Determining the role of WT and mutant FOXO1 in the transcriptional regulation of DLBCL

项目摘要 弥漫性大B细胞淋巴瘤(DLBCL)占非霍奇金淋巴瘤(NHL)的40%，有 对于对护理标准没有反应的患者，几乎没有治疗选择。转录因子FOXO1 在DLBCL亚型中反复突变(MFOXO1)，并在复发/难治患者中丰富。不像 其他肿瘤类型，DLBCL FOXO1突变似乎正在激活。而Foxo1在小鼠体内的缺失 证明了它是B细胞正常发育所必需的，这是假定的激活突变的后果 还没有被调查。重要的是，细胞系中的基因缺失和RNAi研究不能区分 由于缺乏时间分析(分析数天或数周)，直接和间接转录效应之间存在差异 稍后)。此外，先前确定FOXO1控制基因表达的机制的研究是 结果支离破碎，范围有限，结果相互矛盾，细胞类型特定。要研究FOXO1在 在DLBCL的背景下，我们已经使用CRISPR/Cas9开发了体外和体内模型，这将回答许多问题 关于FOXO1生物学的长期存在的问题。我们设计了WT和mFOXO1的内源基因座 使得FOXO1与FKBP12F36V和2XHA表位标签融合。这些细胞系将使我们能够迅速 灭活内源性FOXO1(FKBP12F36V)，并在2小时内进行新生转录分析 药物添加后确定mFOXO1的直接转录靶点。一个表位的结合 标记到内源FOXO1中还将允许我们生成健壮的Cut&Run库，以明确定义 结合的基因组基因座。我们将使用这些细胞的异种移植来确定突变的FOXO1是否具有治疗作用 活体靶标。我们还建立了一个在FOXO1和DLBCL相关突变的生殖系小鼠模型 将这些小鼠与Pten条件缺失小鼠进行杂交。这些小鼠将允许我们直接检验这一假说 MFOXO1逃逸激活的PI3K/AKT信号，维持生发中心的增殖。在目标1中， 我们将使用希伯特实验室中已经采用的技术和信息学平台来确定直接 FOXO1的转录靶点，决定FOXO1调控转录的机制，以及 评估FOXO1是否为治疗靶点。我们已经建立了含有mFOXO1的小鼠模型 并确定它对B细胞的发育造成了干扰。目标2将进一步询问 MFoxo1对生发中心反应的影响，并确定mFoxo1是否促进淋巴肿大。这项建议 将检验这样一种假设，即FOXO1突变使FOXO1成为一种结构性活性形式，这 持续激活生发中心亮区的靶基因，即使面对高PI3K 信号，以触发淋巴肿大。此外，该项目有可能发现新的治疗方法 MFOXO1 DLBCL中的目标。

Project Summary Diffuse large B-cell lymphoma (DLBCL) accounts for up to 40% of non-Hodgkins lymphoma (NHL) and there are few therapeutic options for patients who don’t respond to the standard of care. The transcription factor FOXO1 is recurrently mutated (mFOXO1) across DLBCL subtypes and enriched in relapsed/refractory patients. Unlike other tumor types, DLBCL FOXO1 mutations appear to be activating. While deletion of Foxo1 in mice demonstrated that it is required for proper B-cell development, the consequences of putative activating mutations have not been investigated. Importantly, genetic deletion and RNAi studies in cell lines cannot distinguish between direct and indirect transcriptional effects due to the lack of temporal analysis (analyzed days or weeks later). Furthermore, prior studies determining the mechanisms by which FOXO1 controls gene expression are fragmented, limited in scope, and the results are contradictory and cell type specific. To study FOXO1 in the context of DLBCL, we have developed in vitro and in vivo models using CRISPR/Cas9 that will answer a number of long-standing questions about FOXO1 biology. We engineered the endogenous loci for WT and mFOXO1 such that FOXO1 is fused to FKBP12F36V and a 2XHA epitope tag. These cell lines will allow us to rapidly inactivate the endogenous FOXO1 (FKBP12F36V) and use the analysis of nascent transcription over a 2hr time course after drug addition to identify the direct transcriptional targets of mFOXO1. The incorporation of an epitope tag into the endogenous FOXO1 will also allow us to generate robust Cut&Run libraries to unambiguously define the bound genomic loci. We will use xenografts of these cells to determine if mutant FOXO1 is a therapeutic target in vivo. We have also created a germline mouse model with a DLBCL-associated mutation in FOXO1 and crossed these mice with Pten-conditional deletion mice. These mice will allow us to directly test the hypothesis that mFOXO1 escapes activated PI3K/AKT signaling to maintain proliferation in the germinal center. In Aim 1, we will use techniques and informatic platforms already employed in the Hiebert lab to identify the direct transcriptional targets of FOXO1, determine the mechanism through which FOXO1 regulates transcription, and assess whether FOXO1 is a therapeutic target. We have already established mouse models containing mFOXO1 and determined that it caused a perturbation in B-cell development. Aim 2 will further interrogate the effects of mFOXO1 on the germinal center reaction and determine if mFoxo1 enhances lymphomagenesis. This proposal will test the hypothesis that FOXO1 mutations make a constitutively active form of FOXO1, which continuously activates target genes in the germinal center light zone, even in the face of high PI3K signaling, to trigger lymphomagenesis. Additionally, this project has the potential to identify novel therapeutic targets in mFOXO1 DLBCL.