TP73基因启动子甲基化对老年急性髓系白血病患者化疗敏感性和预后的影响及机制研究

More than half of acute myeloid leukemia (AML) patients are older than 60 years of age, many of them are unable to receive intensive chemotherapy with a median overall survival of only 4 to 6 months. p73 is one of the family members of tumor suppressor p53, different isoforms such as TAp73 and DNp73 have opposite function in cancer progression. Our previous study found that hypermethylation on TP73 promoter and higher ΔNp73/TAp73 ratio are associated with poor drug efficacy of AML patient with standard chemotherapy. We also found that TP53 mutations which are more frequently appeared in senile AML were associated with the upregulation of TP73 promoter methylation and downreglation of its mRNA expression in TCGA AML datasets. Therefore, the present study is firstly designed to make clear the influence of TP53 mutations, TP73 promoter methylation and ΔNp73/TAp73 ratio on the chemosensitivity and survival of senile AML patients in the clinical study. Secondly, cell lines with overexpressed TP53 mutation or TP53-siRNA and isolated CD34+ stem cell will be used to identify whether TP53 mutations can change the sensitivity of cytosine arabinoside and hypomethylating agent decitabine through the reglulation of TP73 promoter methylation. Thirdly, through interfering TAp73 expression, the interactions of TAp73, mutant p53 and ubiquitin are detected by co-immunoprecipitation to explore whether decitabine can increase TAp73 expression and then induce the ubiquitination degradation of mutant p53. The present study will provide new evidence on the personalized therapy of senile AML.

老年患者占急性髓系白血病（AML）的一半以上，多数无法耐受高强度化疗，中位生存期仅4-6个月。p73是抑癌基因p53家族的成员之一，不同亚型TAp73与DNp73在肿瘤形成中发挥相反的功能，本项目前期研究发现TP73启动子高甲基化、ΔNp73/TAp73高比值与AML疗效较差相关；TCGA数据库挖掘发现老年AML患者TP53突变高发，与TP73启动子甲基化上调和mRNA下调相关。本研究拟进行临床研究查明TP53突变、TP73启动子甲基化和ΔNp73/TAp73比值对AML药物反应、预后的影响；构建过表达TP53突变或干扰TP53表达的细胞株和原代培养CD34+干细胞，查明TP53突变是否调控TP73启动子甲基化、影响阿糖胞苷和地西他宾疗效；干扰TAp73表达，通过免疫共沉淀探索地西他宾是否通过上调TAp73表达，诱导突变型p53泛素化降解。本项目旨在为老年AML个体化治疗提供理论依据。

60岁以上患者占急性髓系白血病（AML）的一半以上，部分患者合并其他严重疾病，无法耐受标准治疗方案，通常采用低剂量阿糖胞苷（AraC）或支持疗法，其中染色体核型异常的患者通常无法达到完全缓解。前期对TCGA数据库挖掘发现TP53突变发生率在60岁以上患者中显著增加，与高风险分层密切相关，与TP73基因启动子甲基化上调和mRNA下调相关。地西他滨（DAC）是一种去甲基化药物，近年来被批准用于无法耐受标准治疗方案的AML患者。本项目旨在探索低剂量DAC和AraC对携带TP53突变AML细胞的抑制作用及其机制。研究成果包括：1.相较于携带TP53突变的AML细胞（Kasumi-1、KG-1和THP-1），携带TP53野生型的OCI-AML3细胞对DAC耐受；2.低剂量的AraC和DAC联用在携带TP53突变的AML细胞中有协同作用，在Kasumi-1和KG-1细胞中能进一步诱导细胞凋亡，但单次给药对THP-1细胞生长的抑制作用很小；3.模拟临床治疗方案每天给药、连续5天，除了对OCI-AML3细胞的停药7天的长期抑制作用基本消失，低剂量AraC和DAC单药或联用都能显著抑制3种细胞短期和长期生长；4.低剂量DAC可能通过下调突变p53、上调TAp73、下调DNp73表达或下调PI3K/AKT/mTOR通路来抑制细胞增殖、诱导凋亡、减弱AraC耐药；5.构建稳定表达TP53突变R248Q的THP-1细胞株，发现低剂量DAC能协同AraC抑制细胞生长，可能通过抑制突变型p53合成实现；6.构建稳定干扰TP73表达的THP-1细胞株，发现对AraC和DAC的敏感性降低，提示p73在p53缺失时能代替p53发挥类似作用。本项目发现低剂量DAC能调节p53家族表达，与AraC联用具有协同作用，提示两药联用治疗携带TP53突变AML的可能性。下一步工作，继续深入研究DAC抑制突变型p53合成的具体分子机制，并在动物实验或临床标本中验证。