Biology and Prognostic Implications of FLT3 Mutations in AML

描述（由申请人提供）：AML是一种高度侵袭性的白血病类型，由其基因组复杂性定义。FLT 3基因突变（FLT 3/ITD或FLT 3/ALM）是AML中最常见的体细胞突变，FLT 3/ITD患者的失败风险高，预后差。我们已经确定了FLT 3突变在AML中的临床意义，并使用FLT 3突变状态实施了基于风险的治疗分配。尽管FLT 3抑制剂最初可用作靶向FLT 3突变的定向治疗，但耐药性的快速演变以及由此导致的FLT 3抑制剂缺乏生存获益，需要评价FLT 3突变患者的失败原因和耐药性机制。确定耐药机制并设计策略来规避或治疗耐药表型将有助于将这种最具侵略性的AML类型转化为可治愈的AML。我们对FLT 3突变的生物学和预后意义的研究涉及几种耐药介质，包括获得性单亲二体性（aUPD）的演变，新获得的继发性突变，以及ITD的结构变异（ITD长度和新型FLT 3转录变体），需要进行评估和确认。这项资助的总体目标是建立在过去5年产生的数据的基础上，进一步了解FLT 3生物学和抗病机制，以便设计出更好地靶向该基因用于治疗目的的方法。我们已经创建了特定的实验工具并收集了患者资源来帮助这一奋进，包括生成两种索拉非尼耐药细胞系，克隆不同ITD片段长度的FLT 3/ITD，以及创建对FLT 3抑制剂或常规化疗难治的原发性患者标本库。在这项提案中，我们的目标是初步研究AML中新发现的疾病相关FLT 3转录变体。这些新变体（隐藏和跳跃外显子）具有改变基因功能并影响其生物学和临床意义的显著潜力，并且对FLT 3抑制剂有反应。此外，我们将利用实验室产生的耐药细胞系以及复发/难治患者标本，通过全基因组测序，筛选可能导致耐药性出现的新基因组改变。使用高通量shRNA筛选将鉴定可用于规避耐药性或使耐药细胞再致敏的基因和途径。最后，我们已经证明，在FLT 3/ITD患者中，aUPD和ITD长度与复发高度相关。我们将使用AAV介导的基因靶向以杂合方式敲入不同长度的ITD，并确定FLT 3/ITD的存在是否足以进化aUPD。此外，使用设计的慢病毒载体，我们将评估ITD长度在赋予耐药性中的作用。该提案的数据将为FLT 3突变型AML的耐药机制提供重要见解，并可能导致新的工具纳入临床试验和识别可用于治疗目的的生物学途径。

DESCRIPTION (provided by applicant): AML is a highly aggressive type of leukemia that is defined by its genomic complexity. Mutations of the FLT3 gene (FLT3/ITD or FLT3/ALM) are the most common somatic mutations in AML, and those with FLT3/ITD have a high risk of failure and poor outcome. We have established the clinical implications of FLT3 mutations in AML and have implemented risk-based therapy allocation using FLT3 mutation status. Despite initial utility of FLT3 inhibitors as directed therapy targeting FLT3 mutations, rapid evolution of drug resistance and resultant lack of survival benefit of FLT3 inhibitors requires evaluation of cause of failure and mechanism of drug resistance in patients with FLT3 mutations. Defining the mechanism of drug resistance and devising strategies to circumvent or treat the resistant phenotype would help convert this most aggressive type of AML into a curable one. Our studies into the biology and prognostic significance of FLT3 mutations have implicated several mediators of resistance, including evolution of acquired uniparental disomy (aUPD), newly acquired secondary mutations, as well as structural variation of the ITD (ITD length and novel FLT3 transcript variants) that need to be evaluated and confirmed. The overarching goal of this grant is to build on the data generated in the last 5 years to further our understanding of FLT3 biology, and mechanism of disease resistance in order to devise means of better targeting this gene for therapeutic purposes. We have created specific experimental tools and collected patient resources to aid in this endeavor, including generation of two sorafenib resistant cell lines, cloning of FLT3/ITDs of different ITD fragment lengths and creation of repository of primary patients specimens refractory to FLT3 inhibitors or to conventional chemotherapy. In this proposal, we aim to initially investigate the newly discovered disease-associated FLT3 transcript variants in AML. These novel variants (cryptic and skipped exons) have significant potential to alter the function of the gene and impact its biologic and clinical significance and is response to FLT3 inhibitors. In addition, we will utilize our laboratory generated resistant cell lines as well as our relapsed/refractor patient specimens and thru whole genome sequencing, screen for novel genomic alterations that may have led to the emergence of drug resistance. Using high throughput shRNA screening will identify genes and pathways that can be used to circumvent drug resistance or re-sensitize the resistant cells. Finally, we have demonstrated that in patients with FLT3/ITD, aUPD and ITD length are highly associated with relapse. We will use AAV-mediated gene targeting to knock in ITDs of different lengths in a heterozygous fashion and determine whether presence of FLT3/ITD is sufficient for evolution of aUPD. In addition, using designed lentiviral vectors, we will assess the role of ITD length in conferring drug resistance. The data from this proposal will provide significant insight into the mechanism of drug resistance in FLT3 mutant AML and is likely to lead to new tools for incorporation into clinical trials and identification of biologic pathways that can be targeted for therapeutic purposes.