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）的高达40%，并且存在以下情况： 对标准治疗无效的患者的治疗选择很少。转录因子FOXO 1 在DLBCL亚型中反复突变（mFOXO 1），并在复发/难治性患者中富集。不像 在其他肿瘤类型中，DLBCL FOXO 1突变似乎是激活的。当小鼠Foxo 1基因缺失时， 证明了它是B细胞正常发育所必需的， 还没有被调查。重要的是，细胞系中的基因缺失和RNA干扰研究无法区分 由于缺乏时间分析（分析的天数或周数）， 稍后）。此外，先前确定FOXO 1控制基因表达的机制的研究是 碎片化，范围有限，并且结果是矛盾的和细胞类型特异性的。为了研究FOXO 1在 在DLBCL的背景下，我们已经开发了使用CRISPR/Cas9的体外和体内模型， 关于FOXO 1生物学的长期问题。我们设计了WT和mFOXO 1的内源性基因座， 使得FOXO 1与FKBP 12 F36 V和2XHA表位标签融合。这些细胞系将使我们能够迅速 扩增内源性FOXO 1（FKBP 12 F36 V），并使用2小时内的新生转录分析 药物添加后的过程，以确定mFOXO 1的直接转录靶点。表位的掺入 标记到内源FOXO 1中也将使我们能够生成强大的Cut&Run文库， 结合的基因位点。我们将使用这些细胞的异种移植物来确定突变FOXO 1是否是一种治疗方法。 体内靶向。我们还建立了一个在FOXO 1中具有DLBCL相关突变的种系小鼠模型， 将这些小鼠与Pten条件性缺失小鼠杂交。这些老鼠将使我们能够直接测试假设 mFOXO 1逃避激活的PI 3 K/AKT信号以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已经使用的技术和信息平台来识别直接的 FOXO 1的转录靶点，决定FOXO 1调节转录的机制，以及 评估FOXO 1是否是治疗靶点。我们已经建立了含有mFOXO 并确定它引起了B细胞发育的扰动。目标2将进一步探讨 mFOXO 1对生发中心反应的影响，并确定mFOXO 1是否增强淋巴瘤发生。这项建议 将测试FOXO 1突变使FOXO 1的组成型活性形式的假设， 持续激活生发中心光区的靶基因，即使面对高PI 3 K 信号传导，从而引发淋巴瘤此外，该项目有可能确定新的治疗方法， mFOXO 1 DLBCL中的靶点。

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.