Transcriptional Determinants of L-Asparaginase Response in Leukemia

项目概要/摘要 L-天冬酰胺酶是一种细菌酶，在急性白血病中消耗血清天冬酰胺以抑制白血病细胞生长。 淋巴母细胞白血病一些患者对L-门冬酰胺酶无反应， 人们对无反应的根本原因仍知之甚少。在缺乏天冬酰胺的情况下，细胞激活一种 转录程序称为氨基酸剥夺反应，是由激活介导的 转录因子4（ATF 4）。该程序最终导致天冬酰胺合成酶（ASNS）的表达，即 负责合成天冬酰胺的酶。除了ATF 4之外，参与转录调控的转录因子 这种反应定义不明确。 为了鉴定参与对L-天冬酰胺酶反应的转录基因，我开发了一种CRISPR-Cas9基因， 基因筛选方法，以单独敲除基因组中的每个转录相关基因。这 这种方法允许确定ALL细胞系对L- 天冬酰胺酶。在我的初步基因筛选中，得分最高的基因是一个描述得很差的转录 因子，ZBTB 1。ZBTB 1的敲除使该ALL细胞系对L-天冬酰胺酶治疗敏感。初步 工作表明ZBTB 1富集ASNS的启动子以促进转录。我假设 ZBTB 1和ASNS表达的调控可能是一个合适的药物靶点， 白血病然而，由于缺乏对ZBTB 1在介导转录中的机制作用的了解， 调查这个假设。 在这个建议中，基于我的初步工作，我将测试ZBTB 1正调控的假设， ASNS的转录，并且可能是L-天冬酰胺酶抗性ALL的治疗靶点。在目标1中，我将 探讨ZBTB 1调控ASNS表达的机制。在目标2中，我将确定 ZBTB 1敲除是否使人ALL细胞系和原代人ALL对L- 体内天冬酰胺酶。我预计这些研究将确定：1）ZBTB 1在 白血病细胞中对L-天冬酰胺酶的反应和2）ZBTB 1作为L-天冬酰胺酶治疗靶点的潜力。 天冬酰胺酶耐药ALL。这项工作将在Kivanc Birsoy博士的实验室完成， 罗伯特·罗德博士在洛克菲勒大学的演讲。本提案中概述的培训计划是 旨在最好地准备我的职业生涯作为一个独立的医生，科学家以下居住， 血液学和肿瘤学研究金培训。

Project Summary/Abstract L-asparaginase is a bacterial enzyme that depletes serum asparagine to inhibit leukemic cell growth in acute lymphoblastic leukemia. Some patients do not respond to L-asparaginase and the cellular mechanisms underlying non-response remain poorly understood. Under asparagine deprivation, cells activate a transcriptional program known as the amino acid deprivation response that is mediated by activating transcription factor 4 (ATF4). This program culminates in the expression of asparagine synthetase (ASNS), the enzyme responsible for the synthesis of asparagine. Other than ATF4, the transcriptional regulators involved in this response are poorly defined. To identify transcriptional genes involved in the response to L-asparaginase, I developed a CRISPR-Cas9 genetic screening approach to individually knock out each transcription-related gene in the genome. This approach allowed determination of genes that were required for the resistance of an ALL cell line to L- asparaginase. The top-scoring gene in my preliminary genetic screen was a poorly described transcription factor, ZBTB1. Knockout of ZBTB1 sensitized this ALL cell line to treatment with L-asparaginase. Preliminary work suggested that ZBTB1 enriches in the promoter of ASNS to promote transcription. I hypothesize that ZBTB1 and the regulation of ASNS expression may be a suitable drug target in asparaginase resistant leukemia. A lack of insight into the mechanistic role of ZBTB1 in mediating transcription, however, precludes investigation of this hypothesis. In this proposal, building on my preliminary work, I will test the hypothesis that ZBTB1 positively regulates the transcription of ASNS and may be a therapeutic target for L-asparaginase resistant ALLs. In Aim 1, I will investigate the mechanism by which ZBTB1 regulates the expression of ASNS. In Aim 2, I will determine whether ZBTB1 knockout sensitizes human ALL cell lines and primary human ALLs to treatment with L- asparaginase in vivo. I anticipate that these studies will determine: 1) the mechanistic role of ZBTB1 in the response to L-asparaginase in leukemic cells and 2) the potential of ZBTB1 as a therapeutic target in L- asparaginase resistant ALL. This work will be completed in the laboratory of Dr. Kivanc Birsoy with the co- advisement of Dr. Robert Roeder at the Rockefeller University. The training plan outlined in this proposal is designed to best prepare me for a career as an independent physician-scientist following residency and fellowship training in hematology and oncology.