Genome-wide pan-haematopoietic transposon screening for oncogenic network discovery in mice

近年来，人们对癌症遗传学的理解取得了巨大的进步。例如，下一代测序工作正在创建主要针对所有癌症类型的体细胞突变目录。然而，这些新颖的见解也揭示了驱动肿瘤发生的分子过程的复杂性仍然远未被理解。例如，缺乏系统地搜索突变的人类癌症基因下游的靶点或效应通路的工具，或者精确定位癌症驱动因素的工具，这些基因没有突变，但属于癌细胞中数千个表观遗传、转录或转录后异常基因中的一个。为了解决这些障碍，我们开发了在小鼠身上的iggyBac转座子工具，并展示了它们在体内遗传筛选和癌症基因发现中的应用。在初步实验中，我们使用专门为在造血系统中进行筛选而设计的iggyBac小鼠系，能够诱发各种类型的血液病，包括T细胞、B细胞、髓系或未分化肿瘤等。我们建议现在扩展这种方法，对不同类型的血液病恶性肿瘤中的癌症驱动因素进行系统的全基因组调查。我们的试点筛选已经揭示了一些实体的新癌症基因，包括低分化癌症中的剪接因子MBNL1。从这些筛选中筛选出的MBNL1和其他几个新的癌症基因将使用我们实验室开发的新的未发表的CRISPR/Cas9载体/小鼠在体外和体内进行验证和功能表征。我们在Hoxb8细胞系统中使用RNAi和基于CRISPR/Cas9的基因编辑的初步实验表明，MBNL1在分化控制中的作用将得到进一步探索。最后，将使用大的白血病/淋巴瘤患者队列进行跨物种的鼠/人分析，以确定新的候选癌症基因的临床相关性。这些全基因组筛查将发现其他癌症基因组分析方法无法识别的癌症驱动因素，因此将补充基于测序的人类血液恶性肿瘤癌症基因普查。

Recent years have brought tremendous advances in the understanding of cancer genetics. Next generation sequencing efforts, for example, are creating catalogues of somatic mutations for principally all cancer types. Such novel insights are however also revealing that the complexity of molecular processes driving tumorigenesis is still far from being understood. For example, there is a lack of tools to systematically search for targets or effector pathways downstream of mutated human cancer genes, or to pinpoint cancer drivers that are not mutated but are among the thousands of epigenetically, transcriptionally or post-transcriptionally dysregulated genes in a cancer cell. To address these obstacles, we have developed PiggyBac transposon tools in mice and showed their application for in vivo genetic screening and cancer gene discovery. Using PiggyBac mouse lines specifically designed for screening in the haematopoietic system we were able to induce various types of haematologic malgnanices in pilot experiments, including T cell, B cell, myeloid or undifferentiated neoplasms and others. We propose to now extend this approach to perform systematic genome-wide surveys for cancer drivers in various types of heamatologic malignancies. Our pilot screens have already revealed novel cancer genes for some entities, including the splicing factor Mbnl1 in poorly differentiated cancers. Mbnl1 and few other selected novel cancer genes emerging from these screens will be validated and functionally characterized in vitro and in vivo using novel unpublished CRISPR/Cas9 vectors/mice developed in our lab. Our initial experiments using RNAi and CRISPR/Cas9-based gene editing in the Hoxb8 cell system of bone-marrow derived progenitor cells point towards a role of Mbnl1 in differentiation control, which will be explored further. Finally, cross-species mouse/human analyses will be performed using large leukaemia/lymphoma patient cohorts in order to establish the clinical relevance of novel candidate cancer genes. These genome-wide screens will discover cancer drivers that cannot be identified with other approaches to cancer genome analysis, and will thus complement the sequencing-based census of human cancer genes across haematologic malignancies.