Synthetic Lethal Targeting of p53 in Myelodysplasia

描述(申请人提供)：骨髓发育不良(MDS)是一种恶性前造血干细胞疾病，除非接受异基因骨髓移植治疗，否则通常是致命的，这一选择对60岁以上的人来说并不普遍，因为他们最常受到这种疾病的影响。一个迫在眉睫的领域是开发针对MDS患者的分子靶向治疗，这些患者是由复杂异常核型和p53突变(MDS-CAK，约占所有MDS病例的20%)的高频突变所定义的。在MDS-CAK中，p53突变阻止细胞凋亡，以应对遗传毒性药物和全身照射引起的DNA损伤，使MDS干细胞对这些药物产生抗药性。理想情况下，人们希望确定抗体或小分子对内源性蛋白的抑制与p53突变细胞的生存不相容，但不影响表达野生型蛋白的细胞。我们的建议试图通过利用斑马鱼实验系统来填补这一空白，该系统提供了一个忠实的人类骨髓生成模型，并唯一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(基于吗啉的)候选基因方法。我们的基本假设是，识别对P53突变‘综合致死’的基因[S/(P53)基因]将揭示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将为MDS-CAK和其他与P53突变相关的病理条件提供有用的新的治疗靶点。我们最近发现的Chk1是一种S/(P53)基因，它的Morolino敲除抑制突变的p53e7/E7相关的对DNA损伤诱导的髓系祖细胞凋亡的抵抗，而不影响未照射的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。具体地说，在p53突变的斑马鱼中，基于吗啉的基因打靶将被用于确定基因作用的机制和补偿凋亡途径，在目标2中，我们开发了所需的方法学和专业知识，利用乙基亚硝脲(END)突变和吖啶橙胚胎试验在斑马鱼中进行无偏见、正向遗传、合成致死的筛选。在AIMS 1和AIMS 2中发现的sl(P53)变异体/突变体将通过与各种髓系特异性探针的原位杂交以及使用我们最近开发的PU在体内测试斑马鱼髓系的合成致死性。1条GFP/p53e7/E7线。AIMS 1和AIMS 2的成功完成将产生一组完全有效的sl(P53)基因，长期目标是他们的野生型蛋白产品将体现出符合抗MDS-CAK人类I期临床试验条件的精致靶点。综述：MDS是一种血液干细胞紊乱，预后非常差。这项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细胞被选择性地杀死，而不是体内的正常细胞。

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.