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

项目摘要/摘要 这项研究建议旨在提供博士后和博士后培训，以发展必要的 作为一名癌症生物学独立调查员的职业技能。长期的研究重点是发展 通过1)确定人类的遗传、表观遗传和转录变化来有效的癌症治疗 肿瘤治疗前后，2)研究肿瘤细胞转录变化对治疗的影响 疗效，以及3)利用化学免疫联合治疗人类癌症的新方法。 CRISPR/CAS9.我的论文研究(F99阶段)的目的是确定APE1的乙酰化作用 (AcAPE1)在肿瘤发生和化疗耐药中的作用，目标是将AcAPE1建立为一种新的治疗方法 目标。APE1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。 参与维持基因组的完整性和基因表达的调节。我们发现 APE1 N-末端的多个赖氨酸残基(赖氨酸6、7、27、31和32)可通过乙酰化修饰 (AcAPE1)，它调节APE1和AcAPE1的转录调节功能，在细胞中是必不可少的 生死存亡。目前尚不清楚APE1的N-末端乙酰化如何调节细胞的增殖和存活。 了解APE1乙酰化的功能重要性是至关重要的。我们最近有了几项发现 与这一概念相关。首先，APE1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 APE1的转录偶联乙酰化在DNA碱基损伤的优先修复中起关键作用 AP通过增强APE1的DNA修复活性，在转录的基因区进行定位。此外，这种损伤修复 促进转录。基于这些和其他数据，我们假设AcAPE1介导的修复 转录活性区域促进肿瘤的发生并促进肿瘤细胞的化疗耐药。 作为推论，我们预测靶向肿瘤细胞中APE1的乙酰化位点将提高化疗敏感性。 我们的研究将利用不同的化疗耐药癌细胞株来定义致癌 AcAPE1的损伤修复作用及其活性对转录谱的影响。我们还将 用体外和体内模型测定乙酰化缺陷APE1的治疗活性。要扩大规模 根据未来的研究方向和为独立奠定基础，我将继续进行博士后培训 在癌症免疫治疗方面。拟议的K00博士后培训的目标是获得当前T-T- 基于细胞的免疫治疗策略，促进对这种免疫治疗的耐药性的机制， 杀手T细胞衰竭背后的分子生物学和治疗新组合疗法的发展 癌症。最终，拟议的F99/K00培训将为R01资金提供坚实的智力基础 这将确立我的独立性，并将提供成为有效员工所需技能的专业培训 派和导师。

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.