectrometer necessary for distinguishing N-terminal methylation from acetylation. The UVA Flow Cytometry Core will provide the training necessary for all fluorescent-activating cell sorting experiments. The UVA Gene Targeting and Transgenic Facility will create the NRMT knockout mouse. The Research Histology Core will paraffin embed and make slides of all normal mouse tissue and tumors. My advisory committee member Dr. Amy Bouton will aid in interpretation of the histology. The expertise and reagents of advisory committee member Dr. Todd Stukenberg will aid in characterization of the NRMT multi-spindle phenotype. The UVA W.M. Keck Center for Cellular Imaging supplies state-of-the art imaging facilities for immunofluorescent imaging of mouse tissue samples and training in FRET analysis. The Macara lab currently has three expert mouse biologists available for training in mouse handling and surgical techniques. In addition, UVA offers numerous courses aimed at career development/training, including the UVA transgenic methods and applications workshop, the Flow Cytometry Training Workshop, the Workshop on FRET Microscopy, and a monthly career development seminar series offered by the Office of Postdoctoral Professional Development. PUBLIC HEALTH RELEVANCE: The biological roles of many genes involved in tumorigenesis remain unknown. We need to study their functions in order to achieve a complete understanding of cancer progression and obtain an inclusive list of potential druggable targets. One such protein, NRMT, the newly identified mammalian N-terminal methyltransferase, is under-expressed in breast cancers, and its knockdown results in mitotic defects often considered to contribute to cancer progression. The goal of this proposal is to understand how NRMT is regulated and how its misregulation contributes to tumorigenesis. The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the "Critique" section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The "Resume and Summary of Discussion" above summarizes the final opinions of the committee.