DNA甲基转移酶DNMT3A R882突变等位负荷对AML化疗敏感性和预后的影响及机制研究

DNMT3A mutation is considered as a driver of acute myeloid leukemia (AML). Our previous detection of DNMT3A R882 mutation as a poor prognostic predictor of AML, and its role may be related to the reduction of DNMT3A mRNA expression. lncRNA widely participated in the occurrence and development of tumors. Pre-experimental results showed that R882 mutations could promote the proliferation of leukemia cells by up-regulating the expression of lncRNA AC022182.3. However, the relationship between the allelic burden of R882 mutations and the efficacy and prognosis of AML standard chemotherapy has not been reported. In this study, clinical case analysis combined with molecular biology experiments are designed to explore R882 mutant effect on AML prognosis whether there is difference of mutant allelic burden; and by using cultured human umbilical cord blood hematopoietic stem cells (HSCs) and leukemia cell lines, through intervention DNMT3A expression and the use of RIP combined with high-throughput sequencing, to investigate the role of DNMT3A-lncRNA interaction in leukemic cell proliferation, differentiation and hematopoiesis. The aim of this project is to clarify the new mechanism of DNMT3A R882 mutations influencing the prognosis of AML and to provide a novel perspective for promoting prognostic prediction and precise treatment of AML.

DNMT3A突变被认为是急性髓细胞白血病（AML）发生的一种驱动因素。申请人前期发现DNMT3A R882突变作为AML不良预后的预测因子，其作用可能与降低DNMT3A mRNA表达有关。lncRNA广泛参与肿瘤的发生发展。预实验结果发现R882突变可通过上调lncRNA AC022182.3表达促进白血病细胞的增值。然而，R882突变等位负荷与AML化疗疗效、预后的关系未见报道。本研究拟通过临床研究结合细胞分子生物学实验，探究R882突变对AML预后的影响是否存在突变等位负荷差异；并利用分离培养的人脐带血造血干细胞（HSCs）和白血病细胞系，通过干预DNMT3A表达以及利用RIP结合高通量测序，探讨DNMT3A-lncRNA互作在白血病细胞增值、分化及造血中的作用。本项目旨在阐明R882突变影响AML预后的新机制，为促进AML的预后预测和精准治疗提供新视角。

R882突变作为DNMT3A最主要的突变形式对成人急性髓系白血病(AML)预后的影响目前尚存在争议。进一步细化研究R882突变影响AML预后的表观遗传新机制具有重要临床意义。项目执行内容与项目申请书内容基本一致，主要包括：（1）DNMT3A R882 突变等位负荷对AML患者临床化疗疗效和预后影响；（2）DNMT3A突变对人白血病细胞系细胞增殖、周期的影响；（3）R882突变状态对DNMT3A和lncRNA AC022182.3表达的影响；（4）干扰AC022182.3对携带R882突变AML细胞系的细胞增殖、周期的影响；（5）AC022182.3在AML患者中的临床意义。.在本项目的资助和支持下，发表SCI论著5篇。项目负责人受邀在《Frontiers in Oncology》发表非编码RNA方向综述1篇。同时，项目负责人参与国际学术会议一次，提交的lncRNA相关成果被Cancer Research的增刊收录为会议摘要论文。此外，项目负责人参与获得两项国家发明专利授权。在本项目的资助下，还获得了一些重要实验结果，主要有：（1）lncRNA AC022182.3在多种肿瘤组织中高表达，而在AML中表达倍数最高；（2）与DNMT3A野生型细胞系OCI-AML5相比，DNMT3A突变细胞系OCI-AML2和OCI-AML3中AC022182.3表达显著升高，且在R882突变细胞系OCI-AML3更高；（3）DNMT3A突变可能部分通过调控其本身表达进而上调AC022182.3水平；（4）细胞功能实验表明，携带R882C突变的OCI-AML3细胞增殖能力显著强于DNMT3A 野生型的OCI-AML5细胞，其机制与进细胞周期从G1向S期转化能力增强有关。.简言之，本项目围绕R882突变对AML疗效预后的影响是否存在突变等位负荷或等位基因比值差异，并从lncRNA视角探讨其表观遗传新机制。我们首次发现DNMT3A R882突变对AML预后的影响由突变等位基因比值决定，等位基因比值越高，预后越差；R882突变可通过上调lncRNA AC022182.3表达促进白血病细胞恶性增殖和细胞周期进程。本项目为制定更为精准的AML预后风险分层策略提供了直接研究证据。