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: 8092579
• 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 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8092579
• 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; 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，除非采用同种异体骨髓移植治疗，否则通常是致命的，而同种异体骨髓移植通常不适用于 60 岁以上的人，因为他们最常受到这种疾病的影响。一个非常紧迫的领域是为 MDS 患者子集开发分子靶向治疗，这些患者具有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%）。在 MDS-CAK 中，P53 突变会阻止细胞凋亡，以响应基因毒性药物和全身辐射诱导的 DNA 损伤，从而使 MDS 干细胞对这些药物产生耐药性。理想情况下，人们希望鉴定出内源性蛋白质，其被抗体或小分子的抑制与p53突变细胞的存活不相容，但不影响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细胞生成的忠实模型，并且特别适合识别关键途径，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，鉴定出对 p53 突变体“合成致死”的基因 [s/(p53) 基因] 将暗示 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状况提供有用的新治疗靶点。我们最近发现 chk1 作为 s/(p53) 基因，其吗啉代敲低可抑制突变 p53e7/e7 相关的对 DNA 损伤诱导的骨髓祖细胞凋亡的抵抗力，同时不影响未照射的 p53+/+ 胚胎，证明了这种方法的可行性。在 Aim1 中，我们建议通过应用可用于斑马鱼研究的体内遗传上位性和细胞生物学技术来阐明 p53 和 chk1 之间合成致死关系的机制基础。具体来说，p53突变斑马鱼中基于吗啉基的基因靶向将用于定义基因作用机制和补偿性凋亡途径，在目标2中，我们开发了所需的方法和专业知识，使用乙基亚硝基脲（END）诱变和吖啶橙胚胎测定在斑马鱼中进行无偏倚、前向遗传、合成致死筛选。目标 1 和 2 中鉴定的 sl(p53) morphant/突变体将通过与各种骨髓特异性探针进行原位杂交以及使用我们最近生成的 pu 进行体内测试，以测试斑马鱼骨髓谱系中的合成致死率。 1 gfp/p53e7/e7 线。成功完成目标 1 和 2 将产生一组经过充分验证的 sl(p53) 基因，其长期目标是其野生型蛋白产品将体现符合 MDS-CAK 人类 I 期临床试验资格的精致靶点。简单总结：MDS 是一种血液干细胞疾病，预后极差。该提案旨在改善携带特定类型基因缺陷的 MDS 的治疗。计划中的研究利用斑马鱼动物模型系统来识别其蛋白质产物可以被抗体或小分子抑制的基因，从而导致MDS细胞被选择性杀死，从而不伤害体内的正常细胞。

项目成果

PIDD death-domain phosphorylation by ATM controls prodeath versus prosurvival PIDDosome signaling.

• DOI: 10.1016/j.molcel.2012.06.024
• 发表时间: 2012-09-14
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Ando, Kiyohiro;Kernan, Jennifer L.;Liu, Peter H.;Sanda, Takaomi;Logette, Emmanuelle;Tschopp, Jurg;Look, A. Thomas;Wang, Jianlong;Bouchier-Hayes, Lisa;Sidi, Samuel
• 通讯作者: Sidi, Samuel