RUNX1-ETO白血病融合基因转录与翻译调控机制的研究

Leukemia fusion proteins (LFP), the protein products of leukemia fusion genes (LFG, chimerae of two genes), are the proximate cause of leukemia in these patients. Targeting the inactivation of LFP has been suggested to be the most effective strategy for treating LFG+ leukemia. Most previous studies were directed toward treating leukemia by inhibiting the activity, partner interaction, or target genes/signaling of LFP. We propose that inhibition of the production of LFP might be a novel way to treat leukemia. We want to use RUNX1-LFG+ acute myeloid leukemia cells (LC hereafter) as a model system to test this idea because: 1) the promoters and 5'UTRs of RUNX1 have been well documented; 2) RUNX1 is a key transcriptional regulator of hematopoiesis and it is commonly targeted by leukemic chromosomal translocations; 3) the expression of the leukemogenic allele of LFG generated from most such translocations is controlled by the RUNX1 promoter. As a consequence, translation of their mRNA transcripts is regulated by the 5'UTR of RUNX1 transcripts. RUNX1 gene has P1 and P2, 2 promoters, which produce mRNAs with 2 distinct 5'UTRs. In adult hematopoietic cells, RUNX1 expression is controlled by P1 which produces a mRNA with a <0.45 kb short 5'UTR. The expression of RUNX1-LFGs in LC is primarily controlled by P2 which mRNA transcript has a 1.6kb long 5'UTR. The translation of P1 and P2 RUNX1 mRNAs are regulated by a cap-dependent and a cap-independent mechanisms respectively. We generated a RE-GFP reporter cell line by fusing GFP to the last exon of RUNX1-ETO gene in Kasumi1 AML cell. The GFP intensity in this cell faithfully represents the levels of endogenous RUNX1-LFPs. We also generated a P2-GFP reporter cell line by transducing Kasumi1 cells with p2-5'UTR-d2GFP expressing virus. In this reporter cell line, expression of a d2GFP is controlled by P2-5'UTR of RUNX1 gene which intensity well represents the cap-independent translation of P2-5'UTR. In this proposed studies, we want to identify the key regulatory factors for the transcription/translation of RUNX1-LFGs by transducing RE-GFP and p2-GFP reporter cell with shRNA and/or cDNA library to unbiased screen the genes required for the production of RUNX1-LFPs. Such key molecular regulators will be used as targets for developing novel types of antileukemia drugs for RUNX1 leukemia.

t(8;21)染色体重排所产生的RUNX1-ETO为一种最为常见的白血病融合基因（LFG）。这种LFG是导致这类AML发生的直接原因。同时这类AML细胞的增殖和生存依赖于RUNX1-ETO的蛋白产物的RUNX1-ETO-白血病融合蛋白（LFP）。研究表明，抑制LFP的活性和功能是治疗白血病的最有效手段。以前的大量研究试图通过如下几种途径抑制LFP：1） 诱导LFP降解；2）抑制LFP和其相关蛋白的结合；3）抑制LFP与DNA的结合及抑制下游基因的表达。 我们认为抑制LFP的产生可能为一种治疗AML的新思路。本课题采用我们独特的GFP报告基因系统，通过先进的随机大规模的shRNA和cDNA文库筛选的方法，鉴定调节RUNX1-ETO LFP合成的信号分子和关键基因。寻找治疗RUNX1-ETO相关白血病新型药物的分子靶点。

本课题采用报告基因的策略，采用shRNA和cDNA文库筛选技术鉴定调控RUNX1-ETO白血病融合基因转录与翻译调控分子。共筛出15个调控RUNX1-ETO蛋白表达的关键基因。其中12个为调控转录的转录因子，3个为调控翻译的因子。 对其中9个做了功能鉴定，其它6个的功能鉴定继续进行。结论：1）RUNX1-ETO的转录主要受第二个启动子和远端的一个关键增强子调控；2）翻译主要受cap非依赖性的非经典途径调控；3）抑制这些转录和翻译的调控可以抑制RUNX1-ETO白血病的发生和发展。 同时鉴定出两个抑制RUNX1-ETO翻译的寡核苷酸。这两个寡核苷酸可以特异性的抑制RUNX1-ETO白血病细胞的生长。已发表两篇SCI论文。另有一篇文章正在送审，一篇文章在整理中。