Identification of Selective Enhancer Dependencies in MYC-Intact and MYC-Rearranged Germinal Center B-cell Diffuse Large B-cell Lymphoma [4C-seq]. Identification of Selective Enhancer Dependencies in MYC-Intact and MYC-Rearranged Germinal Center B-cell Diffuse Large B-cell Lymphoma [4C-seq]

High expression of MYC and its target genes define a subset of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) associated with poor outcomes. Half of these high-grade cases show chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, while focal deletions of the adjacent non-coding gene PVT1 are enriched in MYC-intact cases. To identify genomic drivers of MYC activation, we used high-throughput CRISPR-interference (CRISPRi) profiling of candidate enhancers in the MYC locus and rearrangement partner loci in GCB-DLBCL cell lines and mantle cell lymphoma (MCL) comparators that lacked common rearrangements between MYC and immunoglobulin (Ig) loci. Rearrangements between MYC and non-Ig loci were associated with unique dependencies on specific enhancer subunits within those partner loci. Notably, fitness dependency on enhancer modules within the BCL6 super-enhancer (BCL6-SE) cluster regulated by a transcription factor complex of MEF2B, POU2F2, and POU2AF1 was higher in cell lines bearing a recurrent MYC::BCL6-SE rearrangement. In contrast, GCB-DLBCL cell lines without MYC rearrangement were highly dependent on a previously uncharacterized 3’ enhancer within the MYC locus itself (GCBME-1), that is regulated in part by the same triad of factors. GCBME-1 is evolutionarily conserved and active in normal germinal center B cells in humans and mice, suggesting a key role in normal germinal center B cell biology. Finally, we show that the PVT1 promoter limits MYC activation by either native or heterologous enhancers and demonstrate that this limitation is bypassed by 3’ rearrangements that remove PVT1 from its position in cis with the rearranged MYC gene. Overall design: 4C-Seq chromatin topology mapping for the MYC promoter and BCL6 promoter and super-enhancer in three DLBCL cell lines

MYC及其靶基因的高表达可界定一类生发中心B细胞型弥漫性大B细胞淋巴瘤（germinal center B-cell diffuse large B-cell lymphoma, GCB-DLBCL）亚群，该亚群与不良预后相关。此类高级别病例中，半数存在MYC基因座与携带异源增强子的基因座之间的染色体重排，而邻近非编码基因PVT1的局灶性缺失在MYC完整（未发生重排）的病例中更为富集。为鉴定MYC激活的基因组驱动因素，我们对GCB-DLBCL细胞系以及缺乏MYC与免疫球蛋白（immunoglobulin, Ig）基因座常见重排的套细胞淋巴瘤（mantle cell lymphoma, MCL）对照细胞系中的MYC基因座及重排伴侣基因座的候选增强子进行了高通量CRISPR干扰（CRISPR-interference, CRISPRi）筛选。MYC与非Ig基因座的重排，与这些伴侣基因座内特定增强子亚基的独特依赖性相关。值得注意的是，携带复发性MYC::BCL6超级增强子（BCL6 super-enhancer, BCL6-SE）簇重排的细胞系，对由MEF2B、POU2F2及POU2AF1组成的转录因子复合物调控的BCL6-SE簇内增强子模块的适应性依赖性更高。与之相反，未发生MYC重排的GCB-DLBCL细胞系，高度依赖MYC基因座自身内一个此前未被表征的3'端增强子（GCBME-1），该增强子部分受上述三因子复合物调控。GCBME-1在人类与小鼠的正常生发中心B细胞中具有进化保守性且活性活跃，提示其在正常生发中心B细胞生物学过程中发挥关键作用。最后，我们证实PVT1启动子可通过内源或异源增强子限制MYC的激活，并证明将PVT1从其与重排后MYC基因的顺式位置移除的3'端重排，可绕过这一限制机制。整体实验设计：对3株DLBCL细胞系中的MYC启动子、BCL6启动子及超级增强子进行4C-Seq染色质拓扑结构图谱分析。