Transcriptional Determinants of L-Asparaginase Response in Leukemia

白血病 L-天冬酰胺酶反应的转录决定因素

• 批准号: 10316164
• 负责人: Robert Thomas Williams
• 金额: $ 3.52万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-13 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10316164
• 关键词: Acute Lymphocytic Leukemia; Acute T Cell Leukemia; Address; Affect; Amino Acids; Asparagine; Aspartate-Ammonia Ligase; BTB/POZ Domain; Binding; Biological Assay; CRISPR/Cas technology; Cancer cell line; Cell Line; Cells; ChIP-seq; Childhood; Chromatin; DNA Binding; DNA-Directed RNA Polymerase; Drug Targeting; Electrophoretic Mobility Shift Assay; Enzymes; Exhibits; Face; Fellowship; Genes; Genetic Screening; Genetic Transcription; Genome; Guide RNA; Hematology; Human; Immunocompromised Host; Immunoprecipitation; Impairment; In Vitro; Individual; Investigation; Knock-out; Laboratories; Leukemia Acute Lymphoblastic Chemotherapy; Leukemic Cell; Libraries; Luciferases; MLL gene; Malignant Childhood Neoplasm; Malignant neoplasm of lung; Mass Spectrum Analysis; Massive Parallel Sequencing; Mediating; Mus; Oncology; Patients; Physicians; Play; Prognostic Marker; Proteins; RNA Polymerase II; Recombinants; Regulation; Relapse; Reporter; Residencies; Resistance; Resistance development; Risk; Role; Scientist; Serum; Subgroup; Survival Rate; T-Lymphocyte; Testing; Training; Transcription Elongation; Transcription Initiation; Transcriptional Regulation; Transposase; Universities; Up-Regulation; Work; Zinc Fingers; activating transcription factor 4; acute lymphoblastic leukemia cell; asparaginase; career; cell growth; chromatin immunoprecipitation; clinically relevant; deprivation; design; experimental study; genome-wide; high risk; histone modification; in vivo; insight; leukemia; malignant breast neoplasm; pancreatic cancer cells; programs; promoter; recruit; resistance mechanism; response; therapeutic target; transcription factor

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.

项目摘要/摘要 L-天冬酰胺酶是一种细菌酶，可耗尽血清天冬酰胺以抑制急性的白血病细胞生长 淋巴细胞白血病。一些患者对L-天冬酰胺酶和细胞机制不反应 潜在的无响应仍然知之甚少。在天冬酰胺剥夺下，细胞激活A 转录程序称为氨基酸剥夺反应，该反应是通过激活而介导的 转录因子4（ATF4）。该程序最终在天冬酰胺合成酶（ASNS）的表达中达到顶点 负责天冬酰胺的合成的酶。除了ATF4，参与的转录调节器 这种响应的定义很差。 为了识别参与对L-天冬酰胺酶反应的转录基因，我开发了CRISPR-CAS9 基因组中分别淘汰每个转录相关基因的基因筛选方法。这 方法允许确定所有细胞系对L-抗性所需的基因 天冬酰胺酶。我的初步遗传筛选中的得分最高的基因是一个较差的转录 因素，ZBTB1。 ZBTB1的敲除使所有细胞系对用L-天冬酰胺酶进行处理。初步的 工作表明，ZBTB1在ASN的启动子中富集以促进转录。我假设这一点 ZBTB1和ASNS表达的调节可能是抗天冬证的合适药物靶标 白血病。但是，缺乏对ZBTB1在介导转录中的机理作用的见解，但是，排除了 研究该假设。 在此提案中，在我的初步工作的基础上，我将测试ZBTB1积极调节的假设 ASN的转录和可能是抗L-天冬酰胺酶耐药性的治疗靶标。在AIM 1中，我会 研究ZBTB1调节ASN的表达的机制。在AIM 2中，我将确定 ZBTB1敲除是否使人类所有细胞系和原代人类敏感到用L-治疗 体内天冬酰胺酶。我预计这些研究将确定：1）ZBTB1在 对白血病细胞中L-天冬酰胺酶的反应，2）ZBTB1作为L-的治疗靶标的潜力 全部抗白天素酶。这项工作将在Kivanc Birsoy博士实验室完成 洛克菲勒大学罗伯特·罗德（Robert Roeder）博士的建议。该提议中概述的培训计划是 旨在为我做好准备使我成为居住之后的独立医师科学家的职业和 血液学和肿瘤学研究金培训。

项目成果

Dietary thiamine influences l-asparaginase sensitivity in a subset of leukemia cells.

• DOI: 10.1126/sciadv.abc7120
• 发表时间: 2020-10
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Guarecuco R;Williams RT;Baudrier L;La K;Passarelli MC;Ekizoglu N;Mestanoglu M;Alwaseem H;Rostandy B;Fidelin J;Garcia-Bermudez J;Molina H;Birsoy K
• 通讯作者: Birsoy K