Fig. 2: IKAROS regulates CD19 and CD22 surface expression.
A–C Relative IKAROS and CD19 median levels in B-ALL cell lines (697, NALM6, NALM16, NALM20, REH, RS4;11, SUP-B15) transduced with lentivirus expressing scrambled or short hairpin RNA (shRNA) against IKZF1 (A; n from left to right: 20, 20, 8, 25, 25, 12), treated with DMSO or 10 µM lenalidomide (B; n = 8), or combining shRNA knockdown with or without lenalidomide treatment (C, n = 9). Proteins were measured by flow cytometry and normalized to scrambled transduced (A), DMSO-treated (B), or scrambled transduced and DMSO-treated (C) cells. RFI = relative fluorescence intensity. N represents independent biological replicates. D CD19 variance-stabilized transformed (vst) counts in IKAROS WT or KD B-ALL cells. E Accessibility profile of CD19 promoter and gene from one representative IKAROS WT and KD B-ALL cell line pair. Other cell lines and their biological replicates can be found in Supplementary Fig. 5A. F Gene set enrichment analysis (GSEA) results for RNA splicing (GO: 0008380) gene signature in IKAROS WT and KD B-ALL cells. G, H Frequency of intron 10 retention in CD19 mRNA in IKAROS WT or KD B-ALL cells (G) or pediatric B-ALL patients treated with 19.BBz CAR T cells11,14 (n = 11), adult B-ALL patients treated with blinatumomab13 (n = 2), and adult LBCL patients treated with 19.28z CAR T cells29,30 (n = 6) (H). I GSEA results for Zheng Cord Blood C6 HSC/MPP gene signature80 in IKAROS WT and KD B-ALL cells. J Cell type enrichment analysis of genes with differentially accessible promotors in IKAROS WT or IKAROS KD B-ALL cells. K CD22 vst counts in IKAROS WT or KD B-ALL cells. L Relative CD22 median levels in isogenic IKAROS WT or KD B-ALL cells (n = 7; independent biological replicates). Values were measured by flow cytometry or CyTOF and normalized to IKAROS WT condition. M Paired pre-CART22 and post-CD22low relapse cohort of leukemic patient samples analyzed by CyTOF. IKAROS median levels in pro-BII-like tumor cells. N represents individual patient samples. Schematic representation of the patient cohort created in BioRender81. RNA-seq and ATAC-seq experiments (D–G, I–K) were performed in 3 cell lines (NALM6, REH, and SUP-B15) with two biological replicates per cell line. Bar plots in (A–D), (G, H), and (K, L) show mean ± SEM. Boxplots in (M) extend from the 25th to the 75th percentiles, with a line in the middle representing the median and whiskers extending from the minimum to the maximum values. Statistical tests used were two-way ANOVA followed by Šidák’s multiple comparisons test (A–C); DESeq’s Wald test followed by BH correction (D) and (K); multivariate analysis of transcript splicing (rMATS) followed by FDR correction (G); and two-sided paired t-test (L) and (M). Not significant (n.s.), P > 0.05.
