Community Outreach and Education Core

FLT3 突变在 AML 中的作用评估已取得重大进展。这些包括单独分析 FLT3-ITD 表达，以及与 PML-RARa 等合作等位基因组合的分析。项目 2 与项目 1 合作，在小鼠疾病模型中显示疗效，并从小鼠疾病模型的治疗试验中生成数据，从而在 FLT3 抑制剂的临床前开发中发挥了重要作用。在具体目标 1 中，我们将探索 FLT3-ITD 和激活环等位基因的体内活性，并尝试了解 FLT3-ITD 对骨髓谱系疾病和 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以下假设：这些等位基因对多能祖细胞阶段（MPP 或 LMPP）的细胞命运决定具有不同的影响，其中 FLT3 在造血发育过程中高度表达。我们将尝试了解与 FLT3 WT 相比，FLT3ITD 是 STAT5 的有效激活剂的机制，利用突变消除这种活性。在具体目标 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作用。这些模型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。在具体目标 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，并作为测试新型 JAK2 抑制剂的平台，用于项目 5 中临床试验的开发。总体而言，这是一个高度互动的项目，将建立在髓系新疗法的成功和临床前开发的成功记录之上 恶性肿瘤。 SA 1.使用敲入策略由突变FLT3介导的准确的白血病模型的生成和表征。我们将描述每个等位基因的表型。 一个。 FLT3-ITD 条件敲入等位基因的生成和表征 b. FLT3 D835Y 和 I836del 条件敲入等位基因的生成和表征 c.生成 FLT3-ITD Y589F/Y598F 条件敲入等位基因，该等位基因向 STATS 发出信号有缺陷 SA 2. 表征 FLT3-ITD 介导疾病的这些精确基因型模型与其他种系等位基因杂交的协同效应 a. FLT3-ITD KI 与组织蛋白酶 G PML-RARalpha 和 C/EBPalpha 敲入等位基因杂交（与 Tenen Project 3 的相互作用） b. FLT3-ITD I836del KI 与 MIL 融合等位基因杂交（与 Armstrong 相互作用，项目 4） c. FLT3-ITD KI 与 AML1-ETO 条件 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 SA 3. 开发 JAK2V617F 介导的 MPD 的精确小鼠模型 a.开发并表征 JAK2V617F 疾病的逆转录病毒转导模型 b.生成并表征 JAK2V617F 条件敲入等位基因 c.表征 JAK2V617F 疾病（包括 MPL）的新型增强突变 d.表征项目 1 中开发的小鼠模型中的新型 JAK2 抑制剂

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