Identification of the MYBL2-mediated tumor suppression pathway and the essential cooperating mutations in myeloid malignancies

骨髓增生异常综合征（MDS）是一组异质性造血疾病，其特征是由于具有获得性干细胞特性的突变细胞克隆扩增和分化潜力受损而导致造血效率低下。一个杂合共缺失区（CDR）位于20号染色体的长臂上，预计含有肿瘤抑制基因。引人注目的是，在患有20q CDR的患者样本中，未发现该CDR中完整的20号染色体上的等位基因突变。因此，需要双等位基因失活的经典肿瘤抑制因子似乎不太可能存在。相反，单等位基因缺失可能会充分降低基因表达水平，从而促进细胞转化。为了寻找满足这些标准的基因，我们确定转录因子MYBL2是位于20q CDR中的基因剂量依赖性肿瘤抑制因子。令人惊讶的是，三分之二核型正常的MDS患者也受到MYBL2下调的影响，这表明了一种常见的疾病机制。事实上，通过RNAi下调小鼠Mybl2显示出强大的克隆优势和受影响的造血细胞的扩增。然而，由于缺乏其导致MDS克隆优势的机制影响的知识，这一发现对于MDS的治疗仍然是无关紧要的。因此，在这项拨款申请中，我们的目标是(1)确定转录因子MYBL2下调的功能后果，(2)确定MDS发病机制所需的MYBL2相互作用途径。我们发表的模型的一个关键特征是使用稳定的RNAi在体内模拟患者样本中的下调Mybl2。我们现在已经开发了一种可逆的，多西环素（dox）依赖性Mybl2 RNAi载体，并在体外进行了测试。利用该载体，我们可以通过敲低Mybl2使原代小鼠HSPCs永活。重要的是，这些细胞在还原去除后停止增殖。我们将使用该系统在体外和体内确定在具有和不具有dox的免疫表型相等的细胞群中负责这种表型的Mybl2靶基因。此外，我们将使用条件Mybl2敲除动物来定义所有Mybl2靶基因。直接靶基因将通过ChIP-Seq研究确定。在目标2中，我们将确定支持MDS发展的基因，与Mybl2的下调合作，从而开发新的MDS小鼠模型。我们将主要利用我们实验室已经建立的Crispr/ cas9介导的基因组工程，敲除8种不同的MDS抑癌基因。其中一个或两个的失活将用可逆的Mybl2敲低来模拟。由此产生的新的MDS小鼠模型将允许解决关键问题，包括维持疾病的低Mybl2水平的要求。最终，这些模型将有助于识别新的药物靶点。

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders characterized by inefficient hematopoiesis due clonal expansion of mutated cells with acquired stem cell properties and impaired differentiation potential. One heterozygously common deleted region (CDR) is located on the long arm of chromosome 20 and predicted to harbor a tumor suppressor gene. Strikingly, in samples of patients with the 20q CDR, mutations of alleles on the intact chromosome 20 within this CDR had not been identified. Hence, the presence of a classical tumor suppressor requiring biallelic inactivation appeared to be unlikely. Instead, the monoallelic loss by deletion may sufficiently reduce gene expression levels to promote cell transformation.In search of a gene fulfilling these criteria, we identified the transcription factor MYBL2 as gene-dosage dependent tumor suppressor located within the 20q CDR. Surprisingly, two thirds of MDS patients with a normal karyotype are also affected by MYBL2 downregulation indicating a frequent disease mechanism. Indeed, downregulation of Mybl2 in mice by RNAi revealed a strong clonal advantage and expansion of the affected hematopoietic cells. However, this discovery has remained inconsequential for the therapy of MDS due to the lack of knowledge of its mechanistic impact leading to the dominance of the MDS clone.Thus, in this grant proposal, we aim (1) to identify the functional consequences of the downregulation of the transcription factor MYBL2 and (2) to determine the MYBL2-interacting pathways required for the pathogenesis of MDS. A key feature of our published model was the downregulation of Mybl2 using stable RNAi in vivo mimicking the downregulation in patient samples. We have now developed a reversible, doxycycline (dox)-dependent Mybl2 RNAi vector and tested it in vitro. Using this vector, we could immortalized primary murine HSPCs by Mybl2 knockdown. Importantly, these cells cease proliferation upon dox removal. We will use this system to determine, in vitro and in vivo, those Mybl2 target genes that are responsible for this phenotype in immunophenotypically equal cell populations with and without dox. In addition, we will use conditional Mybl2 knockout animals to define all Mybl2 target genes. Direct target genes will be determined by ChIP-Seq studies. In aim 2, we will identify genes that support the development of MDS in cooperation with the downregulation of Mybl2 leading to the development of new MDS mouse models. We will mainly use Crispr/Cas9-mediated genomic engineering, which has been established in our lab, to knockout 8 different MDS tumor suppressor genes. Inactivation of one or two of them will be modeled with the reversible Mybl2 knockdown. The resulting new MDS mouse model will allow to address key questions including the requirement for low Mybl2 levels for the maintenance of the disease. Ultimately, these models will facilitate the identification of novel drug targets.