Synthetic Lethal Targeting of p53 in Myelodysplasia

骨髓增生异常中 p53 的合成致死靶向

• 批准号: 7858453
• 负责人: A. THOMAS LOOK
• 金额: $ 42.75万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7858453
• 关键词: Acridine Orange; Acute Myelocytic Leukemia; Address; Adult; Adverse effects; Affect; Age; Age-Years; Alleles; Allogeneic Bone Marrow Transplantation; Allogenic; Animal Model; Animals; Antibodies; Apoptosis; Apoptotic; Area; Biological; Biological Assay; Biological Models; Biological Process; Candidate Disease Gene; Cell Death; Cell Line; Cells; Cessation of life; Chromosome Deletion; Complement; Complex; Critical Pathways; DNA Damage; DNA damage checkpoint; Defect; Detection; Development; Disease; Dissection; Dose; Drug Delivery Systems; Dysmyelopoietic Syndromes; Embryo; Ethylnitrosourea; Exhibits; Failure; Fertilization; Frequencies; Gamma Rays; Gene Targeting; Genes; Genetic; Genetic Epistasis; Genetic Screening; Goals; Hand; Hematological Disease; Hematopoietic; Hematopoietic stem cells; Hour; Human; In Situ Hybridization; Incidence; Laboratories; Left; Life; Malignant - descriptor; Malignant Neoplasms; Mediating; Methodology; Missense Mutation; Modeling; Molecular; Mutagenesis; Mutation; Myelogenous; Myeloid Cells; Myelopoiesis; Nature; Neurons; Normal Cell; Pathologic; Pathway interactions; Patients; Penetrance; Persons; Pharmaceutical Preparations; Pharmacotherapy; Phase I Clinical Trials; Phenocopy; Phenotype; Phosphorylation; Point Mutation; Precancerous Conditions; Premalignant; Process; Proteins; Radio; Research; Resistance; Signal Transduction; Staining method; Stains; Stem cell transplant; Stem cells; Study Section; System; TP53 gene; Techniques; Testing; Therapeutic; Therapeutic antibodies; Transgenic Organisms; Whole Organism; Whole-Body Irradiation; Zebrafish; aggressive therapy; base; cell type; drug candidate; improved; in vivo; interest; killings; knock-down; mutant; new therapeutic target; novel; outcome forecast; progenitor; radiation resistance; response; small molecule; stem; therapy development; tumor; tumorigenesis; zebrafish genome

DESCRIPTION (provided by applicant): Myelodysplasia (MDS), a premalignant disorder of hematopoietic stem cells, is usually fatal unless treated with allogeneic bone marrow transplantation, an option not generally available for persons over age 60, who are most often affected by this disease. An area of great urgency is the development of molecularly targeted therapy for a subset of MDS patients defined by a high frequency of complex aberrant karyotypes and p53 mutations (MDS-CAK, ~20% of all MDS cases). In MDS-CAK, P53 mutations block apoptosis in response to DNA damage induced by genotoxic drugs and total-body irradiation, rendering the MDS stem cells resistant to these agents. Ideally, one would like to identify endogenous proteins whose inhibition by antibodies or small-molecules is incompatible with the survival of p53 mutant cells but spares cells expressing the wild- type protein. Our proposal seeks to fill this gap by exploiting the zebrafish experimental system, which affords a faithful model of human myelopoiesis and is uniquely suited to identify critical pathways, using 1) large-scale forward genetic screens and 2) rapid (morpholino-based) candidate-gene approaches. Our underlying hypothesis is that identification of genes 'synthetically lethal' to p53 mutants [s/(p53) genes] will implicate novel pathways through which p53-defective cells evade apoptosis; hence, the wild-type products of such genes should provide useful new therapeutic targets in MDS-CAK and other pathologic conditions associated with p53 mutations. The feasibility of this approach is demonstrated by our recent discovery of chk1 as an s/(p53) gene whose morpholino knockdown suppresses mutant p53e7/e7 -associated resistance to DNA damage-induced apoptosis in myeloid progenitors, while not affecting non-irradiated p53+/+ embryos. In Aim1 we propose to elucidate the mechanistic basis of the synthetic-lethal relationship between p53 and chk1, by applying in-vivo genetic epistasis and cell biological techniques available for zebrafish research. Specifically, morpholino-based gene targeting in p53 mutant zebrafish will be used to define mechanisms of gene action and compensatory apoptotic pathways, in Aim 2 we have developed the required methodology and expertise to conduct an unbiased, forward-genetic, synthetic-lethal screen in zebrafish using ethylnitrosourea (END) mutagenesis and an acridine-orange embryonic assay. The sl(p53) morphants/mutants identified in Aims 1 and 2 will be tested for synthetic lethality in the zebrafish myeloid lineage by in situ hybridization with a variety of myeloid specific probes, as well as in vivo using our recently generated pu. 1 gfp/p53e7/e7 line. Successful completion of Aims 1 and 2 will yield a group of fully validated sl(p53) genes, with the long-range goal that their wild-type protein products will embody exquisite targets eligible for human phase I clinical trials against MDS-CAK. Lay summary: MDS is a disorder of blood stem cells with a very poor prognosis. This proposal seeks to improve therapy for MDS carrying a particular type of gene defect. The planned research uses the zebrafish animal model system to identify genes whose protein products can be inhibited with antibodies or small molecules, thus causing the MDS cells to be selectively killed, sparing normal cells in the body.

描述（由申请人提供）：骨髓增生异常（MDS）是一种造血干细胞癌前病变，除非接受异基因骨髓移植治疗，否则通常是致命的，而异基因骨髓移植通常不适用于60岁以上的患者，他们最常受这种疾病的影响。一个非常紧迫的领域是为一部分MDS患者开发分子靶向治疗，这些患者由高频率的复杂异常核型和p53突变（MDS-CAK，约占所有MDS病例的20%）定义。在MDS-CAK中，P53突变阻断细胞凋亡以响应由遗传毒性药物和全身照射诱导的DNA损伤，使得MDS干细胞对这些药剂具有抗性。理想地，人们希望鉴定内源性蛋白质，其被抗体或小分子抑制与p53突变细胞的存活不相容，但保留表达野生型蛋白质的细胞。我们的建议旨在通过利用斑马鱼实验系统来填补这一空白，该系统提供了人类骨髓生成的忠实模型，并且独特地适合于识别关键途径，使用1）大规模正向遗传筛选和2）快速（基于吗啉代）候选基因方法。我们的基本假设是，鉴定对p53突变体“合成致死”的基因[s/（p53）基因]将涉及p53缺陷细胞逃避凋亡的新途径;因此，这些基因的野生型产物应该为MDS-CAK和与p53突变相关的其他病理条件提供有用的新治疗靶点。我们最近发现chk 1作为s/（p53）基因，其吗啉敲低抑制突变型p53 e7/e7相关的抗DNA损伤诱导的髓系祖细胞凋亡，而不影响非辐射p53+/+胚胎，证明了这种方法的可行性。在目标1中，我们建议阐明p53和chk 1之间的合成-致死关系的机制基础，通过应用体内遗传上位性和细胞生物学技术可用于斑马鱼的研究。具体而言，p53突变斑马鱼中基于吗啉代的基因靶向将用于定义基因作用和补偿性凋亡途径的机制，在目标2中，我们已经开发了所需的方法和专业知识，使用乙基亚硝基脲（END）诱变和吖啶橙胚胎试验在斑马鱼中进行无偏倚的、正向遗传的、合成致死的筛选。在目的1和2中鉴定的sl（p53）变体/突变体将通过与多种骨髓特异性探针的原位杂交以及使用我们最近产生的pu的体内试验来测试斑马鱼骨髓谱系中的合成致死性。1株gfp/p53 e7/e7细胞系。目标1和2的成功完成将产生一组完全验证的sl（p53）基因，其长期目标是它们的野生型蛋白质产物将体现有资格用于针对MDS-CAK的人类I期临床试验的精致靶标。简单总结：MDS是一种预后极差的造血干细胞疾病。该建议旨在改善对携带特定类型基因缺陷的MDS的治疗。这项计划中的研究使用斑马鱼动物模型系统来识别可以用抗体或小分子抑制其蛋白质产物的基因，从而导致MDS细胞被选择性杀死，而不伤害体内的正常细胞。