biostudies-arrayexpressVirginia de YébenesHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16241Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-cell lymphoma. Although many patients respond well to R-CHOP immunochemotherapy, those with the activated B-cell (ABC) subtype are often refractory or relapse. Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib have improved outcomes, but acquired resistance limits their long-term efficacy. Here, we modeled the development of ibrutinib resistance in ABC-DLBCL and investigated whether the BCR-signaling regulator microRNA-28 (miR-28) can block this process. Using flow cytometry–based competition assays, multicolor clonal barcoding, transcriptomic profiling, and xenograft models, we found that miR-28 expression impairs the emergence of ibrutinib-resistant ABC-DLBCL cells. Mechanistically, miR-28 interferes with the clonal selection process triggered by ibrutinib treatment and rewires transcriptional programs by downregulating mitochondrial and mTOR signaling pathways critical for resistance development. Furthermore, the miR-28–repressed gene signature associated with ibrutinib resistance correlates with improved survival in ibrutinib-treated patients from the PHOENIX trial cohort with the MCD genetic subtype, which is associated with ABC-DLBCL. Finally, the targeted therapeutic delivery of miR-28 via aptamer-guided nanoparticles suppresses ibrutinib-resistant tumor growth in vivo. These findings identify miR-28 as an effective inhibitor of ibrutinib resistance, underscoring its translational potential as an adjunct strategy in ABC-DLBCL therapy.biostudies-arrayexpressSequencing - Total RNA was extracted from fluorescence-activated cell sorted (FACS) RFP⁺ MD-901 multicolor-labeled (MCL) clones after three weeks of exposure to gradually increasing doses of ibrutinib (ranging from 10-fold below to 2.5-fold above the GR50). RNA extraction was performed using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA integrity and size distribution were evaluated using an Agilent Bioanalyzer. Poly(A)+ RNA was enriched with poly-T oligo-attached magnetic beads, followed by fragmentation, cDNA synthesis, end repair, adaptor ligation, uracil excision, and PCR amplification. Barcoded libraries were sequenced on a DNBSEQ platform (BGI, Shenzhen) to generate 150 bp paired-end reads. FASTQ files were produced by BGI for downstream bioinformatic analysis.Nucleic Acid Extraction - Total RNA was extracted from sorted cells using the miRNeasy Mini Kit (Qiagen) following the manufacturer’s protocol.Sample Collection - MD-901 multicolor-labeled (MCL) clones were cultured and exposed for three weeks to gradually increasing doses of ibrutinib (ranging from 10-fold below to 2.5-fold above the GR50). After treatment, three groups were collected: ibrutinib-sensitive (scr week 5 < week 1, n = 4), ibrutinib-resistant (scr week 5 > week 1, n = 4), and miR-28–expressing (n = 5) clones. At week 3, RFP⁺ cells were isolated by fluorescence-activated cell sorting (FACS) using a FACS Aria cell sorter for subsequent RNA extraction.Library Construction - RNA libraries for RNA-seq were prepared using DNBSEQ Low Input Smart-Seq Eukaryotic mRNA library following manufacturer’s protocols.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw RNA-seq reads were processed by BGI Genomics using their standard RNA-seq analysis pipeline. Reads were filtered with SOAPnuke to remove adaptors and low-quality sequences, and then aligned to the reference genome using HISAT2. Gene expression levels were quantified with RSEM and normalized as TPM (Transcripts Per Million). Processed data files contain normalized gene expression matrices (TPM values) in tab-delimited format. Annotation tables provide corresponding gene identifiers (Ensembl ID, gene symbol, and gene type).UnknownTranscriptomicsGenomicsProteomicsDNBSEQ-G400RNA-seq of coding RNAHomo sapiensVirginia de YébenesEmigdio Álvarez-CorralesfalseTargeting of ibrutinib resistance–driving pathways by miR-28 in ABC-DLBCLDiffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-cell lymphoma. Although many patients respond well to R-CHOP immunochemotherapy, those with the activated B-cell (ABC) subtype are often refractory or relapse. Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib have improved outcomes, but acquired resistance limits their long-term efficacy. Here, we modeled the development of ibrutinib resistance in ABC-DLBCL and investigated whether the BCR-signaling regulator microRNA-28 (miR-28) can block this process. Using flow cytometry–based competition assays, multicolor clonal barcoding, transcriptomic profiling, and xenograft models, we found that miR-28 expression impairs the emergence of ibrutinib-resistant ABC-DLBCL cells. Mechanistically, miR-28 interferes with the clonal selection process triggered by ibrutinib treatment and rewires transcriptional programs by downregulating mitochondrial and mTOR signaling pathways critical for resistance development. Furthermore, the miR-28–repressed gene signature associated with ibrutinib resistance correlates with improved survival in ibrutinib-treated patients from the PHOENIX trial cohort with the MCD genetic subtype, which is associated with ABC-DLBCL. Finally, the targeted therapeutic delivery of miR-28 via aptamer-guided nanoparticles suppresses ibrutinib-resistant tumor growth in vivo. These findings identify miR-28 as an effective inhibitor of ibrutinib resistance, underscoring its translational potential as an adjunct strategy in ABC-DLBCL therapy.2026-03-10T00:00:00Z2026-03-10T11:17:55.868Z2025-11-21T14:51:47.15ZE-MTAB-16241ERP185471EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184