ASXL1去泛素化复合体功能缺失在肿瘤发生发展中的作用及机制研究

Cancer genomics studies have found frequent loss-of-function mutations of ASXL1 in diversity of tumors, especially the hematopoietic malignancies. The patients with ASXL1 mutations tend to have a poor prognosis. We previously found that ASXL1 forms a protein complex with the deubiquitinase BAP1, which specifically removes ubiquitin from Lysine 119 on histone H2A (H2AK119ub1). Therefore we would like to clarify importance of the loss of ASXL1 in tumor initiation and progression (leukemia as an example) and to understand the underlying mechanisms. Here we will overexpress ASXL1 in the ASXL1-mutated leukemia cells and knock down ASXL1 expression in ASXL1 wild-type leukemia cells to observe the effects on cell proliferation, differentiation and colony formation. Taking a KRas transgenic mouse model, we will investigate how the depletion of Asxl1 affects the leukemia progression. Based on the functional studies, we will go on to identify the ASXL1 target genes by ChIP-sequencing and explore the changes of transcriptional profiles in ASXL1-depleted leukemia cells by RNA-sequencing. Then we will follow to measure the changes of histone modifications such as H2AK119ub1 and H3K27me3 at the specific locus on the deregulated target genes. Taken together we will demonstrate the epigenetic mechnisms for ASXL1 depletion in tumor initiation and progression. By collecting clinical leukemia patient samples with or without ASXL1 mutations, we will verify the function of ASXL1 and the regulatory effects on the key target genes. Our study will hopefully provide important therapeutic strategies against tumors with ASXL1 mutations.

ASXL1功能丧失型突变常见于多种肿瘤尤其是血液系统恶性疾病，携带该突变的白血病患者预后通常较差。我们前期研究发现ASXL1与去泛素化酶BAP1形成复合体，特异地移除组蛋白H2AK119位点的单泛素化修饰，因此有必要进一步明确ASXL1缺失在肿瘤发生发展中的作用及其分子机制。本项目拟以白血病为着眼点，在ASXL1突变型和野生型白血病细胞中分别过表达和敲低ASXL1，观察对细胞增殖、分化、克隆形成能力等表型的影响，探索Asxl1缺失对小鼠白血病进展的影响；在此基础上，进一步鉴定白血病细胞中ASXL1的靶基因，检测ASXL1缺失造成的基因表达变化，并分析差异表达的靶基因上H2AK119ub1等相关组蛋白修饰水平的变化，阐明ASXL1去泛素化复合体功能缺失在白血病发生发展中的表观遗传学转录调控机制；然后在临床白血病患者骨髓细胞中进行功能和关键靶基因表达的验证，为相应类型肿瘤的治疗提供策略指导。

我们和德国Jurg Muller实验室在2009年分别在哺乳动物和果蝇中鉴定出ASXL和BAP1所组成的H2A去泛素化复合体，他们在果蝇中很快证实了该复合体功能缺失导致的发育异常，而我们基于ASXL1、BAP1在多种肿瘤中发生突变，利用多种体外、体内肿瘤生物学研究模型，逐步揭示了该复合体功能缺失通过H2AK119ub1失调导致肿瘤发生的分子生物学机制。我们多年来的研究结果不仅仅证实了ASXL1是一个抑癌基因和阐释了其抑癌机制，也明确了H2AK119ub1这一组蛋白修饰动态调控的重要性，基于这些表观遗传调控机制及其所导致的信号通路失调，我们正在探索ASXL1或者BAP1突变肿瘤可能的靶向治疗方案。