Community Outreach and Education Core

在评估FLT3突变在AML中的作用方面取得了重大进展。其中包括FLT3-ITD单独表达的分析，以及与PML-RARa等合作等位基因的结合分析。与项目1合作，项目2在FLT3抑制剂的临床前开发中发挥了重要作用，通过显示在小鼠疾病模型中的有效性，从小鼠疾病模型的治疗试验中产生数据。在Specific Aim 1中，我们将探讨FLT3- itd和激活环等位基因的体内活性，并试图了解FLT3- itd在髓系疾病中的相对倾向，以及FLT3激活环等位基因在淋巴系疾病中的相对倾向。我们将使用多参数流式细胞术来验证这些等位基因在造血发育过程中FLT3高度表达的多能祖细胞阶段（MPP或LMPP）对细胞命运决定有不同影响的假设。我们将尝试了解FLT3ITD的机制，与FLT3 WT相比，FLT3ITD是STAT5的有效激活剂，使用的突变可以消除这种活性。在Specific Aim 2中，我们将与项目3和项目4合作，探索FLT3-ITD介导疾病的这些精确基因型模型的协同效应。这些反过来将作为有用的体内模型，用于测试项目1中开发的新型联合疗法。在Specific Aim 3中，我们将开发由JAK2V617F等位基因介导的骨髓增生性疾病的小鼠模型，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，用于项目5的临床试验。总的来说，这是一个高度互动的项目，将建立在成功的记录和髓系新疗法的临床前开发上

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