Murine models of activated FLT3 receptor tyrosine kinase

描述（由申请人提供）：FLT 3是急性髓性白血病（AML）病例中最常见的突变基因。约30-35%的患者在质膜（JM）结构域中具有内部串联重复（FLT 3-ITD）或在FLT 3的活化环中具有突变，其结果是酪氨酸激酶的组成性活化。在急性淋巴细胞白血病中也描述了FLT 3激活突变，包括携带11 q23重排的亚群，表明其与更广泛的造血肿瘤的发病机制有关。值得注意的是，这些突变在大多数回顾性研究中赋予患者不良的临床预后，为理解FLT 3-ITD介导的疾病的发病机制以及开发和测试可以特异性靶向FLT 3分子的新疗法提供了令人信服的基础。 这项为期5年的提案中描述的研究概述了具体目标，旨在通过表征我们使用的新型FLT 3-ITD敲入突变小鼠模型来研究激活FLT 3-ITD突变在整个动物系统中的体内影响。标准转基因和胚胎（ES）细胞基因靶向技术的组合。在这些动物中，从内源性鼠FLT 3启动子表达活化的FLT 3提供了与逆转录病毒转导模型相比的明显优势，在逆转录病毒转导模型中，活化的FLT 3的表达水平的差异可能影响疾病表型，并避免了由逆转录病毒整合引入的潜在突变。这些动物模型不仅可以深入了解激活的FLT 3如何促进白血病的发病机制，还可以了解该基因在正常造血中的生物学作用。此外，这些小鼠应提供可用于评估靶向药物治疗对FLT 3诱导的白血病的疗效的强大试剂，包括使用目前建立的FLT 3抑制剂和新一代高亲和力FLT 3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型的突变如何在急性白血病的发展中相互合作。具体目标包括：（1）表征FLT 3-ITD敲入小鼠模型，包括（a）评估内源性FLT 3启动子的FLT 3-ITD表达对造血祖细胞发育的影响和（B）单等位基因和双等位基因FLT 3-ITD敲入表型的详细表征;（2）评估小分子FLT 3酪氨酸激酶抑制剂在FLT 3-ITD小鼠系的动物药物试验中的治疗功效;（3）通过将FLT 3-ITD小鼠与其他致白血病小鼠模型（包括PML/RAR α、AML 1/ETO和C/EBPalpha p30转基因小鼠系）杂交来研究急性白血病的协作模型，并评估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.