Functions of the TG-Interacting Transcription Factor (TGIF) in Hematopoiesis

描述(申请人提供)：我们发现，干细胞表达的转录抑制因子转化生长干扰因子(TGIF)的定量表达是急性髓细胞白血病(AML)患者生存的最具区分性和最强大的预测因子。较低的肿瘤生长因子水平与较短的生存期相关，而较高的水平预示较长的生存期(p=0.00001)。为了了解我们临床发现的生物学基础，我们研究了TGIF在髓系祖细胞系和TG/‘f基因敲除小鼠模型中的功能。在髓系祖细胞系，如HL60中，TGIF基因敲除导致显著的生长抑制、相对分化障碍和髓系转录程序中断。对TG/7基因缺失小鼠的造血干细胞(HSC)功能的分析表明，与野生型HSC相比，在竞争性移植研究中，HSC产生的克隆形成单位更少，并且具有生长优势。这些数据表明，Tg/f基因敲除可能改变了HSC的静止、分化和自我更新等关键功能之间的平衡。因此，我们假设TGIF在HSC功能中起着基本的调节作用，并将通过1)更彻底地定义TGIF的HSC和祖细胞功能的作用；2)确定TGIF对HSC作用的细胞学基础；以及3)确定TGIF对HSC作用的分子基础来检验这一假说。这些目标将通过使用各种标准和最先进的分子技术进行一系列实验，包括限制稀释竞争再种群分析、连续移植、体内细胞周期分析，以及使用自然发生的TGIF突变和体外HSC转导进行结构-功能表征。除了目前使用的TGIF基因敲除模型外，我们还将采用新的条件性TGIF基因敲除等位基因，这将为我们的实验设计提供极大的灵活性。这项提案还概述了一项旨在帮助候选人在学术医学领域发展成功的计划。应聘者拥有范德比尔特大学微生物学和免疫学博士学位，并已完成儿科住院医师培训和遗传学研究员培训。候选人将在斯蒂芬·勃兰特和斯科特·希伯特博士的指导下扩大他在造血和白血病发生方面的知识。指导委员会将定期提供额外的指导。相关性(见说明)：在急性白血病中，包括急性髓系白血病，造血干细胞静止、自我更新和分化之间的正常平衡被破坏。我们的初步数据表明，TGIF可能调节这种平衡，它也作为AML的预后指标具有临床作用。因此，我们对TGIF如何调控HSC关键功能的研究将为HSC生物学提供新的见解，并增加我们对AML发病机制的理解，从而更好地诊断和治疗这种毁灭性的疾病。

DESCRIPTION (provided by applicant): We have discovered that quantitative expression of the stem cell expressed transcriptional represser Transforming-growth-interfering factor (TGIF) is the most discriminating and powerful predictor of patient survival identified in acute myelogenous leukemia (AML). Lower levels of TGIF correlated with shorter survival, whereas higher levels predicted longer survival (p=0.00001). To understand the biological basis for our clinical finding, we investigated TGIF's function in myeloid progenitor cell lines and in a Tg/'f knockout mouse model. TGIF knockdown in myeloid progenitor cell lines, such as HL60, resulted in significant growth inhibition, a relative differentiation block, and disruption of the myeloid transcription program. Analysis of hematopoietic stem cell (HSC) function in Tg/7-null mice showed that the HSCs produced fewer colony- forming units and had a growth advantage compared to wild-type HSCs in competitive transplant studies. These data suggest that Tg/f knockout may alter the balance between the key HSC functions of quiescence, differentiation and self-renewal. We thus hypothesize that Tgif plays a fundamental regulatory role in HSC function and will examine this hypothesis by 1) defining more thoroughly the role of Tgif'm HSC and progenitor cell function; 2) determining the cellular basis of Tgif's effects on HSCs; and 3) determining the molecular basis of Tgif's effects on HSCs. These aims will be carried out in a series of experiments using a variety of standard and state-of-the-art molecular techniques, including limiting dilution competitive repopulation analysis, serial transplantation, in vivo cell cycle analysis, and structure-function characterizations using naturally occurring TGIF mutations and ex vivo HSC transduction. In addition to the Tgif knockout model currently in use, we will also employ a new conditional Tgif knockout allele, which will offer us great flexibility in our experimental design. This proposal also outlines a plan designed to help the candidate develop a successful career in academic medicine. The candidate has a Ph.D. in Microbiology and Immunology from Vanderbilt University and has completed residency training in Pediatrics and fellowship training in Genetics. The candidate will expandhis knowledge of hematopoiesis and leukemogenesis under the mentorship of Drs. Stephen Brandt and Scott Hiebert. A mentoring committee will provide added guidance at regular intervals. RELEVANCE (See instructions): In acute leukemias, including AML, the normal balance between quiescence, self-renewal and differentiation of HSCs is disrupted. Our preliminary data suggest that Tgif may regulate this balance and that it also has a clinical role as a prognostic indicator in AML. Thus, our investigations of how Tgif regulates key HSC functions will provide new insights into HSC biology as well as increase our understanding of AML pathogenesis, leading to better diagnosis and treatment of this devastating disease.