PROJECT SUMMARY Diffuse Large B cell Lymphoma (DLBCL) is incurable in ~30% of patients and, despite recent advances in CAR- T cell therapies, remains a significant clinical challenge. One barrier to rationally targeted new therapies is the remarkable heterogeneity of these tumors, which leaves as many as 20-50% of cases unclassified based on cell-of-origin or more recent genetic-based classifications. This may be due in part to the fact that current taxonomies are limited to the analysis of coding regions, representing only 3% of the genome, while further genetic complexity of pathogenetic relevance may reside in the non-coding regulatory portion of the genome. To this end, we recently investigated whether critical regulatory domains such as enhancers and super-enhancers (SEs) could be the site of functionally relevant mutations in DLBCL. We found that regions corresponding to active SEs are highly and specifically hypermutated in 97% of DLBCL cases, as compared to the same loci when not active as SE. Such aberrant SE hypermutation displays signatures of AID activity and is linked to genes encoding B cell regulators and well-established oncogenes. As evidence of oncogenic relevance, we showed that the hypermutated SEs linked to the BCL6, BCL2, and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (BCL6) and the steroid-receptor NR3C1 (BCL2 and CXCR4). Of note, CRISPR/Cas9-mediated correction of the SE hotspot mutation restored target gene regulation and impaired cell growth, indicating a key role for the SE mutation in maintaining the transformed phenotype (Bal et al., Nature 2022). Overall, these findings identify a highly pervasive, pathogenetically relevant, mutational mechanism that is likely to significantly influence the current understanding of the somatic genetic landscape of DLBCL. The overall goal of this research program will be to: i) identify and mechanistically dissect the top recurrently mutated/functionally relevant SEs and associated target genes; ii) understand the role of the glucocorticoid receptor pathway, which appears to be commonly targeted by the SE hypermutation mechanism as well as by direct coding mutations, in normal B cell biology and lymphomagenesis. We anticipate that this new layer of genetic alterations will identify novel mechanisms of dysregulation for known oncogenes, as well as new dysregulated genes and pathways, with implications for precision classification and therapeutic targeting of DLBCL.

NARRATIVE Diffuse-Large-B-cell-lymphoma (DLBCL) is the most common B-cell non-Hodgkin lymphoma and remains incurable in ~30% of patients. Coding-genome sequencing efforts identified several genes/pathways altered in this disease, including new potential therapeutic targets; however, the non-coding genome of DLBCL remains largely unexplored. This proposal builds on our recent discovery of a highly pervasive, pathogenetically relevant mutational mechanism targeting super-enhancers in DLBCL that is likely to significantly influence the current understanding of the somatic genetic landscape of this disease, by identifying novel mechanisms of dysregulation for known oncogenes, as well as new dysregulated genes and pathways, with implications for precision classification and therapeutic targeting of DLBCL.