Cancer Therapeutic Resistance: Implication of AcAPE1 Mediated DNA Repair and T-Cell Based Immunotherapy

项目总结/摘要 本研究建议旨在提供博士前和博士后培训，以开发必要的 作为癌症生物学独立研究者的职业技能。长期的研究重点是发展 通过1）鉴定人类中的遗传、表观遗传和转录组学变化， 2）研究肿瘤细胞中的转录变化对治疗前和治疗后的癌症的影响， 疗效，和3）开发新的组合疗法对人类癌症使用化学免疫， CRISPR/Cas9.我的论文研究（F99阶段）的目的是确定APE 1乙酰化的作用 （AcAPE 1）在肿瘤发生和化疗耐药性中的作用，目的是将AcAPE 1确立为一种新的治疗药物。 目标APE 1是一种普遍存在的多功能蛋白，是碱基切除修复（BER）途径中的关键酶， 参与基因组完整性的维持和基因表达的调节。我们发现 APE 1的N-末端结构域中的多个赖氨酸残基（Lys 6、7、27、31和32）可以通过乙酰化修饰 （AcAPE 1），它调节APE 1的转录调控功能，AcAPE 1在细胞内是必需的 生存目前尚不清楚APE 1的N端乙酰化如何调节细胞增殖和存活。 了解APE 1乙酰化的功能重要性至关重要。我们最近有了几个发现 与这个概念有关。首先，APE 1在与染色质中的AP位点结合后被乙酰化， 这一过程只发生在转录活性基因区域。重要的是，随后的研究表明， APE 1的转录偶联乙酰化在受损DNA碱基的优先修复中起关键作用， 转录基因区域的AP位点，通过增强APE 1的DNA修复活性。此外，这种损害-修复 便于转录。基于这些和其他的数据，我们假设AcAPE 1介导的细胞凋亡的修复， 转录活性区促进肿瘤发生并促进肿瘤细胞的化学抗性。 作为推论，我们预测靶向肿瘤细胞中APE 1的乙酰化位点将促进化疗敏感性。 我们的研究将利用不同的化疗药物耐药癌细胞系来定义一个致癌的 AcAPE 1的损伤修复作用以及AcAPE 1活性对转录谱的影响。我们还将 使用体外和体内模型确定乙酰化缺陷APE 1的治疗活性。扩大 根据未来的研究方向和建立独立的基础，我将继续博士后培训 癌症免疫疗法拟议的K 00博士后培训的目标是获得当前T- 基于细胞的免疫策略，促进对这种免疫疗法的抵抗的机制， 杀手T细胞耗竭背后的分子生物学，以及新的组合疗法的发展，以治疗 癌最终，拟议的F99/K 00培训将为R 01资助提供坚实的知识基础 这将建立我的独立性，并将提供专业培训所需的技能，是一个有效的 PI和导师。

Project Summary / Abstract This research proposal is intended to provide predoctoral and postdoctoral training to develop the necessary skills for a career as an independent investigator in cancer biology. The long-term research focus is development of effective cancer therapies through 1) identification of genetic, epigenetic and transcriptomic changes in human cancers before and after therapy, 2) studying the effects of transcriptional changes in tumor cells on therapeutic efficacy, and 3) developing novel combinatorial therapies against human cancers using chemo-immuno- CRISPR/Cas9. The objective of my dissertation research (F99 phase) is to define the role of acetylation of APE1 (AcAPE1) in oncogenesis and chemoresistance, with the goal of establishing AcAPE1 as a novel therapeutic target. APE1 is a ubiquitous multifunctional protein, a key enzyme in Base Excision Repair (BER) pathway, involved in the maintenance of genomic integrity and in the regulation of gene expression. We discovered that multiple Lysine residues (Lys 6, 7, 27, 31, &32) in N-terminal domain of APE1 can be modified by acetylation (AcAPE1), which modulates the transcriptional regulatory function of APE1 and AcAPE1 is essential in cell survival. It remains unclear how N-terminal acetylation of APE1 regulates cell proliferation and survival. Knowledge of functional importance of acetylation of APE1 is critical. We have recently made several discoveries relevant to this concept. First, APE1 is acetylated after binding to the AP sites in the chromatin and the acetylation process occurs only at transcriptionally active gene regions. Importantly, subsequent studies revealed that transcription-coupled acetylation of APE1 plays a key role in the preferential repair of damaged DNA bases or AP sites in transcribed gene regions, via enhancing DNA repair activity of APE1. Moreover, this damage –repair facilitates transcription. Based on these and additional data we hypothesize that AcAPE1 mediated- repair of transcriptionally active regions promotes oncogenesis and facilitates chemo-resistance in tumor cells. As a corollary, we predict that targeting acetylation sites of APE1 in tumor cells will promote chemosensitivity. Our research will utilize different chemotherapeutic drug resistant cancer cell lines to define an oncogenic damage-repair role of AcAPE1 and the effect of AcAPE1 activity on the transcriptional profile. We will also determine the therapeutic activity of acetylation defective APE1 using in vitro and in vivo models. To expand upon the future research direction and to build the foundation for independence, I will pursue postdoctoral training in cancer immunotherapy. The goal of the proposed K00 postdoctoral training is to gain expertise on current T- cell based immunotherapeutic strategies, the mechanisms that promote resistance to such immunotherapy, the molecular biology behind killer T-cell exhaustion, and development of novel combinatorial therapeutics to treat cancer. Ultimately, the proposed F99/K00 training will provide a strong intellectual foundation for R01 funding that will establish my independence and will provide professional training in the skills required to be an effective PI and mentor.