Selective Enhancer Dependencies in MYC-Intact and MYC-Rearranged Germinal Center B-cell Diffuse Large B-cell Lymphoma

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. Key points CRISPR-interference screens identify a conserved germinal center B cell MYC enhancer that is essential for GCB-DLBCL lacking MYC rearrangements. Functional profiling of MYC partner loci reveals principles of MYC enhancer-hijacking activation by non-immunoglobulin rearrangements.

[1]  Michael G. Kharas,et al.  BTG1 mutation yields supercompetitive B cells primed for malignant transformation , 2023, Science.

[2]  M. Chiang,et al.  ETV6 Deficiency Unlocks ERG-Dependent Microsatellite Enhancers to Drive Aberrant Gene Activation in B-Lymphoblastic Leukemia. , 2022, Blood cancer discovery.

[3]  Patrick C. Fiaux,et al.  Systematic discovery and functional dissection of enhancers needed for cancer cell fitness and proliferation , 2022, Cell reports.

[4]  Lei S. Qi,et al.  Nested epistasis enhancer networks for robust genome regulation , 2022, Science.

[5]  Ryan D. Morin,et al.  Super-enhancer hypermutation alters oncogene expression in B cell lymphoma , 2022, Nature.

[6]  G. Ciriello,et al.  Histone acetylation dynamics modulates chromatin conformation and allele-specific interactions at oncogenic loci , 2021, Nature Genetics.

[7]  Lisa E. Wagar,et al.  Integrated single-cell transcriptomics and epigenomics reveals strong germinal center-associated etiology of autoimmune risk loci , 2021, bioRxiv.

[8]  O. Elemento,et al.  Unique Immune Cell Coactivators Specify Locus Control Region Function and Cell Stage. , 2020, Molecular cell.

[9]  M. Yaspo,et al.  The Leukemogenic TCF3-HLF Complex Rewires Enhancers Driving Cellular Identity and Self-Renewal Conferring EP300 Vulnerability. , 2019, Cancer cell.

[10]  Ryan D. Morin,et al.  --The double hit signature identifies double-hit diffuse large B-cell lymphoma with genetic events cryptic to FISH. , 2019, Blood.

[11]  Neva C. Durand,et al.  Activity-by-Contact model of enhancer-promoter regulation from thousands of CRISPR perturbations , 2019, Nature Genetics.

[12]  Ryan D. Morin,et al.  Double-Hit Gene Expression Signature Defines a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma. , 2019, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[13]  S. Barrans,et al.  Molecular High-Grade B-Cell Lymphoma: Defining a Poor-Risk Group That Requires Different Approaches to Therapy , 2018, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[14]  Kendall R. Sanson,et al.  Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities , 2018, Nature Communications.

[15]  Ryan D. Morin,et al.  High-resolution architecture and partner genes of MYC rearrangements in lymphoma with DLBCL morphology. , 2018, Blood advances.

[16]  Stefano Monti,et al.  Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes , 2018, Nature Medicine.

[17]  Roland Schmitz,et al.  Genetics and Pathogenesis of Diffuse Large B‐Cell Lymphoma , 2018, The New England journal of medicine.

[18]  Andreas Trumpp,et al.  A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies , 2018, Nature.

[19]  Shawn M. Gillespie,et al.  A B Cell Regulome Links Notch to Downstream Oncogenic Pathways in Small B Cell Lymphomas. , 2017, Cell reports.

[20]  A. McKenna,et al.  FlashFry: a fast and flexible tool for large-scale CRISPR target design , 2017, bioRxiv.

[21]  Phillip G. Montgomery,et al.  Defining a Cancer Dependency Map , 2017, Cell.

[22]  R. Dalla‐Favera,et al.  Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia , 2017, Proceedings of the National Academy of Sciences.

[23]  Sharon R Grossman,et al.  Systematic mapping of functional enhancer–promoter connections with CRISPR interference , 2016, Science.

[24]  Olivier Elemento,et al.  Multi-tiered Reorganization of the Genome during B Cell Affinity Maturation Anchored by a Germinal Center-Specific Locus Control Region. , 2016, Immunity.

[25]  David M Sabatini,et al.  Single Guide RNA Library Design and Construction. , 2016, Cold Spring Harbor protocols.

[26]  A. Sabò,et al.  MYC: connecting selective transcriptional control to global RNA production , 2015, Nature Reviews Cancer.

[27]  Shawn M. Gillespie,et al.  Detection of Enhancer-Associated Rearrangements Reveals Mechanisms of Oncogene Dysregulation in B-cell Lymphoma. , 2015, Cancer discovery.

[28]  S. Spicuglia,et al.  [The European Blueprint project: towards a full epigenome characterization of the immune system]. , 2015, Medecine sciences : M/S.

[29]  Yijun Ruan,et al.  B Cell Super-Enhancers and Regulatory Clusters Recruit AID Tumorigenic Activity , 2014, Cell.

[30]  Jun S. Liu,et al.  MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens , 2014, Genome Biology.

[31]  Paolo Vineis,et al.  Genome-wide association study identifies multiple susceptibility loci for diffuse large B-cell lymphoma , 2014, Nature Genetics.

[32]  Teresa Palomero,et al.  A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia , 2014, Nature Medicine.

[33]  R. Spang,et al.  A recurrent 11q aberration pattern characterizes a subset of MYC-negative high-grade B-cell lymphomas resembling Burkitt lymphoma. , 2014, Blood.

[34]  Ming Yu,et al.  Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation , 2013, Genes & development.

[35]  M. Nussenzweig,et al.  The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry , 2012, Nature Immunology.

[36]  Amos Tanay,et al.  Robust 4C-seq data analysis to screen for regulatory DNA interactions , 2012, Nature Methods.

[37]  A. Ashworth,et al.  A genome-wide association study of Hodgkin Lymphoma identifies new susceptibility loci at 2p16.1 (REL), 8q24.21, and 10p14 (GATA3) , 2010, Nature Genetics.

[38]  W. Chan,et al.  BCL6 promoter interacts with far upstream sequences with greatly enhanced activating histone modifications in germinal center B cells , 2010, Proceedings of the National Academy of Sciences.

[39]  G. K. Davis,et al.  Phenotypic robustness conferred by apparently redundant transcriptional enhancers , 2010, Nature.

[40]  E. Campo,et al.  Common variants at 2q37.3, 8q24.21, 15q21.3, and 16q24.1 influence chronic lymphocytic leukemia risk , 2010, Nature Genetics.

[41]  F. Alt,et al.  Long-range Oncogenic Activation of IgH/c-myc Translocations by the IgH 3’ Regulatory Region , 2009, Nature.

[42]  D. Koller,et al.  The Immunological Genome Project: networks of gene expression in immune cells , 2008, Nature Immunology.

[43]  Katia Basso,et al.  Mutations of the BCL6 proto-oncogene disrupt its negative autoregulation in diffuse large B-cell lymphoma. , 2003, Blood.

[44]  Ash A. Alizadeh,et al.  Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling , 2000, Nature.

[45]  M. Neuberger,et al.  A second B cell-specific enhancer 3' of the immunoglobulin heavy-chain locus , 1990, Nature.

[46]  J. Banerji,et al.  A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes , 1983, Cell.