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

针对突变 FLT3 分子开发新型抗白血病疗法的基础研究。

• 批准号: 14570973
• 负责人: KIYOI Hitoshi
• 金额: $ 2.3万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570973/
• 关键词: FLT3; tyrosinekinase; molecular target therapy; leukemia; genetic mutation

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.

本研究通过对突变型FLT 3激酶信号转导机制的研究，筛选出抑制突变型FLT 3激酶白血病细胞增殖或诱导其分化的治疗药物，取得了以下研究结果.我们将FLT 3的胞内区域分成若干片段，并将每个片段在Cos 7细胞中表达，发现JM和TK区域之间存在分子内关联.利用双杂交系统，我们发现了几个特异性结合突变或野生型FLT 3 JM区的分子.我们通过对髓系分化相关基因表达水平的定量分析，分析了突变型FLT 3表达的32 D细胞抑制G-CSF诱导的髓系分化的生物学机制。在突变型FLT 3表达细胞中，髓系分化的阻滞是由于MPO、C/EBP-α和CIEBP-ε基因的抑制所致.我们分析了FLT 3转录本的表达水平，并与181例初发AML病例中的几种基因改变进行了定量比较。在AML细胞中，FLT 3转录物的平均表达水平为20，203拷贝/μgRNA，而每个水平在0至2，322，706拷贝/μgRNA之间变化。FLT 3的高表达水平与FLT 3/ITD（p= 0.0020）、MLL-TD（p= 0.0121）和FLT 3/D835 Mt（p= 0.0463）相关，但与N-RAS或p53突变无关。过表达的FLT 3显示了自身磷酸化，并且对FLT 3抑制剂具有与FLT 3/ITD相同的敏感性。在91例无FLT 3/ITD的AML病例中，FLT 3过表达（超过200，000拷贝/μgRNA）是总生存期的不利预后因素。这些结果表明，FLT 3的过表达可能在没有FLT 3突变的AML中区分新的疾病实体，并作为FLT 3抑制剂的治疗靶点。

项目成果

Kazuoki Osumi: "Rapid screening of leukemia fusion transcripts in a cute leukemia by Real-time PCR"Leuk Lymphoma. 43. 2291-2299 (2003)

Kazuoki Osumi：“通过实时 PCR 快速筛选可爱白血病中的白血病融合转录本”白血病淋巴瘤。

Yosuke Minami: "Selective apoptosis of tandemly duplicated FLT3-transformed leukemia cells by Hsp9O inhibitors"Leukemia. 16. 1535-1540 (2002)

Yosuke Minami：“Hsp9O 抑制剂对串联复制的 FLT3 转化白血病细胞的选择性凋亡”白血病。

Manabu Ninomiya: "Retinoic acid syndrome in NOD/scid mice induced by injecting an acute promyelocytic leukaemia cell line"Leukemia. 18. 442-448 (2004)

Manabu Ninomiya：“注射急性早幼粒细胞白血病细胞系诱导 NOD/scid 小鼠出现视黄酸综合征”白血病。

Kazuoki Osumi: "Rapid screening of leukemia fusion transcripts in acute leukemia by Real-time PCR."Leuk Lymphoma. 43. 2291-2299 (2002)

Kazuoki Osumi：“通过实时 PCR 快速筛查急性白血病中的白血病融合转录本。”白血病淋巴瘤。

Minami Y: "Different anti-apoptotic pathways between wild-type and mutated FLT3 : insights into therapeutic targets in leukemia."Blood. 102. 2969-2975 (2003)

Minami Y：“野生型和突变型 FLT3 之间的不同抗凋亡途径：深入了解白血病的治疗靶点。”血液。