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

急性髓系白血病是一组起源于造血干/祖细胞(HSPC)的异质性克隆性疾病，通过循序渐进地获得协作性基因病变而转化。由于缺乏适当的技术来在白血病中发现的复杂程度上修改人类细胞的基因组，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。即使在小鼠中，这些修饰是可行的，但组合遗传学中两个以上的修饰是耗时和昂贵的。我们最近开发了一个基于慢病毒的CRISPR-Cas(簇状规则间隔短回文重复(CRISPR)-CRISPR相关)系统，它允许我们在单个HSPC中同时修改其内源基因位点上的多达5个基因。在这里，我们将利用这一新的基因组修改工具来描绘唐氏综合征(ML-DS)儿童髓系白血病的白血病演变。5-30%的DS患儿被诊断为一过性异常骨髓生成()，具有典型的白血病特征。在大多数没有接受抗白血病治疗的婴儿中，的病情会恶化，然而，在这些儿童中，有20%-30%的儿童在出生后的头四年内出现了急性髓系白血病。和ML-DS几乎普遍(95%)都存在造血转录因子GATA1的突变，导致截短蛋白(GATA1)的独占表达，并被认为是导致DS婴儿的原因。为了确定向ML-DS转变过程中的基因异常，我们加入了一个国际联盟，对22例配对的/ML-DS患者的外显子进行了测序，然后对400例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外，特别高比例的患者出现了粘附素复合体相关基因或组蛋白修饰物的突变，这与儿童非DS AML的突变谱形成了鲜明对比。利用我们的慢病毒CRISPR-Cas基因组编辑工具和经典慢病毒cDNAs在Gata1s小鼠和患者的原始样本中的表达，我们将询问向ML-DS转化过程中粘附素复合体和组蛋白修饰物突变的功能和分子后果。因此，我们将揭开在GATA1和21三体的背景下引导白血病转化的分子过程。在基于CRISPR-CAS的功能丧失筛查中，我们将利用这一关于ML-DS转化的知识来确定新的治疗靶点，从而能够通过靶向和减毒治疗来根除ML-DS，或者甚至开辟新的场所来消除白血病前期克隆，从而将转化为ML-DS。

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.