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细胞和祖细胞（HSPC）的异质性克隆性疾病，其通过逐步获得协同遗传病变而转化。由于缺乏适当的技术来修改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一直是不切实际的建模和询问在人类系统。即使在小鼠中，这些修饰是可行的，超过两个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（成簇规则间隔短回文重复序列（CRISPR）-CRISPR相关）系统，该系统允许我们在单个HSPC中的内源基因座上同时修饰多达5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演变。5-30%患有DS的婴儿被诊断为一过性异常骨髓生成（TAM），其具有白血病的典型特征。大多数未经抗白血病治疗的婴儿的TAM都会消退，然而，其中20-30%的儿童在出生后的前四年内就会患上AML。TAM和ML-DS几乎普遍（95%）在造血转录因子GATA 1中存在突变，导致截短蛋白（GATA 1 s）的排他性表达，并已被确定为DS婴儿中诱导TAM的致病性病变。然而，GATA 1 s突变是不足以导致ML-DS和合作的病变仍然unknow.To确定从TAM到ML-DS的过渡过程中的遗传畸变，我们已经加入了一个国际财团和测序22配对TAM/ML-DS患者的外显子组，随后在400 ML-DS样本的候选突变的靶向测序。除了酪氨酸激酶和细胞因子受体信号传导相关基因突变的高患病率外，尤其是高百分比的患者出现粘着蛋白复合物相关基因或组蛋白修饰物突变，这与儿科非DS AML中观察到的突变谱形成对比。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Gata 1 s小鼠和原发性TAM患者样本中-我们将询问TAM转化为ML-DS中粘着蛋白复合物和组蛋白修饰剂突变的功能和分子后果。因此，我们将解开的分子过程，指导白血病转化的背景下，GATA 1 s和三体21。在基于CRISPR-Cas的功能丧失筛选中，我们将利用关于ML-DS转化的这一知识来鉴定新的治疗靶点，这些靶点将允许用靶向和降低毒性的治疗来根除ML-DS，或者甚至打开新的途径来消除白血病前TAM克隆，从而将TAM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.