Identification of BCL2A1 as a resistance factor to OAsIs combination. (A) scRNA-seq analysis of BCL2A1 gene expression at inclusion (incl.) or relapse (Rel.) in all OAsIs samples available (left panel) or longitudinal samples from patient MCL34. Each dot represents a cell. Wilcoxon-sign-rank test; ∗∗∗∗P < .0001. (B) Bulk RNA-seq analysis of BCL2A1 gene expression in PB MCL cells (PB, n = 63) or MCL cell lines (n = 7). (C) BCL2A1 protein expression in primary PB MCL cells (n = 8), MAVER, and NTS3 cell lines, and in OAsIs samples (MCL 24 and MCL36) was evaluated by immunoblot. (D) Survival of Mino_ct and Mino_A1 (supplemental Figure 5) treated with single-agent venetoclax BCL2-i at indicated doses or with OAsIs combination (ibrutinib, 500 nM; obinutuzumab 500 ng/mL) for 48 hours. Cell viability was measured by the lack of annexin-V staining. ANOVA test; ∗∗∗∗P < .0001. The mean and standard deviation of 3 independent experiments are represented. (E) BCL2A1 specific BH3-profiling was performed using the selective peptide FS2 (20 μM) in venetoclax-sensitive Mino cells (left panel) and venetoclax-resistant SP53 cells (right panel). Mino cells were pretreated or not with venetoclax (BCL2-i) at indicated doses. SP53 cells were pretreated or not with venetoclax (BCL2-i, 500 nM), S63845 (MCL1-i, 500 nM) and A1155463 (BCLxL-i, 500 nM) for 6 hours before cytochrome-C release measurement. ANOVA test; ∗∗∗P < .001. The mean and standard deviation of 3 independent experiments are represented. ANOVA, analysis of variance.