Bertoni, Francesco Munz, Nicolas 2025 info:eu-repo/date/embargoEnd/2028-03-17 Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma. The R-CHOP immunochemotherapy is the current standard treatment, but at least one-third of patients do not respond or relapse after an initial response. The contribution of long non-coding RNAs (lncRNAs), such as enhancer-derived lncRNAs (eRNAs), to treatment failure and disease progression is poorly characterized. Here, we investigated the contribution of eRNAs in the response of DLBCL cells to R-CHOP using a CRISPR interference (CRISPRi) screening. We defined a DLBCL-specific eRNA collection using a newly developed bioinformatic approach for the reliable identification of eRNA in de novo reconstructed transcripts from RNA-seq data of 27 DLBCL cell lines, integrating patient expression data. A customized paired guide RNAs (pgRNAs) library was designed to target transcription start sites (TSS, n = 828) of eRNAs, including negative (n = 33) and positive controls (n = 90). We performed a CRISPRi screening in dCas9-ZIM3-KRAB stably expressing SU-DHL-16 DLBCL cells, which were exposed to an in vitro R-CHOP equivalent or DMSO for 9 and 14 days. Among pgRNAs showing differential enrichment between time zero, day 9, and 14, we identified U47924.31, a novel eRNA, which also showed prognostic impact in R-CHOP-treated DLBCL patients (HR = 0.79, 95% CI: = 0.64–0.97, p = 0.022, n = 300). The R-CHOP sensitizing activity of U47924.31 was further validated in additional DLBCL models and downregulation was identified in two out of three R-CHOP-resistant DLBCL cell lines derived from DoHH2, HBL-1, and OCI-Ly-10, compared to their parental counterparts, underscoring the importance of the eRNA in R-CHOP response and development of therapy resistance. Incorporating the differentially expressed genes of DLBCL cell lines and R-CHOP-treated DLBCL patients, the deregulation of U47924.31 was associated with gene sets mainly in cell cycle, metabolic, and immune response pathways, potentially indicating the R-CHOP sensitizing role of U47924.31 acting via the regulation of genes within these processes. Integrating publicly available data, a non-canonical open reading frame (ncORF) at the 5' end of U47924.31 was identified and associated with a small peptide product. Combinational treatment of DLBCL cells with the associated small peptide, together with R-CHOP, resulted in an increased therapeutic response, adding another layer of complexity to U47924.31 function and challenging the current definition of such transcripts as non-coding RNAs. Due to the function of U47924.31, we renamed the eRNA as REACTORID (R-CHOP-related enhancer-associated and coding-potential transcript optimizing response in DLBCL) and suggest its potential as clinical biomarker in DLBCL patients. application/pdf https://localhost:5000/ark:/12658/srd1335005 https://susi.usi.ch/global/documents/335005 https://susi.usi.ch/documents/335005/files/2025BIOMED008.pdf eng info:eu-repo/semantics/altIdentifier/ark/12658/srd1335005 info:eu-repo/semantics/altIdentifier/urn/urn:nbn:ch:rero-006-123786 info:eu-repo/semantics/embargoedAccess License undefined Lymphoma DLBCL ERNAs R-CHOP Therapy resistance Predictive biomarker NcORF Small peptide REACTORID info:eu-repo/classification/udc/61 Genetic CRISPRi screening identifies functional roles of enhancer RNAs in refractory diffuse large B cell lymphoma http://purl.org/coar/resource_type/c_db06