BTG2 SUPER‐ENHANCER MUTATIONS DISRUPT TFAP4 BINDING AND DYSREGULATE BTG2 EXPRESSION IN DIFFUSE LARGE B‐CELL LYMPHOMA

Introduction: Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy, remains incurable in ∼40% of patients. Coding-genome sequencing efforts identified several genes/pathways altered in this disease, as well as genetic subgroups of potential clinical relevance. However, the large non-coding portion of the genome remained largely unexplored. We recently identified a pervasive hypermutation mechanism targeting active super-enhancers (SEs) in >90% of DLBCL and leading to dysregulation of multiple genes, including well-known lymphoma oncogenes (Bal et al., Nature 2022). As evidence of oncogenic relevance, we demonstrated that mutational hotspots in the BCL6, BCL2 and CXCR4 SEs impair the binding of specific transcriptional repressors, preventing the gene negative regulation and creating oncogenic dependencies in DLBCL cells. Here we aimed to define the pathogenic role of mutations targeting the intragenic SE (iSE) of the BTG2 gene, the second most commonly mutated in DLBCL. BTG2 encodes a member of the B-cell translocation gene (BTG)/TOB family involved in transcriptional co-activation and modulation of mRNA abundance. BTG2 is also a recurrent target of somatic missense mutations (6%–11% of DLBCL), suggesting a major role in the pathogenesis of this disease. Methods: We screened 243 DLBCL cases for the presence of mutational hotspots within the BTG2-iSE, and combined in silico prediction, DNA-binding assays (Reverse-ChIP, electromobility-shift assay, ChIP-qPCR), RNA-seq and CRISPR/Cas9 editing approaches in isogenic BTG2 mutant versus WT DLBCL cell lines to identify transcription factors bound to the SE and disrupted by the mutation. Results: We identified a recurrent mutational cluster affecting the BTG2-iSE in 52/243 (21%) DLBCLs, with preferential enrichment in ST2 subgroup. CRISPR-Cas9 mediated correction of the mutation in 3 DLBCL cell lines led to counter selection and reduced BTG2 expression, consistent with oncogenic addiction. In silico motif prediction and in vitro DNA-binding assays, followed by validation in multiple isogenic DLBCL cell lines, identified TFAP4 as a major transcription factor that binds to the WT, but not to the mutated site. TFAP4 is an important regulator of B-cell proliferation and cell fate decisions, which can function as a transcriptional activator or repressor in germinal center B-cells, and acts downstream of/in parallel with c-MYC. Of note, introduction of SE hotspot mutations in WT DLBCL cells was associated with increased BTG2 expression, confirming a direct link between SE mutations and deregulated gene expression through escape from TFAP4-mediated suppression. Conclusions: These findings suggest a major role for BTG2 deregulation by SE aberrant somatic hypermutation in the pathogenesis and heterogeneity of DLBCL, with implications for precision classification and potential therapeutic targeting of DLBCL. Keywords: aggressive B-cell non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

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