BTK is expressed in AML and promotes cell survival and proliferation. (A) Immunohistochemical analysis of BTK expression in 28 bone marrow specimens of AML patients. For comparison, bone marrow–derived samples from healthy individuals and from diffuse large B-cell lymphoma (DLBCL) patients were stained by antibodies against BTK. (B) Cleared cellular lysates of the lymphoma cell line DG75 and various AML cell lines (left panel) or primary patient-derived AML cultures (right panel) were subjected to immunoblotting with antibodies against phosphorylated BTK (pBTK; upper panels) and BTK (middle panels). Protein loading was monitored by anti-actin immunoblotting (lower panels). (C-D) Growth curves of the AML cell lines as well as the patient-derived AML cultures FFM04, -05, -12, -21, -40, and -41 that were treated with ibrutinib at final concentrations of 10 nM (red lines), 50 nM (green lines), or 250 nM (purple lines) or with dimethylsulfoxide (DMSO) as a control (blue lines.). Data from 4 independent experiments are shown (mean ± standard deviation [SD]). *P < .05 between the different treatment groups and the control cells using Student t test. (E) Relative cell numbers of primary AML cultures that were treated with 250 nM ibrutinib for 3 cycles. Each cycle consisted of a 1-hour ibrutinib treatment followed by a 23-hour washout period. Results (mean ± SD) are from 4 independent experiments.*P < .05 between the different treatment groups and the control cells using Student t test. (F) Immunoblots of cleared cellular lysates derived from KG-1 and MV4-11 control cells (lane 1) or the respective BTK knockdown cells (lane 2) with antibodies recognizing BTK (upper panels) or actin (lower panels). (G) KG-1 and MV4-11 cells were transduced either with lentiviral vectors encoding BTK-specific shRNAs and GFP or with unspecific control shRNAs and GFP. GFP expression was subsequently monitored in the respective cell batches by flow cytometry 1 day after lentiviral transduction (day 1) and 7 days thereafter (day 7). The outlined dot plots (representative for all 3 experiments) and histograms summarizing data from 3 independent experiments (mean ± SD) show the relative abundance of GFP-expressing cells on day 1 and day 7 for the respective BTK knockdown or control cell batches, as determined by flow cytometry. *P < .05 between the BTK knockdown and the control cells (nsp) using Student t test. (H) Flow cytometric cell cycle and (I) apoptosis analysis of KG-1 and MV4-11 BTK knockdown (BTK KD) cells or control cells (nsp) at day 2 after transduction. Results (mean ± SD) are from 4 independent experiments. *P < .05 between the BTK knockdown and the control cells using Student t test.