Identifying Functional Drivers of MYC Activation via Developmental Enhancers in Diffuse Large B-cell Lymphoma

项目摘要/摘要 癌基因MYC的过度表达是大多数高危弥漫性大B细胞淋巴瘤的特征，并且是 对淋巴瘤模型的生存至关重要。然而，只有一半的DLBCL被分配到高危亚组 生发中心样蛋白(GCB)-DLBCL的基因表达谱研究 在另一个常见的高危亚型ABC-DLBCL中，MYC和MYC重排的频率甚至更低。 目前对导致MYC转录失调的机制了解很少。 缺乏MYC和免疫球蛋白基因之间激活的基因组重排的DLBCL 基因座(MYC-IG)。这项建议的总体目标是定义远端MYC调控的新机制 增强剂。我们的长期目标是利用这种理解来开发改进的治疗策略和/或 DLBCL患者的预测生物标志物。我们的工作假设是MYC的转录激活 维持DLBCL所需的依赖于少数必需远端的顺式调节活动 位于更大的“超级增强器”区域内的增强器模块。这些模块位于 在8q24上MYC基因座的3‘区域内(在DLBCL中没有MYC重排)，或在基因组中 重排伙伴基因座，如3q27，这是最常见的非IG MYC重排之一 合伙人。我们将利用高吞吐量CRISPR干扰分析来执行可靠的功能询问 在缺乏MYC的DLBCL细胞系中，存在于8q24和3q27中的复杂的、多模块的“超级增强子” 重排(目标1)，或分别承担t(3；8)(q27；q24)重排(目标2)。在确定了 MYC激活所需的离散末端增强子元件，我们将利用一组互补的 实验方法，包括增强子序列的基因缺失或突变，染色质 免疫沉淀、报告分析、体外DNA-蛋白质结合分析以及反式因子和 上游途径，以表征这些元素的调节。我们的初步数据表明， 3‘MYC增强子区域内的增强子模块对于具有不同像差的DLBCL是必不可少的 影响选择性结合基本增强子模块的反式因子。这些像差包括不同的 激活核因子-kB因子的信号和遗传事件，或者是共激活因子OCA的过度激活- B和一组协同作用的发育转录因子。我们将评估在多大程度上 T(3；8)重排背景下MYC的激活依赖于MEF2B调节的“劫持” 增强子模块，通常负责癌基因BCL6的激活。成功后 完成拟议的研究后，我们预计将严格定义多种不同的机制，通过这些机制 增强子和相关的反式因子可以驱动DLBCL中MYC的转录激活。我们的创新 通过复杂的多模块末端识别癌基因激活的关键机制 增强剂可以作为在多种癌症类型中进行类似研究的模型。

PROJECT SUMMARY / ABSTRACT Overexpression of the oncogene MYC is characteristic of most high-risk diffuse large B-cell lymphomas, and is essential for the survival of lymphoma models. However, only half of DLBCLs assigned to high-risk subgroups of germinal center-like (GCB)-DLBCL by gene expression profiling bear activating genomic rearrangements of MYC, and MYC rearrangements are even less frequent in another common high-risk subtype, ABC-DLBCL. There is currently a poor understanding of the mechanisms that lead to MYC transcriptional dysregulation in DLBCL in the absence of an activating genomic rearrangement between MYC and an immunoglobulin gene locus (MYC-IG). The overall objective of this proposal is to define novel mechanisms of MYC regulation by distal enhancers. Our long-term goal is to use this understanding to develop improved therapeutic strategies and / or predictive biomarkers for DLBCL patients. Our working hypothesis is that transcriptional activation of MYC required to sustain DLBCL is dependent on the cis-regulatory activity of a small number of essential distal enhancer modules, located within much larger “super-enhancer” regions. These modules are located either within the 3’ region of the MYC locus on 8q24 (in DLBCL without MYC rearrangement), or in a genomic rearrangement partner locus such as 3q27, which is among the most common non-IG MYC rearrangement partners. We will utilize high-throughput CRISPR-interference profiling to perform robust functional interrogation of complex, multi-modular “super-enhancers” present in 8q24 and 3q27 in DLBCL cell lines that lack MYC rearrangements (Aim 1), or bear t(3;8)(q27;q24) rearrangements (Aim 2) respectively. Having identified the discrete distal enhancer elements that are required for MYC activation, we will utilize a complementary set of experimental approaches, including genetic deletion or mutation of enhancer sequences, chromatin immunoprecipitation, reporter assays, in vitro DNA-protein binding assays, and perturbation of trans factors and upstream pathways to characterize the regulation of these elements. Our preliminary data suggests that distinct enhancer modules within the 3’ MYC enhancer region are essential for DLBCLs with different aberrations affecting trans factors that selectively bind the essential enhancer modules. These aberrations include diverse signaling and genetic events that activate NF-kB factors, or alternately, hyperactivation of the coactivator OCA- B and a set of synergistically acting developmental transcription factors. We will evaluate the extent to which MYC activation in the context of a t(3;8) rearrangement is dependent on “hijacking” of MEF2B-regulated enhancer modules that are typically responsible for activation of the oncogene BCL6. Upon successful completion of the proposed research, we expect to rigorously define multiple distinct mechanisms by which enhancers and associated trans-factors can drive MYC transcriptional activation in DLBCL. Our innovative approach for identifying the key mechanisms underlying oncogene activation by complex multi-modular distal enhancers may serve as a model for similar investigations in a wide variety of cancer types.