Murine models of activated FLT3 receptor tyrosine kinase

描述(申请人提供)：Flt3基因是急性髓细胞白血病(AML)中最常见的突变基因。大约30-35%的患者存在膜旁(JM)区域的内部串联复制(Flt3-ITD)或Flt3激活环的突变，其后果是酪氨酸激酶的结构性激活。Flt3激活突变也在急性淋巴细胞性白血病中被描述，包括含有11q23重排的亚群，这与更广泛的造血肿瘤的发病机制有关。值得注意的是，在大多数回顾性研究中，这些突变给患者带来了不良的临床预后，为了解Flt3-ITD介导的疾病的发病机制以及开发和测试能够特异性靶向flt3分子的新疗法提供了令人信服的基础。 在这项为期5年的计划中描述的研究概述了旨在通过描述我们使用标准转基因和胚胎(ES)细胞基因打靶技术建立的新的Flt3-ITD敲入突变小鼠模型的特征，在整个动物系统中研究激活的Flt3-ITD突变的体内效应。在这些动物中表达内源性小鼠Flt3启动子激活的flt3比逆转录病毒转导模型有明显的优势，在逆转录病毒转导模型中，激活的flt3表达水平的差异可能会影响疾病表型，并避免逆转录病毒整合引入的潜在突变。这些动物模型不仅应该为了解激活的Flt3如何在白血病的发病机制中做出贡献，而且还应该为该基因在正常造血中的生物学作用提供见解。此外，这些小鼠应该提供强大的试剂，可用于评估靶向药物治疗对Flt3诱导的白血病的疗效，包括使用目前已建立的Flt3抑制剂和新一代高亲和力Flt3抑制剂。最后，这些小鼠可以与其他已建立的小鼠模型系统一起使用，以进一步了解不同类别的突变如何在急性白血病的发展过程中相互合作。具体目的包括：(1)建立Flt3-ITD基因敲入小鼠模型，包括(A)评估内源性flt3启动子的flt3-itd基因表达对造血祖细胞发育的影响；(B)详细鉴定单等位基因和双等位基因的flt3-itd基因敲入表型；(2)评价小分子flt3酪氨酸激酶抑制剂在动物药物试验中的疗效；(3)将Flt3-ITD小鼠与PML/RARpha、AML1/ETO、C/EBPalpha P30转基因小鼠模型杂交，建立急性白血病的协同模型，并评价新型小分子抑制剂的治疗效果。

DESCRIPTION (provided by applicant): FLT3 is the most frequently mutated gene in cases of acute myelogenous leukemia (AML). About 30-35% of patients have either internal tandem duplications (FLT3-ITD) in the juxtamembrane (JM) domain or mutations in the activating loop of FLT3, the consequences of which are constitutive activation of the tyrosine kinase. FLT3 activating mutations have also been described in acute lymphoblastic leukemias including subsets harboring 11q23 rearrangements implicating it in the pathogenesis of a broader spectrum of hematopoietic neoplasms. Significantly these mutations confer a poor clinical prognosis to patients in most retrospective studies providing a compelling basis for understanding the pathogenesis of FLT3-ITD mediated disease and developing and testing new therapies that can specifically target the FLT3 molecule. The research described in this 5-year proposal outlines specific aims designed to investigate the in vivo effects of an activating FLT3-ITD mutation in a whole animal system through the characterization of a novel FLT3-ITD knock-in mutant mouse model which we have generated using a combination of standard transgenic and embryonic (ES) cell gene-targeting techniques. Expression of activated FLT3 from the endogenous murine FLT3 promoter in these animals provides distinct advantages over retroviral transduction models where differences in expression levels of activated FLT3 may affect disease phenotype and avoids potential mutations introduced by retroviral integration. These animal models should provide insights not only into how activated FLT3 contributes to the pathogenesis of leukemia, but also into the biological role of this gene in normal hematopoiesis. Moreover, these mice should provide powerful reagents that can be used to assess the efficacy of targeted drug therapy against FLT3-induced leukemias including the use of both currently established FLT3 inhibitors and new generation high affinity FLT3 inhibitors. Finally, these mice can be employed with other established mouse model systems to further our understanding of how different classes of mutations can cooperate with one another in the development of acute leukemia. Specific aims include: (1) To characterize a FLT3-ITD knock-in mouse model including (a) assessing effects of FLT3-ITD expression from the endogenous FLT3 promoter on hematopoietic progenitor development and (b) detailed characterization of mono-allelic and bi-allelic FLT3-ITD knock-in phenotypes; (2) To assess the therapeutic efficacy of small molecule FLT3 tyrosine kinase inhibitors in animal drug trials with FLT3-ITD mouse lines; and (3) To investigate a cooperative model of acute leukemia by crossing FLT3-ITD mice with other leukemogenic mouse models including PML/RARalpha, AML1/ETO, and C/EBPalpha p30 transgenic mouse lines and assessing for therapeutic efficacy with novel small molecule inhibitors.