早幼粒细胞白血病蛋白PML调节TET2在肿瘤发生发展中的作用及机制

Deregulation of the DNA epigenetic modification in cancer is one of the research hot spots. Although growing evidence indicates that aberrant TET-mediated DNA modification plays an important role in tumor, the regulatory mechanism and role still remain elusive. Our previous study indicated that PML is regulated by BMK1 cellular signal transduction pathway (Cancer Cell，2010) and involved in the drug resistance (Oncogene，2013). Our pilot experiment indicated that PML WT significantly regulates TET2, while the PML oncogenic mutant--PML-RARa, interacts with TET2 poorly. Based on these, it is hypothesized that PML regulates the epigenetic modification through the recruitment of TET2, while the deregulation of interaction between PML and TET2 plays an important role in tumorigenesis and tumor progression. In this study, NGS (Next generation sequencing), Cas9-mediated knock out cell and mouse model will be carried out. After the specific treatment (such as arsenic trioxide), the subcellular localization and colocalization of TET2, PML and PML-RARa will be investigated to evaluate the role of PML and TET2 in tumorigenesis and tumor progression. Cas9-mediated knock out cell and mouse model will also be applied to verify the role of PML and TET2. Furthermore, using NGS (Next generation sequencing), the epigenetic map will be built to unveil the regulatory mechanism of TET2. We hope that this study could shed some light on the regulatory mechanism and role of the aberrant DNA epigenetic modification in tumorigenesis and tumor progression.

恶性肿瘤中DNA修饰异常是生命科学研究最大热点之一。而甲基胞嘧啶双加氧酶TET2在DNA修饰异常中起到重要作用，但其功能和调控机制尚待研究。申请人前期研究发现早幼粒细胞白血病蛋白PML被有丝分裂原活化蛋白激酶BMK1信号通路调节（Cancer Cell，2010），进而控制肿瘤耐药（Oncogene，2013）。而预实验表明野生型PML，而非突变体PML-RARa，结合并调节TET2。据此，申请人认为PML通过调节TET2，控制表观修饰，参与肿瘤发生发展。本研究拟利用下一代测序、基因敲除细胞和小鼠等手段，通过观察砷化剂等处理下TET2、PML及PML-RARa的分布和细胞生长特征，结合小鼠发病情况，研究PML调节TET2在肿瘤发生发展中的作用；并通过构建表观基因组图谱、表达检测和功能分析，进一步探讨其分子机制和作用靶点。本研究将为阐明DNA表观修饰异常促进肿瘤发生发展的机制奠定基础。

DNA修饰异常在癌症的发生发展中起到关键作用，其中未能激活甲基化沉默的抑癌基因被认为是癌症化疗耐药的重要原因，而化疗药物如何诱发脱去DNA甲基化的机制仍待研究。我们发现化疗药物可通过早幼粒细胞白血病蛋白PML招募甲基胞嘧啶双加氧酶TET2到特定位点，脱甲基激活抑癌基因，进而抑制癌细胞生长。我们的研究表明甲基关键酶TET2是多种化疗药物诱导脱甲基中间体5羟甲基胞嘧啶（5hmC）所必需的。而后利用稳定同位素标记的细胞培养（SILAC）结合质谱技术鉴定出与TET2有紧密相互作用的蛋白，在这些蛋白中PML表现出最强烈的诱导5hmC的能力。进一步研究表明，PML可通过其C端与TET2结合，招募TET2到PML核小体。当PML发生t(15;17)突变，PML的C端被替换为RARa的C端，形成PML-RARa融合蛋白，则破坏了PML与TET2相互作用。而化疗药物可通过PML招募TET2，脱甲基激活沉默的基因，进而抑制癌细胞生长。PML敲除则可抑制化疗药物阿霉素诱导的DNA脱甲基。并且在头颈癌患者中，PML和TET2表达水平与生存期显著正相关。这些结果将为阐明癌症化疗调控DNA表观修饰的机制奠定基础。