MURINE MODELS OF MYELOID MALIGNANCIES

意向书 在评估Flt3突变在AML中的作用方面已经取得了重大进展。这些研究包括单独分析Flt3-ITD的表达，以及与PML-RARA等协同等位基因结合分析。与项目1合作，项目2通过展示在小鼠疾病模型中的疗效，从小鼠疾病模型的治疗试验中产生数据，在Flt3抑制剂的临床前开发中发挥了重要作用。在特定的目的1中，我们将探索Flt3-ITD和激活环等位基因在体内的活性，并试图了解Flt3-ITD对髓系疾病和Flt3激活环等位基因对淋巴系统疾病的相对偏好。我们将使用多参数流式细胞术来检验这一假设，即这些等位基因在多能祖细胞阶段(MPP或LMPP)对细胞命运的决定具有不同的影响，在造血发育过程中，Flt3高表达。我们将试图了解与FLT3 WT相比，FLT3ITD， 是STAT5的有效激活剂，使用突变来消除这一活性。在具体目标2中，我们将与项目3和项目4合作，探索这些精确的Flt3-ITD介导疾病的基因模型的协同效应。这些模型反过来将用作测试项目1中开发的新型组合疗法的体内模型。在具体目标3中，我们将建立由JAK2V617F等位基因介导的骨髓增殖性疾病的小鼠模型，使用这些模型来了解人类疾病的表型多效性，并作为测试新型JAK2抑制剂的平台，以开发项目5中的临床试验。总体而言，这是一个高度互动的项目，将建立在髓系恶性肿瘤新疗法的成功和临床前开发的已证实记录的基础上。 SA 1.使用敲入策略建立由突变的Flt3介导的准确的白血病模型并进行表征。我们将描述每个等位基因的表型。 A.一个Flt3-ITD条件性敲入等位基因的产生和特性 B.Flt3D835Y和I836del条件连锁等位基因的产生及特性 C.对STATS信号有缺陷的条件敲入等位基因Y589F/Y598F的产生 Sa 2.表征这些精确的Flt3-ITD介导的疾病基因模型与其他生殖系等位基因杂交的协同效应 A.Flt3-ITD Ki与组织蛋白酶G PML-RARpha和C/EBPalpha等位基因杂交(与Tenen Project 3交互作用) B.Flt3-ITD I836del Ki与MIL融合等位基因杂交(与阿姆斯特朗相互作用，项目4) C.Flt3-ITD Ki与AML1-ETO条件性Ki等位基因杂交 使用这些模型来测试项目1中描述的联合疗法，其中可能包括联合信号转导抑制剂、ATRA、HDAC抑制剂或HSP抑制剂 建立准确的JAK2V617F介导的MPD小鼠模型 A.JAK2V617F病逆转录病毒转导模型的建立及特性 B.JAK2V617F条件性敲入等位基因的产生和特征 C.鉴定包括MPL在内的JAK2V617F病的新的增强突变 D.在项目1中开发的小鼠模型中表征新型JAK2抑制剂

STATEMENT OF INTENT Significant progress has been made in the assessment of the role of FLT3 mutations in AML. These have included analysis of FLT3-ITD expression alone, and in combination with cooperating alleles such as PML-RARa. Working with Project 1, Project 2 has been instrumental in preclinical development of FLT3 inhibitors by showing efficacy in murine models of disease, generating data from therapeutic trials in murine models of disease. In Specific Aim 1, we will explore the in vivo activity of FLT3-ITD and activation loop alleles, and try to understand the relative predilection of FLT3-ITD for myeloid lineage disease and of the FLT3 actiavtion loopalleles for lymphoid disease. We will use multiparameter flow cytometry to test the hypothesis that these alleleshave differential effect on cell fate determination at the multipotent progenitor stage (MPP or LMPP) where FLT3 is highly expressed during hematopoietic development. We will try to understand the mechanism whereby FLT3ITD, in contrast with FLT3 WT, is a potent activator of STAT5 using mutations that abrogate this activity. In Specific Aim 2, we will explore cooperating effects of these accurate genotypic models of FLT3-ITD mediated disease, working with Projects 3 and 4. These in turn will serve as useful in vivo models for testing novel combination therapies that are developed in Project 1. In Specific Aim 3, we will develop murine models of myeloproliferative disease mediated by the JAK2V617F allele, use these models to understand phenotypicpleiotropy of disease in humans, and as a platform for testing novel JAK2 inhibitors for development of clinical trials in Project 5. Overall, this is a highly interactive Project that will build on a proven track record of success and preclinical development of novel therapies for myeloid malignancies. SA 1. Generation and characterization of accurate models of leukemia mediated by mutated FLT3 using knock-in strategies. We will characterize the phenotype of each of these alleles. a. Generation and characterization of a FLT3-ITD conditional knock-in allele b. Generation and characterization of FLT3 D835Y and I836del conditional knock-in alleles c. Generation of FLT3-ITD Y589F/Y598F conditional knock-in allele that is defective in signaling to STATS SA 2. Characterize the cooperative effects of these accurate genotypic models of FLT3-ITD mediated disease with crosses to other germline alleles a. FLT3-ITD KI crossed with Cathepsin G PML-RARalpha and C/EBPalpha knock-in alleles (Interaction with Tenen Project 3) b. FLT3-ITD I836del KI crossed with MIL fusion alleles (interaction with Armstrong, Project 4) c. FLT3-ITD KI crossed with AML1-ETO conditional KI allele Use these models to test combination therapy delineated in Project 1 that could include combination signal transduction inhibitors, ATRA, HDAC inhibitors, or HSP inhibitors SA 3. Develop accurate murine models of JAK2V617F mediated MPD a. Develop and characterize a retroviral transduction model of JAK2V617F disease b. Generate and characterize JAK2V617F conditional knock-in allele c. Characterize novel potentiating mutations of JAK2V617F disease including MPL d. Characterize novel JAK2 inhibitors in murine models as developed in Project 1