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）：除非接受同种异体骨髓移植的治疗，否则肌发育不全（MDS）是一种造血干细胞前疾病，通常是致命的，这通常是60岁以上的人，他们最常见于这种疾病。一个极大的迫切区域是针对由高频率复杂异常核型和p53突变（MDS-CAK，所有MDS占所有MDS）定义的MDS患者的分子疗法的发展。在MDS-CAK中，p53突变会响应于遗传毒性药物和全体体辐射引起的DNA损伤的凋亡，从而使MDS干细胞具有对这些药物的抗性。理想情况下，人们希望鉴定内源性蛋白质的抗体或小分子抑制与p53突变细胞的存活不符，但助长了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运动的忠实模型，并且使用1）使用1）大规模的远期遗传筛选和2）快速（Morpholino）候选候选者方法非常适合识别关键途径。我们的基本假设是，对p53突变体[s/（p53）基因的基因“合成性致死”的鉴定将暗示p53缺陷细胞逃避凋亡的新途径。因此，此类基因的野生型产品应在MDS-CAK和与p53突变有关的其他病理状况中提供有用的新治疗靶标。我们最近发现CHK1作为S/（p53）基因的可行性证明了这种方法的可行性，其Morpholino敲低抑制了突变体P53E7/E7相关的抗DNA损伤诱导的髓样祖细胞凋亡的抗性，而不会影响非辐照p53+/+胚胎。在AIM1中，我们建议通过应用体内遗传性上学和可用于斑马鱼研究的细胞生物学技术来阐明p53和CHK1之间合成致命关系的机械基础。 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.在AIM 1和2中鉴定的SL（p53）形态/突变体将通过与各种髓样特异性探针以及使用我们最近生成的PU进行原位杂交测试斑马鱼髓样谱系中的合成致死性。 1 GFP/p53e7/e7线。成功完成目标1和2将产生一组全面验证的SL（p53）基因，其远程目标是其野生型蛋白质产品将体现有资格参加针对MDS-CAK的人类I期临床试验的精致目标。摘要摘要：MDS是一种血液干细胞的疾病，预后较差。该建议旨在改善携带特定类型基因缺陷的MD的治疗。该计划的研究使用斑马鱼动物模型系统来鉴定其蛋白产物可以用抗体或小分子抑制的基因，从而导致MDS细胞被选择性地杀死，从而在体内保留正常细胞。

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