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

确定 WT 和突变体 FOXO1 在 DLBCL 转录调控中的作用

• 批准号: 10617282
• 负责人: Hillary Margaret Layden
• 金额: $ 3.27万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2024-02-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10617282
• 关键词: Adopted; Affect; Alleles; Antibodies; Architecture; Attention; B-Cell Development; B-Lymphocytes; BCL6 gene; Binding; Biochemical; Biological; Biology; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cell model; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Coupling; DNA-Binding Proteins; Darkness; Data; Dependence; Development; Diagnosis; Dissection; Drug Targeting; Engineering; Epitopes; Event; FOXO1A gene; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Goals; Heterogeneity; Immunophenotyping; In Vitro; Informatics; Lead; Libraries; Light; Lymphoma; Lymphoma cell; Lymphomagenesis; Malignant lymphoid neoplasm; Mediating; Modeling; Molecular; Morphology; Mus; Mutate; Mutation; N-terminal; Non-Hodgkin' s Lymphoma; Nuclear; Oncogenic; Outcome; PI3K/AKT; PIK3CG gene; Pathogenesis; Patients; Pharmaceutical Preparations; Proliferating; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; Reaction; Recurrence; Refractory; Regulation; Relapse; Role; Running; Sampling; Signal Transduction; Structure of germinal center of lymph node; Tacrolimus Binding Proteins; Techniques; Testing; Therapeutic; Time; Transcriptional Activation; Transcriptional Regulation; Xenograft procedure; cell type; chemical genetics; genetic approach; genomic locus; in vivo; in vivo Model; ineffective therapies; knock-down; large cell Diffuse non-Hodgkin' s lymphoma; lenalidomide; mouse model; mutant; new therapeutic target; prevent; programs; small hairpin RNA; small molecule; standard of care; therapeutic target; transcription factor; tumor

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.

项目概要 弥漫性大 B 细胞淋巴瘤 (DLBCL) 占非霍奇金淋巴瘤 (NHL) 的 40%，并且有 对于对标准护理没有反应的患者来说，治疗选择很少。转录因子FOXO1 在 DLBCL 亚型中反复发生突变 (mFOXO1)，并且在复发/难治性患者中富集。不像 其他肿瘤类型中，DLBCL FOXO1 突变似乎正在激活。当小鼠中 Foxo1 缺失时 证明它是 B 细胞正常发育所必需的，是假定的激活突变的后果 尚未被调查。重要的是，细胞系中的基因删除和 RNAi 研究无法区分 由于缺乏时间分析（分析天或周），直接和间接转录效应之间存在差异 之后）。此外，先前的研究确定了 FOXO1 控制基因表达的机制 碎片化、范围有限、结果相互矛盾且针对细胞类型。研究 FOXO1 在 DLBCL 的背景下，我们使用 CRISPR/Cas9 开发了体外和体内模型，这将回答一些问题 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 FOXO1 与 FKBP12F36V 和 2XHA 表位标签融合。这些细胞系将使我们能够快速 灭活内源性 FOXO1 (FKBP12F36V) 并使用 2 小时内的新生转录分析 添加药物后的过程，以确定 mFOXO1 的直接转录靶标。表位的掺入 将标签添加到内源性 FOXO1 中还将使我们能够生成强大的 Cut&Run 库来明确定义 结合的基因组位点。我们将使用这些细胞的异种移植物来确定突变型 FOXO1 是否具有治疗作用 体内目标。我们还创建了 FOXO1 和 DLBCL 相关突变的种系小鼠模型 将这些小鼠与 Pten 条件缺失小鼠进行杂交。这些小鼠将使我们能够直接检验假设 mFOXO1 逃避激活的 PI3K/AKT 信号传导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 FOXO1 的转录靶标，确定 FOXO1 调节转录的机制，以及 评估 FOXO1 是否是治疗靶点。我们已经建立了含有mFOXO1的小鼠模型 并确定它会扰乱 B 细胞的发育。目标 2 将进一步探讨以下效果： mFOXO1 对生发中心反应并确定 mFoxo1 是否增强淋巴瘤发生。这个提议 将检验 FOXO1 突变形成 FOXO1 的组成型活性形式的假设，该形式 即使面对高 PI3K，也能持续激活生发中心光区的目标基因 信号传导，触发淋巴瘤发生。此外，该项目有可能确定新的治疗方法 mFOXO1 DLBCL 中的目标。