Biology and Prognostic Implications of FLT3 Mutations in AML

FLT3 突变在 AML 中的生物学和预后意义

• 批准号: 8701243
• 负责人: SOHEIL MESHINCHI
• 金额: $ 41.62万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8701243
• 关键词: 13q; Acquired uniparental disomy; Adult Acute Myeloblastic Leukemia; Allogenic; Apoptosis; BAY 54-9085; Biological Assay; Biology; Cell Line; Cell Proliferation; Cells; Characteristics; Childhood Acute Myeloid Leukemia; Clinical; Clinical Trials; Cloning; Collaborations; Data; Development; Diagnosis; Diagnostic; Disease; Disease Resistance; Dose; Drug resistance; Evaluation; Event; Evolution; Exons; Exposure to; FLT3 gene; FLT3 inhibitor; Failure; Foundations; Gene Targeting; Generations; Genes; Genome; Genomics; Goals; Grant; In Vitro; Institutes; Knock-in Mouse; Knowledge; Laboratories; Lead; Length; Lentivirus Vector; Leukemic Cell; Libraries; Mediating; Mediator of activation protein; Molecular; Mono-S; Mutation; Outcome; Pathway interactions; Patients; Phase III Clinical Trials; Phenotype; Population; Prevalence; Protein Isoforms; Refractory; Relapse; Resistance; Resources; Risk; Role; Sampling; Somatic Mutation; Specimen; Stem cell transplant; Testing; Transcript; Variant; base; chemotherapy; clinically significant; cytokine; deep sequencing; design; drug sensitivity; effective therapy; exon skipping; gene function; genome sequencing; high risk; high throughput screening; insight; interest; leukemia; mutant; novel; prognostic; receptor; repository; resistance mechanism; response; screening; small hairpin RNA; structural genomics; therapeutic gene; therapeutic target; tool; transcriptome sequencing; vector

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.

描述（由申请人提供）：AML 是一种高度侵袭性的白血病，由其基因组复杂性决定。 FLT3 基因突变（FLT3/ITD 或 FLT3/ALM）是 AML 中最常见的体细胞突变，患有 FLT3/ITD 的患者失败和预后不良的风险很高。我们已经确定了 FLT3 突变对 AML 的临床影响，并根据 FLT3 突变状态实施了基于风险的治疗分配。尽管FLT3抑制剂最初用作针对FLT3突变的定向治疗，但耐药性的快速演变以及由此导致的FLT3抑制剂缺乏生存益处需要评估FLT3突变患者的失败原因和耐药机制。定义耐药机制并制定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 类型转化为可治愈的类型。我们对 FLT3 突变的生物学和预后意义的研究涉及多种耐药介质，包括获得性单亲二体性 (aUPD) 的进化、新获得的二次突变以及 ITD 的结构变异（ITD 长度和新的 FLT3 转录物变异）需要评估和确认。这笔赠款的总体目标是基于过去 5 年产生的数据，进一步加深我们对 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进行治疗的方法。我们创建了特定的实验工具并收集了患者资源来帮助实现这一目标，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 FLT3/ITD 以及创建对 FLT3 抑制剂或传统药物耐药的原发性患者样本库化疗。在本提案中，我们的目标是初步研究 AML 中新发现的与疾病相关的 FLT3 转录变异体。这些新的变异（隐秘和跳过的外显子）具有改变基因功能并影响其生物学和临床意义的巨大潜力，并且是对 FLT3 抑制剂的反应。此外，我们将利用我们实验室产生的耐药细胞系以及复发/难治患者标本，并通过全基因组测序，筛选可能导致耐药性出现的新基因组改变。使用高通量 shRNA 筛选将识别可用于规避耐药性或使耐药细胞重新敏感的基因和途径。最后，我们证明，在 FLT3/ITD 患者中，aUPD 和 ITD 长度与复发高度相关。我们将使用 AAV 介导的基因靶向以杂合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，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FLT3突变型AML的耐药机制提供重要见解，并可能带来新的工具，用于纳入临床试验和识别可针对治疗目的的生物途径。