Abstract PR04: Characterization of an essential MYC enhancer targeted by focal genomic alterations in diffuse large B-cell lymphoma

Abstract We sought to identify mechanisms that activate MYC expression in germinal center B cell- (GCB) diffuse large B-cell lymphomas (DLBCL) in the absence of MYC rearrangement. High-throughput CRISPR-interference screens in MYC-rearranged (n=4) and non-MYC-rearranged (n=2) GCB-DLBCL cell lines revealed a distal enhancer complex 436 kb downstream of MYC, GME-1, that was uniquely essential in non-rearranged (MYC-intact) cell lines. CRISPR-interference studies confirmed growth inhibition and MYC downregulation upon GME-1 repression in 4 DLBCL cell lines. ChIP-Seq showed that GME-1 subunits were bound by OCT2, OCA-B, and MEF2B, which are known to activate germinal center-specific enhancers, and knockdown of the corresponding genes reduced MYC expression. GME-1 subunits showed GCB-specific acetylation and accessibility across normal human B cell populations in public chromatin datasets. The mouse ortholog of GME-1 showed germinal center B cell-specific accessibility. GME-1 was strongly acetylated in MYC-intact but not MYC-rearranged GCB-DLBCL cell lines and patient biopsies. Hi-C revealed significant looping of GME-1 to the MYC promoter in MYC protein+, MYC-intact DLBCL biopsies (n=2), but not MYC-rearranged DLBCL (n=2). We used sequence conservation and chromatin accessibility data to define four GME-1 subunits (195A/B and196A/B), which were cloned into luciferase enhancer reporter constructs and compared to two subunits of the BCL6 distal super-enhancer complex that had previously been identified as regulated by the OCT2/OCA-B/MEF2B complex. GME-1 subunit 195B was the only strongly transcription-activating element based on luciferase assays across three MYC-intact cells lines, where it showed activity comparable to BCL6 enhancers. In contrast, GME-1 enhancers were minimally active in reporter assays performed in two MYC-rearranged GCB-DLBCL cell lines. Deletion of a conserved OCT2 binding site or knockdown of the POU2F2 and POU2AF1 genes reduced activity of GME-1. Thus GME-1 trans-regulation is divergent from that of previously characterized GCB enhancers, despite shared dependency on the OCT2 ternary complex. Whole-genome sequencing (WGS) revealed that the GME-1 dependent cell line SUDHL5 bears a 176bp tandem duplication within GME-1 195B. This altered enhancer showed stronger luciferase reporter activity across three MYC-intact cell lines. Published WGS datasets showed isolated genomic tandem copy gains of GME-1 in 2/49 biopsies from recurrent/refractory DLBCL and 1/93 unselected DLBCL. In a cohort of 18 DLBCL with the high-risk DZSig gene expression signature, GME-1 amplification was seen in two biopsies with concurrent deletion of the PVT1 promoter (PVT1-P) and no inter-chromosomal MYC locus rearrangement. CRISPRi showed that PVT1-P repression reduced growth and MYC expression in GME-1-dependent DLBCL, but not in cell lines where rearrangements removed PVT-P from its position in cis with the MYC gene. Our findings identify GME-1 as an activator of MYC expression and target of rare but recurrent genomic copy gains in high-risk DLBCL. Citation Format: Aishwarya Gurumurthy, Ashwin R. Iyer, Shih-Chun A. Chu, Marcin P. Cieslik, Russell J. H. Ryan. Characterization of an essential MYC enhancer targeted by focal genomic alterations in diffuse large B-cell lymphoma abstract. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5 (3Suppl): Abstract nr PR04.