Fig 2: Haematopoietic differentiation. (a) Culture condition schema. Embryoid bodies were formed from iPSCs in the presence BMP4, VEGF, bFGF, CHIR99021 and SB431542, and haematopoietic cytokines (Flt3-ligand, SCF and IL-3) over the course of 8 days. A representative image of EBs are shown at right. (b) FACS phenotype of EB-derived CD34. Dissociated EBs were analysed by FACS for CD73 and CXCR4 in the CD34 high-expressing population. (c) Methylcellulose CFU assay. CD34 cells were embedded in methylcellulose, and an image of the dominating CFU-G/M colony types are shown. The analyses/experiments are from at least four different differentiation procedures. FACS, fluorescence-activated cell sorting.

Fig 3: Aberrant cell-to-cell communication signaling associated with therapeutic resistance.a Differentially expressed genes (NR vs. R) in CD4+ and CD8+ T-cells pre- and post-ibrutinib treatment, respectively. Filled circle sizes are proportional to the Log2-scaled fold changes of each gene. Upregulated genes are shown in red; downregulated genes are shown in blue. Pre: pre-treatment; Post: post-treatment. b Representative genes are shown in violin plots. c Alterations (NR vs. R) of ligand-receptor-based cell-to-cell communication networks based in pre- and post-treatment samples. d Flow cytometry validation of upregulated CD69 and CXCR4 expression in ibrutinib nonresponders in comparison to the responders in additional patient cohorts (n = 65 samples collected from 22 patients). The line in the box is the median value. The bottom and top of the box are the 25th and 75th percentiles of the sample. The bottom and top of the whiskers are the minimum and maximum values of the sample. p Values from the two-side Wilcoxon Signed-rank Test are shown. e Reverse correlation between the proportion of PRF1+ CD8 T cells (cytotoxic) and the expression of CXCR4 using scRNA-seq. The bounds of shape correspond to 95% confidence band for the regression line. The Pearson correlation coefficient (r) is shown. p Value corresponds to F test of linear regression model.

Fig 4: Plerixafor-induced proliferation does not require substantial CXCR4 surface expression but is associated with AKT activation. a CXCL12 and plerixafor bind to the CXCR4 receptor. TC-32 and CADO-ES1 cells were grown in standard conditions and treated with CXCL12 (100 ng/ml) and/or plerixafor (1 µM) for 12 h before flow cytometric analysis of CXCR4 signal with a 12G5-CXCR4 antibody. b Significant reduction in CXCR4 mRNA (left panel) and surface protein expression (middle and right panel). Cells were transfected with CXCR4-targeting siRNA (siCXCR4) or non-silencing control (siCtrl) and analyzed by real-time quantitative PCR and flow cytometry after 48 h. c Proliferative plerixafor effects are maintained despite significant reduction in CXCR4 surface expression. 48 h after siRNA transfection, cells from (b) were treated with plerixafor for another 72 h. Relative cell number was measured by WST-1 assay. All graphs of this figure represent means ± SD of three independent experiments. d AKT is activated in response to plerixafor in CXCR4-high and -low cell lines. Cells grown in standard conditions were treated with plerixafor (1 µM) for 12 h or remained untreated. Whole cell lysates were analyzed by Western blotting. Actin was loading control

Fig 5: Plerixafor induces phosphorylation of receptor tyrosine kinases. a Phospho-RTK arrays were probed with whole cell lysates of CXCR4-low A673 and CXCR4-high TC-32 cells that were treated with 1 µM plerixafor or DMSO vehicle for 1 h. b RTKs activated by plerixafor. Mean pixel densities of duplicate RTK capture spots were measured and normalized to mean pixel densities of control spots of respective membranes. RTKs phosphorylated in the presence of plerixafor (mean normalized pixel density above the mean of all RTKs) were defined as active and considered for analysis. Fold changes (fc) of normalized mean pixel densities in plerixafor-treated (grey bars) compared to control cells (black bars) were calculated and RTKs with an activation fold change greater than the mean (Z-score (z) of fold change > 0) were defined as plerixafor-activated. The full data set is available in Additional file 2