Synthetic Lethal Targeting of p53 in Myelodysplasia

描述（由申请人提供）：骨髓发育不良（MDS）是一种造血干细胞的恶性前病变，通常是致命的，除非接受同种异体骨髓移植治疗，这种选择通常不适用于60岁以上的人群，他们最常受到这种疾病的影响。一个非常紧迫的领域是为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%）定义的MDS患者子集开发分子靶向治疗。在MDS- cak中，P53突变阻断细胞凋亡以应对基因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耐药性。理想情况下，人们希望鉴定内源性蛋白，其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，斑马鱼实验系统提供了人类骨髓形成的忠实模型，并且特别适合于识别关键途径，使用1)大规模的前向遗传筛选和2)快速（基于morpholino的）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和其他与p53突变相关的病理状况提供有用的新治疗靶点。我们最近发现chk1是一个s/(p53)基因，其morpholino敲除抑制p53e7/e7突变体对DNA损伤诱导的髓系祖细胞凋亡的抗性，同时不影响未辐照的p53+/+胚胎，这证明了这种方法的可行性。在目的1中，我们建议通过应用斑马鱼体内遗传上位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，在p53突变斑马鱼中，基于morpholino的基因靶向将用于定义基因作用机制和代偿性凋亡途径，在Aim 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公正的、前向遗传的、合成致死筛选。在Aims 1和2中鉴定的sl（p53）突变体/突变体将通过使用多种髓系特异性探针进行原位杂交，以及使用我们最近生成的pu在体内测试斑马鱼髓系谱系的合成致病性。1 gfp/p53e7/e7线。Aims 1和2的成功完成将产生一组完全验证的sl（p53）基因，其长期目标是其野生型蛋白产品将包含适合用于针对MDS-CAK的人类I期临床试验的精致靶点。总结：MDS是一种血液干细胞疾病，预后很差。这一建议旨在改善MDS携带特定类型的基因缺陷的治疗。计划中的研究使用斑马鱼动物模型系统来识别基因，这些基因的蛋白质产物可以被抗体或小分子抑制，从而导致MDS细胞被选择性地杀死，而保留体内的正常细胞。

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.