An oncogenic TLR9/BTK transducer module operates in FLT3-ITD–negative AML. (A) The endogenous BTK interactome was identified in KG-1 cells using quantitative SILAC-based MS. All identified proteins (except contaminants) are plotted according to their signal intensities and their H/L ratio of enrichment on logarithmic scales. Proteins with an H/L ratio > 5 (red) were identified as interaction partners of BTK. The complete list of identified proteins and quantification statistics is provided in supplemental Table 3. (B) Cleared cellular lysates of H2228 cells (negative control, lane 1) and various AML cell lines were subjected to immunoblotting with antibodies against TLR9 (upper panel) and actin (lower panel) as loading controls. (C) KG-1, FFM04, MV4-11, and Molm13 cells were lysed and subjected to immunoprecipitation using anti-TLR9 antibodies (lane 2) or isotype-matched control antibodies (lane 1 [C]). The proteins obtained were analyzed by immunoblotting with antibodies directed against BTK (upper panels). Effective immunoprecipitation of TLR9 was confirmed by immunoblotting using TLR9-specific antibodies (lower panels). (D) FFM04 and FFM12 cells were lysed and subjected to immunoprecipitation using anti-BTK antibodies (lane 2) or isotype-matched control antibodies (lane 1 [C]). The proteins thus isolated were analyzed by immunoblotting with antibodies directed against TLR9 (upper panels). Effective immunoprecipitation of BTK was confirmed by immunoblotting using BTK-specific antibodies (lower panels). (E) Left panel: Immunoblot analyses of lysates derived from KG-1 and FFM04 cells that were left untreated (lane 1) or stimulated by CpG dinucleotides for 15, 30, or 60 minutes (lanes 2 to 4). Right panel: Immunoblot analysis of lysates derived from FFM04 cells that were either left untreated (lane 1), cocultured with bone marrow (BM) stroma cells (lane 2), or cocultured with (Annexin V-positive) apoptotic stroma cells that had been irradiated prior to coculture (lane 3). Immunoblotting was performed by using phosphosite-specific antibodies against pTyr-223 of BTK (upper panels). Protein loading was monitored by immunoblotting of BTK (lower panels). (F) XTT-based proliferation analysis of KG-1 and FFM04 cells left untreated or treated with ibrutinib and 1.5 ng/μL CpG. Results (from 4 independent experiments; mean ± SD) are shown for cells that were treated for 3 days. *P < .05 between the different treatment groups compared with the DMSO control (black bar) using Student t test. (G) KG-1, FFM04, MV4-11, and Molm13 cells were transduced either with lentiviral vectors encoding TLR9-specific shRNAs and GFP or with unspecific control shRNAs (nsp) and GFP. Subsequently, expression of TLR9 and actin was monitored by immunoblotting. (H) GFP expression was monitored in the respective transduced cell batches by flow cytometry 1 day after lentiviral transduction (day 1) and 7 days thereafter (day 7). The outlined diagram summarizes data from 4 independent experiments (mean ± SD) and shows the relative abundance of GFP-expressing cells at day 1 or day 7 for the respective TLR9 knockdown or control cell batches.