Transcriptional Determinants of L-Asparaginase Response in Leukemia

项目摘要/摘要 L-天冬酰胺酶是一种细菌酶，能耗尽血清天冬酰胺以抑制急性白血病细胞的生长。 淋巴细胞性白血病。部分患者对L-天冬酰胺酶无反应及其细胞机制 人们对潜在的无反应仍然知之甚少。在天冬酰胺缺乏的情况下，细胞激活一种 被称为氨基酸剥夺反应的转录程序，通过激活介导 转录因子4(ATF4)。该程序最终表达了天冬酰胺合成酶(ASNS)，即 负责合成天冬酰胺的酶。除ATF4外，参与转录调控的基因 这种反应定义得很不明确。 为了鉴定参与L-天冬酰胺酶反应的转录基因，我开发了一个CRISPR-Cas9 基因筛选方法，分别敲除基因组中的每个转录相关基因。这 方法允许确定ALL细胞系对L的抗性所需的基因- 天冬酰胺酶。在我的初步基因筛查中，得分最高的基因是一个描述不佳的转录 因数，ZBTB1。ZBTB_1基因敲除使ALL细胞对L-天冬酰胺酶敏感。初步 研究表明，ZBTB1富含ASNS启动子，促进转录。我假设 ZBTB1及其对ASNS表达的调控可能是天冬酰胺酶耐药的药物靶点 白血病。然而，由于缺乏对ZBTB1在调节转录中的机制作用的洞察，排除了 对这一假说的调查。 在这个提案中，在我的初步工作的基础上，我将检验ZBTB1正向调节 ASNS的转录，并可能成为治疗L-天冬酰胺酶耐药ALL的靶点。在《目标1》中，我将 探讨ZBTB1调控ASNS表达的机制。在目标2中，我将决定 ZBTB1基因敲除是否使人ALL细胞株和原代人ALL对L治疗敏感-- 体内的天冬酰胺酶。我预计这些研究将确定：1)ZBTB1在 白血病细胞对L-天冬酰胺酶的反应及2)ZBTB-1作为L治疗靶点的潜力- 对天门冬酰胺酶都有抗性。这项工作将在Kivanc Birsoy博士的实验室与合作伙伴一起完成- 洛克菲勒大学罗伯特·罗德博士的建议。这份提案中概述的培训计划是 旨在为我的职业生涯做好最好的准备，成为一名独立的医生-科学家，在实习和 血液学和肿瘤学方面的团契培训。

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.