Non-Coding Mutations of BCL6 in Classical Hodgkin Lymphoma
Authors: Pirosa Maria Cristina , Salehi Matin , Bruscaggin Alessio , Terzi Di Bergamo Lodovico , Jauk Federico , Forestieri Gabriela , Bocchetta Simone , Piffaretti Deborah , Moia Riccardo , Cristaldi Vanessa , di Trani Martina , Galimberti Georgia Alice , Pini Katia , Spina Valeria , Giordano Claudia , Condoluci Adalgisa , Annunziata Salvatore , Bergesio Fabrizio , Boldorini Renzo , Borsatti Eugenio , Bulian Pietro , Chauvie Stephane , Cuzzocrea Marco , Gerber Bernhard , Kurlapski Michal , Larocca Luigi Maria , Rinaldi Andrea , Rodari Marcello , Romanowicz Grzegorz , Sacchetti Gian Mauro , Stathis Anastasios , Stüssi Georg , Zangrilli Ilaria , Calabretta Eleonora , Corrado Francesco , Pinto Antonello , Mazzucchelli Luca , Gattei Valter , Zaucha Jan Maciej , Santoro Armando , Hohaus Stefan , Cavalli Franco , Zucca Emanuele , Gaidano Gianluca , Carlo-Stella Carmelo , Tzankov Alexandar , Ceriani Luca , Rossi Davide 
Published: December 2024

Introduction: Non-coding regions, constituting a substantial part of the human genome, are crucial for gene regulation. In cancer, somatic non-coding regulatory mutations can significantly alter gene expression by either disrupting or creating transcription factor-binding sites. This phenomenon is notably well-known in diffuse large B-cell lymphoma (DLBCL), where activation-induced cytidine deaminase (AID) misfires during the germinal center (GC) reaction, leading to numerous non-coding mutations that disrupt gene regulation and contribute to malignancy. Classical Hodgkin lymphoma (cHL) is the most prevalent lymphoma among young adults. Although cHL originates from GC B cells, research into AID-associated hypermutation affecting non-coding regulatory regions has not yet been carried out.
Methods: We investigated somatic mutations in 155 coding genes and thirty-three genomic regions (mostly non-coding) known to be foci of AID-associated hypermutation and contained within regulatory regions of genes involved in GC B cell biology and transformation. ctDNA was analyzed using LyV4.0 ctDNA CAPP-seq in 297 cHL patients from the IOSI-EMA003-NCT03280394 and FIL-RougeBIO-NCT05066555 studies, and in 235 DLBCL for comparison.
Results: Mutations of in at least one of region of AID-associated hypermutation was detected in 83% of cHL, a prevalence comparable to that observed in DLBCL (88%). The region of AID-associated hypermutation within the BCL6 locus was the most frequently mutated, found in 56% of cHL cases. Other hypermutated regions occurring in &gt;10% of patients mapped in the SOCS1, CD83, RHOH, ST6GAL1, PAX5, BTG1, BCL7A, LRMP and HIST1H1E loci. We then scanned the sequenced non-coding spaces to detect recurrently mutated hotspots. This analysis involved evaluating overlapping 20 bp intervals through a sliding window of one bp while accounting for the nucleotide context. A hotspot located at chr3:187462671-187462703 of BCL6-intron 1 (designated as BCL6 Hotspot 7) was mutated in 30% of cases, a proportion that closely resembled that of DLBCL patients where it is a validated somatic expression quantitative trait locus (seQTL). BCL6 Hotspot 7 aligned with an area of accessible chromatin and heightened H3K27 acetylation in cHL, which was also nominated a super enhancer in 33% cHL cell lines. Moreover, ChIP-seq analysis of BCL6 Hotspot 7 revealed increase in H3K27ac in mutant cHL cell lines that suggested differential binding of transcription factors. BCL6 Hotspot 7 sequence predicted to bind the PRDM1 transcriptional repressor of BCL6, and mutations therein were found to impede BLIMP1 binding in cHL cells. Notably, BCL6 Hotspot 7 mutations were found to co-occur with BCL6 expression in cHL cell lines and HRS cells of primary biopsies, despite the co-expression of PRDM1. BCL6 protein expression ranging from weak to strong was detected in the nucleus of HRS cells of 68% of primary biopsies. The core set of genes that are directly bound and regulated by BCL6 exhibited similar expression levels in GC B cells and in BCL6 expressing cHL cell lines and in primary HRS cells. Furthermore, the core set of genes directly bound and regulated by BCL6 had a nearly identical chromatin accessibility patterns in GC B cells and in BCL6 expressing cHL cell lines. BCL6 protein degradation was observed with BI-3802 in four cHL cell lines expressing BCL6. After BCL6 degradation, the core set of genes directly bound and regulated by BCL6 was similarly derepressed in cHL cell lines as in the SU-DHL4 DLBCL cell line, indicating that BCL6 is involved in their repression in cHL as in DLBCL. Compared to the BI-5372 control molecule, treatment with BI-3802 significantly decreased proliferation in all cell lines where BCL6 degradation was observed, as well as in the BCL6-dependent SU-DHL4 DLBCL cell line, which was used as a control.
Conclusion: This study broadens the understanding of known oncogenic mechanisms in cHL development and identifies BCL6 as a vulnerability in a fraction of cHL and as a potential therapeutic target.