Basic investigation for the development of a novel anti-leukemic therapy targeting to the mutant FLT3 molecule.

在本研究中，我们研究了突变的Flt3激酶所带来的信号转导机制，并筛选了抑制Flt3基因突变的白血病细胞增殖或诱导其分化的治疗药物，取得了以下结果。我们将Flt3的胞内区分成几个片段，分别在Cos7细胞中表达，发现了JM区和TK区之间的分子内关联。利用双杂交系统，我们发现了几个与突变或野生型Flt3JM区域特异结合的分子。我们通过定量检测髓系分化相关基因的表达水平，分析了抑制G-CSF诱导32D细胞髓系分化的生物学机制。在表达突变的Flt3细胞中，髓系分化受阻是由于mpo、C/eBP-α和CIEBP-ε基因的抑制所致。我们定量分析了181例初治AML患者中Flt3转录本的表达水平，并与几种基因改变进行了比较。在急性髓系白血病细胞中，Flt3转录本的平均表达水平为20,203拷贝/μgRNA，而每个水平在0到2,322,706拷贝/μgRNA之间变化。Flt3高表达与Flt3/ITD(p=.0020)、MLL-TD(p=.0121)和Flt3/D835Mt(p=.0463)有关，与N-RAS或p53突变无关。过表达的Flt3具有自身磷酸化作用，对Flt3抑制剂的敏感性与Flt3/ITD相同。在91例无Flt3/μ的急性髓系白血病患者中，Flt3基因的过度表达(>200,000拷贝/ITDgRNA)是影响总体生存的不良预后因素。这些结果表明，在无Flt3突变的AML中，Flt3的过表达可能是一种新的疾病实体，并可作为Flt3抑制剂的治疗靶点。

In this study, we investigated the signal-transduction mechanism brought by the mutant FLT3 kinase and screened the therapeutic agents which inhibit the proliferation or induce the differentiation in leukemia cells harboring FLT3 mutations, then obtained the following results.1. We divided the intracellular region of the FLT3 into several segments and expressed each segment to Cos7 cells, then found the intramolecular association between the JM and TK regions.2. Using the two-hybrid system, we found several molecules which specifically bind to the mutated or wild-type FLT3 JM region.3. We analyzed the biological mechanism for inhibiting the G-CSF induced myeloid differentiation in mutant FLT3 expressing 32D cells by means of quantitating the expression level of myeloid differentiation associated genes. In mutant FLT3 expressing cells, the block of myeloid differentiation was shown to be resulted from the inhibition of MPO, C/EBP-α and CIEBP-ε genes.4. We analyzed the expression level of the FLT3 transcript quantitatively in comparison with several gene alterations in 181 de novo AML cases. In AML cells, the mean expression level of the FLT3 transcript was 20,203 copies/μgRNA, while each level varied from 0 to 2,322,706 copies/μgRNA. A high expression level of FLT3 was related to FLT3/ITD (p=.0020), MLL-TD (p=.0121) and FLT3/D835Mt (p=.0463), but not to N-RAS or to p53 mutations. Overexpressed FLT3 revealed auto-phosphorylation, and had the same sensitivity to the FLT3 inhibitor as FLT3/ITD. Overexpression of FLT3 (over 200,000 copies/μgRNA) was an unfavorable prognostic factor for overall survival in 91 AML cases without FLT3/ITD. These results indicated that overexpression of FLT3 might distinguish a novel disease entity in AML without FLT3 mutations and serve as a therapeutic target for FLT3 inhibitors.