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。Flt3基因突变(Flt3/ITD或Flt3/ALM)是AML中最常见的体细胞突变，携带Flt3/ITD的患者失败风险高，预后差。我们已经确定了急性髓细胞白血病中Flt3突变的临床意义，并根据Flt3突变状态实施了基于风险的治疗分配。尽管最初使用Flt3抑制剂作为针对Flt3突变的定向治疗，但耐药的快速演变以及由此导致的Flt3抑制剂缺乏生存益处需要对Flt3突变患者的失败原因和耐药机制进行评估。明确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袭性的AML转变为可治愈的AML。我们对Flt3突变的生物学和预后意义的研究已经涉及到几个耐药的介体，包括获得性单亲二体(AUPD)的进化，新获得的次级突变，以及ITD(ITD长度和新的Flt3转录本变体)的结构变异，这些都需要评估和确认。这项资助的主要目标是建立在过去5年中产生的数据的基础上，以加深我们对Flt3生物学和抗病机制的理解，以便设计出更好地针对该基因的治疗目的。我们已经创建了专门的实验工具并收集了患者资源来帮助这一努力，包括产生两个对索拉非尼耐药的细胞系，克隆不同ITD片段长度的Flt3/ITD，以及创建对Flt3抑制剂或传统化疗无效的原发患者标本资料库。在这项提案中，我们的目标是初步研究在AML中新发现的与疾病相关的Flt3转录变体。这些新的变异体(隐含的和跳过的外显子)有很大的潜力改变基因的功能，影响其生物学和临床意义，并对Flt3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/折射患者的样本，通过全基因组测序，筛选可能导致耐药出现的新的基因组变化。使用高通量shRNA筛选将识别可用于规避耐药或使耐药细胞重新敏感的基因和途径。最后，我们已经证明，在FLT3/ITD患者中，aUPD和ITD长度与复发高度相关。我们将使用AAV介导的基因打靶，以杂合的方式敲入不同长度的ITD，并确定Flt3/ITD的存在是否足以促进aUPD的进化。此外，利用设计的慢病毒载体，我们将评估ITD长度在增加耐药性方面的作用。这项提案的数据将为了解Flt3突变AML的耐药机制提供重要的见解，并可能导致新的工具，用于纳入临床试验和识别可用于治疗目的的生物通路。

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.