CARD11 gain of function upregulates BCL2A1 and promotes resistance to targeted therapies combination in B-cell lymphoma [scRNA-seq]

Strategy combining targeted therapies is effective in B-cell lymphomas such as mantle celllymphoma (MCL), but acquired resistances remain a recurrent issue. Herein, we performed integrative longitudinal genomic and single-cell RNA-seq analyses of MCL patients treated with targeted therapies against CD20, BCL2 and BTK (i.e., OAsIs trial). We revealed the emergence of subclones with a selective advantage against OAsIs combination in vivo and showed that resistant cells were characterized by BCR-independent overexpression of NFkB1 target genes, especially due to CARD11 mutations. Functional studies demonstrated that CARD11 gain of function not only resulted in BCR independence, but also directly increase the transcription of the antiapoptotic BCL2A1, leading to venetoclax and OAsIs combination resistance. Based on the transcriptional profile of OAsIs-resistant subclones, we designed a 16-gene resistance signature that was also predictive for MCL patients treated with conventional chemotherapy, underlying a common escape mechanism. Overall design: 7 bone marrow samples from 6 patients included in the OAsIs trial were analyzed in single cell RNA sequencing (scRNA-seq). Briefly, live MCL cells were first enriched by Ficoll-Paque Plus and anti-human CD19 magnetic beads. Depletion of human erythroid precursors and erythrocytes was then performed using CD235a (Glycophorin-A) magnetic beads. 15,000 cells from the resulting suspensions were loaded on a Chromium controller to generate single-cell partitions following the manufacturer's protocol (10X genomics). scRNA-seq libraries were constructed using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 and sequenced using the NovaSeq 6000 system (Illumina).