Identifying Selective Enhancer Dependencies in MYC-intact and MYC-Rearranged Germinal Center B-cell Diffuse Large B-cell Lymphoma
Creators: name: Iyer, Ashwin
Publisher: name: My University
Year: 2023
Subjects: subject: CRISPRi screens
subject: Enhancer hijacking rearrangements
subject: Germinal Center B-cell Diffuse Large B-cell Lymphoma
subject: Pathology
subject: Health Sciences

description: A recently defined subgroup of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) is characterized by strong expression of MYC and MYC-regulated genes, leading to poor clinical outcomes with current treatment protocols. A significant proportion of these cases exhibit large-scale chromosomal rearrangements between the MYC locus and distal enhancer-bearing loci while focal deletions of the adjacent non-coding gene PVT1 are enriched in MYC intact cases. While both the MYC locus and partner loci display broad clusters of accessible and acetylated chromatin elements known as "super-enhancers," the specific elements within these regions that sustain MYC expression and lymphoma fitness remain largely unknown. To investigate enhancer dependencies, we conducted high-throughput CRISPR-interference functional profiling (CRISPRi) focusing on individual enhancer modules centered on nucleosome-free regions (NFRs) across the MYC topological domain (TAD) and rearrangement partner loci. This profiling involved MYC-intact and MYC-rearranged GCB-DLBCL cell lines, as well as comparator mantle cell lymphoma (MCL) cell lines. By quantitatively comparing enhancer fitness effect sizes and examining trans-factor dependencies, we gained insights into the essential elements and regulatory factors involved. This study identified the GCB MYC enhancer 1 (GCBME-1), located downstream of the noncoding RNA PVT1, as the most crucial distal enhancer within the MYC locus in MYC-intact GCB-DLBCL cell lines. Sustained MYC expression in these cell lines relied on the activity of GCBME-1, which was directly bound by essential transcription factors (TFs) such as MEF2B, POU2F2, and POU2AF1. Notably, GCBME-1 exhibited chromatin accessibility and acetylation in primary GCB-DLBCL patient samples and normal germinal center B-cells, suggesting its role in hyper-activating physiological MYC enhancers. In contrast, GCBME-1 was non-essential in MYC-rearranged DLBCL cell lines. We also observed strong dependencies on enhancers within cell-line-specific partner regions in GCB-DLBCL and MCL cell lines with intrachromosomal rearrangements of chromosome 8, supporting the notion of enhancer hijacking by these rearrangements. Furthermore, we identified recurrent t(3;8)(q27;q24) "pseudo-double-hit" rearrangements in three GCB-DLBCL cell lines, connecting the MYC gene to a distal super-enhancer region upstream of BCL6. While distal BCL6 super-enhancers have been implicated in sustaining the essential BCL6 oncogene, our findings revealed weak dependency on these enhancers in MYC-rearranged GCB-DLBCL cell lines lacking t(3;8) rearrangements. In contrast, GCB-DLBCL cell lines with t(3;8) rearrangements showed strong dependency on specific NFRs within the distal BCL6 super-enhancer, as validated by low-throughput CRISPRi assays. These enhancers were crucial for sustaining the expression of both MYC and BCL6 in t(3;8)-bearing cell lines. 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.In summary, our study uncovers novel mechanisms of MYC activation in both MYC-rearranged and non-rearranged GCB-DLBCL, highlighting the critical role of alternate pathways in shaping functional dependencies in GCB-DLBCL.