Cytoplasmic FOXM1 contributes to the higher complete remission and longer overall survival of AML patients after chemotherapy

 描述(由申请人提供)：在过去的30年里，急性髓系白血病(AML)的结果仍然非常糟糕。20%-40%的患者诱导化疗未能达到缓解，50%-70%的完全缓解患者在3年内复发。虽然细胞遗传学是风险分层的支柱，但近50%的AML病例是细胞遗传学正常的(CN-AML)，并且在生物学和临床上是最异质性的群体。核质穿梭蛋白核磷蛋白(NPM1)在50%的CN-AML病例中发生突变，导致该蛋白的核输出。这已经在大量的临床队列中得到证明，可以提供良好的预后，并消除了这些患者进行骨髓移植的需要。在世卫组织2008年血液系统恶性肿瘤分类中，AML的这一子集已被确认为一个独特的诊断类别。突变的NPM1(NPM1mut)在CN-AML中赋予这种优势的机制尚不清楚。以前的假设是，NPM1mut将其他蛋白质伙伴错位到细胞质中，从而影响它们的功能。我们先前已经证明，FOXM1是一种癌基因转录因子，与NPM1在癌细胞中共定位。在这个方案中，我们将使用一种新的多光谱成像设备来研究FOXM1和NPM1在AML原代原始细胞中的细胞相互作用。我们还将利用AML细胞系来研究FOXM1在该病中的定位的功能意义。重要的是，我们将评估胞浆FOXM1错位与AML预后的临床相关性，目的是检验其作为CN-AML预后良好标志物的作用。我们打算证明FOXM1是决定AML预后的致癌驱动因素。NPM1的突变导致其核出口将FOXM1错误地定位到细胞质中，在那里它作为转录因子不活跃。这可能解释了在这一相当大的AML患者亚群中改善结果的原因。在几个AML细胞系中，我们将在体外和体内测试FOXM1基因敲除或FOXM1过表达如何影响化疗药物的敏感性。我们将确定在具有FOXM1核定位的AML细胞系中FOXM1基因敲除是否会使它们对化疗更敏感。将FOXM1基因敲除稳定的AML细胞株、FOXM1基因敲除稳定的OCI-AML3细胞株和FOXM1基因过表达的OCI-AML3细胞系分别接种到裸鼠体内，建立裸鼠移植瘤模型。我们预计，在U937和HL60 AML细胞系中，FOXM1基因敲除将增加对这些AML细胞系诱导的异种移植瘤治疗的敏感性。相比之下，抑制OCI-AML3细胞系中定位于细胞质的FOXM1不会影响该细胞系诱导的异种移植瘤对化疗的敏感性。我们建议中描述的实验将揭示胞浆FOXM1是否是AML的一个新的有利预后因素，以及FOXM1的核表达是否决定了AML细胞系诱导的异种移植瘤对化疗的耐药性。

 DESCRIPTION (provided by applicant): The outcomes for acute myeloid leukemia (AML) have remained abysmal for the past 30 years. 20- 40% of patients fail to achieve remission with induction chemotherapy, and 50-70% of patients who achieve a complete remission relapse within 3 years. While cytogenetics are the backbone of risk-stratification, nearly 50% of AML cases are cytogenetically normal (CN-AML) and, biologically and clinically, the most heterogenous group. The nucleo-cytoplasmic shuttle protein nucleophosmin (NPM1) is mutated in 50% of CN-AML cases resulting in the nuclear export of this protein. This has been shown in large clinical cohorts to confer a favorable prognosis and obviates the need for a bone marrow transplant in these patients. This subset of AML has been recognized in a unique diagnostic category in the WHO 2008 classification of hematologic malignancies. The mechanism by which mutated NPM1 (NPM1mut) confers this advantage in CN-AML is unclear. It has previously been hypothesized that NPM1mut dislocates other protein partners into the cytoplasm and consequently affecting their function. We have previously demonstrated that FOXM1, an oncogenic transcription factor, co-localizes with NPM1 in cancer cells. In this proposal, using a novel multispectral imaging modality we will study the cellular interaction of FOXM1 and NPM1 in AML primary blast cells. We will also investigate the functional significance of FOXM1 localization in this disease using AML cell lines. Importantly, we will assess the clinical correlation of mislocalized cytoplasmic FOXM1 with outcomes in AML with the goal of examining its role as a favorable prognostic marker in CN-AML. We intend to show that FOXM1 is the oncogenic driver dictating prognosis in AML. Mutations in NPM1 resulting in its nuclear export mislocalizes FOXM1 to the cytoplasm where it is inactive as a transcription factor. This may account for the improved outcome in this sizeable subset of AML patients. In several AML cell lines we will test how FOXM1 knockdown or FOXM1 overexpression will affect sensitivity to chemotherapeutic drugs in vitro and vivo. We will determine whether FOXM1 knockdown in AML cell lines with nuclear localization of FOXM1 makes them more sensitive to chemotherapy. Nude mice will be injected with AML cell lines with stable FOXM1 knockdown or OCI-AML3 cell line with stable FOXM1 knockdown or with FOXM1 overexpression, and with control cell lines to establish xenograft tumors. We expect that FOXM1 knockdown in U937 and HL60 AML cell lines will increase sensitivity to treatment in xenograft tumors induced by these AML cell lines. I contrast, suppression of FOXM1, localized in the cytoplasm, in OCI-AML3 cell line would not affect sensitivity to chemotherapy in xenograft tumors induced by this cell line. The experiments described in our proposal will reveal if cytoplasmic FOXM1 is a novel favorable prognostic factor in AML and whether nuclear expression of FOXM1 determines the resistance of xenograft tumors induced by AML cell lines to chemotherapy.