Mechanisms of genomic instability, tumor initiation and progression following the disruption of the RTF2-RNase H2 axis

项目摘要/摘要 核糖核酸在基因组中的异常结合或保留是基因组的常见原因。 不稳定，使其容易被水解和下游突变。核糖核酸酶H2是一种 通过移除这些基因组来防止基因组不稳定的主要机制- 嵌入的RNA。 我们的实验室最近发现了一种调节RNaseH2的新机制，通过揭示这种复制 终止因子2(RTF2)参与了RNaseH2在复制分叉的定位和调节。 需要进一步阐明这种相互作用，以了解调节背后的基本生物学和 RNaseH2如何防止基因组不稳定的功能。有趣的是，RNaseH2的拷贝数丢失是常见的 发现于慢性淋巴细胞白血病(CLL)，在30%以上的病例中。在我的前期工作中，我发展了 RNaseH2和RTF2可以被耗尽的各种细胞模型，我已经表达和纯化了 重组RNaseH2和RTF2，允许进行调节RNaseH2活性的体内研究 通过RTF2和体外研究它们之间的相互作用。此外，我还开发了一种新的检测方法，允许 人类细胞基因组中核糖核苷酸掺入的定量分析。这项化验将被用于 研究RTF2对RNaseH2的调节，并将评估其在预测CLL对 PARP抑制剂。RNaseH2缺失的肿瘤进展背后的直接机制尚未被研究。 在这个提案中，基于我上面的前期工作，我将测试RTF2直接与 并调节复制分叉处的RNaseH2的活性，并研究RNaseH2丢失背后的机制 核糖核酸酶H2损害了基因组的稳定性，并导致肿瘤进展。 我是威尔·康奈尔大学/洛克菲勒大学/斯隆·凯特林三学院的医学博士/博士生，我现在在那里 在洛克菲勒大学阿加塔·斯莫戈尔泽夫斯卡博士的实验室里。我的长期目标是成为一名 内科科学家，作为血液学家和肿瘤学家执业，并管理着一个独立的基础科学实验室 作为一所学术大学医院。这项提案中概述的计划，以及 阿加塔·斯莫戈尔泽夫斯卡博士，我的论文研究委员会，以及三所大学的医学博士项目将对我有所帮助 实现这些职业抱负。

Project Summary/Abstract The aberrant incorporation or retention of ribonucleic acids (RNAs) in the genome is a common cause of genomic instability, rendering it susceptible to hydrolysis and downstream mutagenesis. The enzyme RNase H2 is one of the primary mechanisms protecting against this destabilization of the genome by removing these genome- embedded RNAs. Our lab recently uncovered a novel mechanism of regulation of RNase H2, by uncovering that replication termination factor 2 (RTF2) is involved in localizing and regulating the levels of RNase H2 at the replication fork. Further elucidation of this interaction is required to understand the basic biology behind the regulation and function of how RNase H2 prevents genome instability. Interestingly, copy number loss of RNase H2 is commonly found in Chronic Lymphocytic Leukemia (CLL), in over 30% of cases. In my preliminary work, I have developed various cellular models in which RNase H2 and RTF2 can be depleted, and I have expressed and purified recombinant RNase H2 and RTF2, allowing for both in vivo studies of which RNase H2 activities are regulated by RTF2 and in vitro studies of their interaction. Furthermore, I have developed a novel assay allowing a quantitative analysis of ribonucleotide incorporation in the genomes of human cells. This assay will be used to study the regulation of RNase H2 by RTF2, and will be assessed in its applicability to predict CLL responses to PARP-inhibitors. The direct mechanism behind tumor progression in the loss of RNase H2 has not been studied. In this proposal, building on my above preliminary work, I will test the hypothesis that RTF2 interacts directly with and regulates the activities of RNase H2 at the replication fork and examine the mechanism behind how loss of RNase H2 compromises genomic stability and leads to tumor progression. I am an MD/PhD student at the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional Program, where I am in the laboratory of Dr. Agata Smogorzewska at The Rockefeller University. My long-term goal is to become a physician scientist, practicing as a hematologist-oncologist as well as running an independent basic science lab as an academic university hospital. The plan outlined in this proposal, along with the support and mentorship of Dr. Agata Smogorzewska, my thesis research committee, and the Tri-Institutional MD-PhD program will help me achieve these career aspirations.