Role of a Novel Homeobox Transcription Factor (HLX) in Acute Myeloid Leukemia

描述（由申请人提供）：尽管多种化疗的使用和优化以及新药物的开发可以暂时减轻肿瘤负担，复发仍然是急性髓性白血病（AML）最常见的死亡原因。在目前的治疗方案下，只有不到三分之一的急性髓系白血病患者获得持久缓解，而且预后和风险分层仍然具有挑战性。需要确定新的目标以进行更有效和个性化的治疗干预。我们最近报道，在小鼠AML模型和绝大多数（87%）AML患者（包括分种干细胞）的白血病干细胞中，一种非聚集型同源盒基因HLX过表达，并且较高的HLX水平与AML患者较差的总生存率独立相关。功能研究表明，HLX过表达可导致分化受阻、序列克隆性无限的异常祖细胞形成，HLX敲低对体外和体内AML细胞生长均有抑制作用。然而，HLX的作用机制、其下游途径及其在白血病发生和维持中的作用尚不清楚。基于我们的发现和初步数据，我们假设HLX过表达是AML发病的早期步骤，并且HLX与其他常见的疾病等位基因（包括FLT3-ITD和CBFB-MYH11）协同作用，并通过特定的HLX依赖途径（包括PAK1和BTG1）起作用。我们进一步假设HLX直接转录调控AML中的靶基因，并且靶向HLX或功能关键的下游途径是抑制AML的合适的新方法。我们的具体研究目的是：1)研究HLX过表达在AML起始中的作用，包括HLX过表达与其他疾病等位基因的协同能力；2)研究降低HLX表达对体内AML维持的影响；3)确定AML中与HLX功能相关的直接转录靶点。我们将使用逆转录病毒共表达/移植试验，以及新开发的条件HLX敲入小鼠模型，研究HLX过表达与辅助因子（包括FLT3ITD和CBFB-MYH11）的白血病转化。我们将研究HLX抑制在原代人AML细胞和AML遗传小鼠模型中的抗白血病作用，并确定介导HLX下调白血病抑制作用的功能关键途径。我们将研究HLX是否直接调控候选下游基因PAK1和BTG1的转录。此外，我们将通过ChIP-seq确定全基因组hlx -染色质相互作用，并确定新的hlx调控靶点，并测试AML中的功能相关性。综上所述，基于我们的初步发现，本研究将探讨HLX在AML发病机制中的作用，以及如何利用HLX下调抑制白血病。这项研究的结果将增强我们对AML致病机制的认识，并将HLX及其下游途径定义为AML治疗的新靶点。

DESCRIPTION (provided by applicant): Despite the established use and optimization of polychemotherapy and the development of new agents that transiently reduce the tumor burden, relapse continues to be the most common cause of death in acute myeloid leukemia (AML). Less than one third of patients with AML achieve durable remission with current treatment regimens, and prognostication and risk stratification remain challenging. New targets need to be identified for more effective and individualized therapeutic intervention. We have recently reported that a non- clustered homeobox gene, HLX, is overexpressed in leukemic stem cells in a mouse AML model and in the vast majority (87%) of AML patients, including in sorted stem cells, and that higher HLX levels are independently associated with poor overall survival of AML patients. Functional studies showed that HLX overexpression leads to the formation of aberrant progenitors with blocked differentiation and unlimited serial clonogenicity, and that HLX knockdown has an inhibitory effect on AML cell growth in vitro and in vivo. However, the mechanism of action of HLX, its downstream pathways, and its role in leukemia initiation and maintenance are unknown. Based on our findings and preliminary data we hypothesize that HLX overexpression is an early step in AML pathogenesis and that HLX acts in concert with other common disease alleles, including FLT3-ITD and CBFB-MYH11, and through specific HLX-dependent pathways, including PAK1 and BTG1. We further hypothesize that HLX directly transcriptionally regulates target genes in AML, and that targeting HLX or functionally critical downstream pathways is a suitable, novel approach for the inhibition of AML. Our specific research aims are: 1) To study the role of HLX overexpression in the initiation of AML, including the ability of HLX overexpression to cooperate with other disease alleles; 2) To investigate the effect of reducing HLX expression on AML maintenance in vivo; 3) To identify functionally relevant, direct transcriptional targets of HLX in AML. We will study leukemic transformation by HLX overexpression in concert with cofactors, including FLT3ITD and CBFB-MYH11, using retroviral co-expression/transplantation assays, as well as a newly developed conditional Hlx knockin mouse model. We will study the anti-leukemic effects of HLX inhibition in primary human AML cells and a genetic mouse model of AML, and identify functionally critical pathways which mediate the leukemia-inhibitory effect of HLX downregulation. We will study whether HLX directly regulates transcription of candidate downstream genes PAK1 and BTG1. In addition, we will determine genome-wide HLX-chromatin interactions by ChIP-seq, and identify new HLX-regulated targets and test for functional relevance in AML. In summary, based on our initial discovery this study will investigate the function of HLX in AML pathogenesis, and how HLX downregulation can be utilized to inhibit leukemia. The results of this study will enhance our knowledge of disease-causing mechanisms in AML, and define HLX and its downstream pathways as novel targets for therapy in AML.