m6A识别蛋白YTHDF2促白血病细胞生长的研究

BCR-ABL可诱发慢性髓细胞白血病（CML），酪氨酸激酶抑制剂（TKI）是CML治疗的一线药物，但复发与耐药仍困扰部分患者。N-6甲基腺苷（m6A）修饰是RNA代谢新的调控方式，但它在CML中作用未知。预研究显示YTHDF2基因（编码m6A识别蛋白）在患者CD34+细胞中表达较对照细胞显著增高，这些细胞的m6A修饰丰度较对照细胞显著下降。基因干预、RNA-seq和MeRIP等显示：YTHDF2促进CML细胞生长、使它们耐受TKI，可降低细胞m6A修饰丰度，并增强某些抑癌基因（如TINAGL1）mRNA的降解。为研究YTHDF2调控CML干/祖细胞生长与TKI反应的功能及其机制，将分析YTHDF2对CML干/祖细胞体内外生长的影响（±TKI）；用RNA-seq/MeRIP-seq和多种生化/细胞方法鉴定YTHDF2的功能靶基因。将揭示m6A修饰在CML中的作用、深化对CML致病机制的认知。

Chronic myeloid leukemia (CML) originates from hematopoietic stem cells acquiring BCR-ABL fusion gene to encode a tyrosine kinase with deregulated activity. Specific tyrosine kinase inhibitors (TKIs, e.g. imatinib mesylate, IM) against BCR-ABL have witnessed great improvement of the disease treatment. However, drug resistance and relapse happen to some patients as a result of the existence of leukemia stem cells from patients even in complete remission (CR), suggesting the insufficiency of TKIs to induce apoptosis of these primitive cells. Therefore, better understanding of the molecular mechanisms for CML stem/progenitor cells to grow and survive is still in need, which likely leads to the identification of novel therapeutic target to improve the disease management..Our data showed that ZC3H13, a component of m6A methyltransferase complex and YTHDF2, a m6A reader protein had significant higher expression in CML CD34+ cells compared with control cells. Meanwhile, the overall m6A modification was significantly lower in CML CD34+ cells than that in normal control cells. Silence of YTHDF2 increased the m6A modification in K562 cells, and inhibited the cell growth and colony-forming cell (CFC) production while sensitized these cells to IM treatment. In contrast, overexpression of YTHDF2 had opposite effects on K562 cells. Importantly, YTHDF2 silencing inhibited the CFC production of CML CD34+ cells as well. To obtain molecular insight of how m6A modification regulates CML cells, RNA-seq data were generated to compare YTHDF2 silenced K562 cells with their control. Based on the indication of RNA-seq data, it was validated that TINAGL1 was upregulated upon YTHDF2 silencing. MeRIP analysis revealed that TINAGL1 transcript had m6A modification. TINAGL1 had significant lower expression in CML CD34+ cells than that in control cells, and overexpression of TINAGL1 inhibited the CFC production of K562 cells. Knockdown of TINAGL1 in YTHDF2 silenced K562 cells “rescued” the inhibited growth and sensitized IM response caused by YTHDF2 silencing. Taken together, our data demonstrated that YTHDF2 modulated the growth and drug response of CML cells. .Herein, we propose to investigate the effect of YTHDF2 silencing on the CFC production (±IM), LTC-IC activity (±IM) and xenoegraftment of purified CML stem/progenitor cells. In contrast, the effect of YTHDF2 overexpression on the growth of BaF3 cells, naïve mice hematopoietic stem/progenitor cells and human CD34+ cells alone or together with BCR-ABL will be studied as well. Using RNA-seq and MeRIP-seq, the functional target genes regulated by YTHDF2 through m6A modification will be identified as well. The project will obtain novel insights of how m6A modification regulates the growth/survival and TKI response of CML stem/progenitor cells, which possibly provides new clues to improve the disease management.