Fig. 5: Combined inhibition of Bcr-Abl and BTK impairs clonogenic potential, reduces proliferation, and enhances apoptosis in nondividing LSCs.

a K562 cells were treated with increasing concentrations of Ibr (Ibrutinib, 0.5–2.5 µM) or IM (Imatinib, 0.5–2.5 µM) for 18 h. Subsequently, protein lysates were analyzed for pBcr-Abl, pSTAT5, pBTK, Bcr-Abl, STAT5, BTK, and GAPDH. b Primary CML MNCs (10,000 cells/ml) were treated for 48 h (c) and CML CD34⁺ cells (1000 cells/ml) for 72 h with Ibr (0.5 µM, 2.5 µM), IM (2.5 µM, 5.0 µM), or the combination of both drugs. Cells were resuspended and plated using methylcellulose-containing cytokines. CFU numbers were counted after 7 days (n = 3, each). d CML CD34⁺ cells were treated for 72 h with 2.5 µM Ibr, 5.0 µM IM, and the combination of both drugs. Protein lysates were analyzed for pSTAT5, pBTK, pERK1/2, STAT5, BTK, and ERK1/2. e Evaluation of cell divisions in CML CD34⁺ cells upon treatment using CFSE combined with FACS analyses. For a clear presentation, the level of significance is given for Ibr + IM vs. IM only. f Annexin V⁺ staining of undivided CFSE^Max cells in treated CML CD34⁺ cells. g Proposed mechanism: elevated FcγRIIb expression in malignant cells persists despite BCR-ABL inhibition and this could be mediated via inflammatory cytokines in the CML microenvironment. FcγRIIb activity results in increased BTK expression and phosphorylation and dual targeting of BCR-ABL and BTK activity induces apoptosis in LSCs. Data are shown as mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001, and n.s. not statistically significant.