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Functional analysis of the leukemic evolution in children with Down Syndrome utilizing CRISPR-Cas genome editing

利用 CRISPR-Cas 基因组编辑对唐氏综合症儿童白血病进化进行功能分析

基本信息

批准号：
276311671
负责人：
Professor Dr. Dirk Heckl
金额：
--
依托单位：
Institut für Experimentelle Pädiatrische Hämatologie und Onkologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/276311671?language=en
关键词：
Functional analysis leukemic evolution children

项目摘要

Acute myeloid leukemias are a heterogeneous group of clonal diseases originating from the hematopoietic stem- and progenitor cells (HSPCs), which transform through stepwise acquisition of cooperating genetic lesions. Due to the lack of appropriate technologies to modify the genomes of human cells at the degree of complexity found in leukemia, this evolutionary process of stepwise transformation has been impractical to model and interrogate in the human system. Even in mice, where these modifications are feasible, combinatorial genetics of more than two modifications are time consuming and expensive. We have recently developed a lentivirus-based CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated) system, which allowed us to simultaneously modify up to five genes at their endogenous loci in a single HSPC. Here, we will leverage this novel genome modification tool to delineate the leukemic evolution of myeloid leukemia in children with Down syndrome (ML-DS). 5-30% of infants with DS are diagnosed with transient abnormal myelopoiesis (TAM), which has typical characteristics of leukemia. TAM regresses in most of the infants without anti-leukemic treatment, however, in 20-30% of these children an AML develops within the first four years of life. Both TAM and ML-DS almost universally (95%) harbor mutations in the hematopoietic transcription factor GATA1, which lead to the exclusive expression of a truncated protein (GATA1s), and have been identified as the causative lesion inducing TAM in infants with DS. Nevertheless, GATA1s mutations are insufficient to cause ML-DS and cooperating lesions remain unknown.To identify the genetic aberrations during the transition from TAM to ML-DS, we have joined an international consortium and sequenced exomes of 22 paired TAM/ML-DS patients, followed by targeted sequencing of candidate mutations in 400 ML-DS samples. Besides high prevalence of mutations in tyrosine kinases and cytokine receptor signaling-associated genes, a particularly high percentage of patients presented with mutations in cohesin-complex-associated genes or histone modifiers, which contrasts the mutational spectrum seen in pediatric non-DS AML. Utilizing our lentiviral CRISPR-Cas genome editing tool and classical lentiviral cDNA expression -both in Gata1s mice and primary TAM patient samples- we will interrogate the functional and molecular consequences of cohesin-complex- and histone modifier mutations in the transformation of TAM to ML-DS. We will thereby unravel the molecular processes that guide leukemic transformation in the context of GATA1s and trisomy 21. In a CRISPR-Cas based loss of function screening we will leverage this knowledge about ML-DS transformation to identify novel therapeutic targets that will allow to eradicate ML-DS with targeted and reduced toxicity therapy, or open even new venues to eliminate preleukemic TAM clones and thereby transformation of TAM to ML-DS.

急性髓系白血病是一组异质性克隆性疾病，起源于造血干细胞和祖细胞（HSPCs），其通过逐步获得合作遗传病变而转化。由于缺乏适当的技术来修饰人类细胞的基因组在白血病中发现的复杂程度，这种逐步转化的进化过程在人类系统中建模和询问是不切实际的。即使在老鼠身上，这些修饰是可行的，两种以上修饰的组合遗传学也是耗时且昂贵的。我们最近开发了一种基于慢病毒的CRISPR- cas（聚集规律间隔短回文重复(CRISPR) - CRISPR相关）系统，该系统允许我们同时在单个HSPC的内源性位点上修饰多达五个基因。在这里，我们将利用这种新的基因组修饰工具来描述唐氏综合征（ML-DS）儿童髓性白血病的白血病进化。5-30%的DS患儿被诊断为短暂性骨髓生成异常（TAM），具有白血病的典型特征。在大多数未接受抗白血病治疗的婴儿中，TAM会消退，然而，在这些儿童中，20-30%的AML在生命的前四年内发展。TAM和ML-DS几乎普遍（95%）存在造血转录因子GATA1突变，导致截断蛋白（GATA1s）的唯一表达，并且已被确定为DS婴儿中诱发TAM的致病病变。然而，GATA1s突变不足以引起ML-DS，其协同病变尚不清楚。为了确定从TAM到ML-DS转变过程中的遗传畸变，我们加入了一个国际联盟，对22对TAM/ML-DS患者的外显子组进行了测序，随后对400个ML-DS样本中的候选突变进行了靶向测序。除了酪氨酸激酶和细胞因子受体信号相关基因突变的高发率外，粘聚物复合物相关基因或组蛋白修饰因子突变的患者比例特别高，这与儿童非ds AML的突变谱形成了对比。利用我们的慢病毒CRISPR-Cas基因组编辑工具和经典的慢病毒cDNA表达——在Gata1s小鼠和原发性TAM患者样本中——我们将探讨在TAM向ML-DS转化过程中，内聚物复合物和组蛋白修饰因子突变的功能和分子后果。因此，我们将揭示在GATA1s和21三体的背景下指导白血病转化的分子过程。在基于CRISPR-Cas的功能丧失筛选中，我们将利用这些关于ML-DS转化的知识来确定新的治疗靶点，这些靶点将允许通过靶向和降低毒性的治疗来根除ML-DS，或者甚至开辟新的场所来消除白血病前期TAM克隆，从而将TAM转化为ML-DS。