Figure 2
Figure 2. BTK acts downstream of FLT3-ITD to induce proliferation in AML cells. (A) Cleared cellular lysates of patient-derived AML cells (12 FLT3-ITD–positive and 12 FLT3-ITD–negative patients) were subjected to immunoblotting with antibodies against pBTK (upper panels) and BTK (middle panels). Protein loading was monitored by anti-actin immunoblotting (lower panels). The right panel shows the BTK phosphorylation normalized to BTK expression based on the signal intensities measured by immunoblotting. (B) The endogenous FLT3-ITD interactome was identified in MV4-11 cells using quantitative SILAC-based MS. All proteins identified that showed a medium (M) vs light (L) ratio (M/L) greater than 4 are plotted according to their M/L ratio and heavy (H) vs light (H/L) ratios of enrichment on logarithmic scales. Proteins with an M/L ratio greater than 4 were identified as interaction partners of FLT3-ITD in DMSO-treated cells. The H/L ratio represents the FLT3-ITD interactome in cells treated with 20 nM quizartinib for 1 hour. The complete list of proteins identified and quantification statistics for 2 independent experiments are provided in supplemental Table 2. (C) Cleared cellular lysates of 32D cells expressing FLT3-ITD and GFP (lane 2) and the respective GFP-expressing control cells (lane 1) were subjected to immunoblotting with antibodies against pBTK (upper panels) and BTK (middle panels). Protein loading was monitored by anti-actin immunoblotting (lower panels). (D) Cleared cellular lysates of 32D cells expressing FLT3-ITD, MV4-11 (middle panel) and Molm13 cells (right panel) that were treated with DMSO (lane 1) and 20 nM quizartinib (lane 2) were subjected to immunoblotting with antibodies against pBTK (upper panels) and BTK (middle panels). Protein loading was monitored by anti-actin immunoblotting (lower panels). (E) Cleared cellular lysates of 32D cells expressing FLT3-wild-type (wt) that were left untreated (lane 1) or were stimulated by FLT3-ligand (FL) for durations indicated (lanes 2 and 3) were subjected to immunoblotting with antibodies against pBTK, BTK, pERK, and actin. (F) Tetrazolium salt (XTT)-based proliferation analysis of 32D cells expressing FLT3-ITD and GFP, or the respective control cells expressing GFP, that were treated with either DMSO or quizartinib/ibrutinib at the final concentrations indicated. Results (from 3 independent experiments; mean ± SD) are shown for cells that had been treated for 3 days. (G-H) XTT-based proliferation analysis of (G) Ba/F3 cells expressing either GFP or FLT3-ITD in combination with GFP and (H) murine myeloid progenitor cells expressing either HOXA9 and MEIS1 in combination with BFP or HOXA9 and MEIS1 in combination with both BFP and FLT3-ITD that have been treated with ibrutinib in DMSO or with DMSO control at the final concentrations indicated. Results are representative for 3 independent experiments and are shown for cells that had been treated for 3 days; mean ± SD are shown for technical replicates. (I) Colony formation assays of 32D cells expressing FLT3-ITD and GFP or the respective control cells in the presence of DMSO or ibrutinib (upper panel) or upon BTK knockdown (lower panel). Results (mean ± SD) are from 4 independent experiments. *P < .05 between the different treatment groups (upper panel: ibrutinib compared with DMSO; lower panel: BTK KD compared with nsp) using Student t test.

BTK acts downstream of FLT3-ITD to induce proliferation in AML cells. (A) Cleared cellular lysates of patient-derived AML cells (12 FLT3-ITD–positive and 12 FLT3-ITD–negative patients) were subjected to immunoblotting with antibodies against pBTK (upper panels) and BTK (middle panels). Protein loading was monitored by anti-actin immunoblotting (lower panels). The right panel shows the BTK phosphorylation normalized to BTK expression based on the signal intensities measured by immunoblotting. (B) The endogenous FLT3-ITD interactome was identified in MV4-11 cells using quantitative SILAC-based MS. All proteins identified that showed a medium (M) vs light (L) ratio (M/L) greater than 4 are plotted according to their M/L ratio and heavy (H) vs light (H/L) ratios of enrichment on logarithmic scales. Proteins with an M/L ratio greater than 4 were identified as interaction partners of FLT3-ITD in DMSO-treated cells. The H/L ratio represents the FLT3-ITD interactome in cells treated with 20 nM quizartinib for 1 hour. The complete list of proteins identified and quantification statistics for 2 independent experiments are provided in supplemental Table 2. (C) Cleared cellular lysates of 32D cells expressing FLT3-ITD and GFP (lane 2) and the respective GFP-expressing control cells (lane 1) were subjected to immunoblotting with antibodies against pBTK (upper panels) and BTK (middle panels). Protein loading was monitored by anti-actin immunoblotting (lower panels). (D) Cleared cellular lysates of 32D cells expressing FLT3-ITD, MV4-11 (middle panel) and Molm13 cells (right panel) that were treated with DMSO (lane 1) and 20 nM quizartinib (lane 2) were subjected to immunoblotting with antibodies against pBTK (upper panels) and BTK (middle panels). Protein loading was monitored by anti-actin immunoblotting (lower panels). (E) Cleared cellular lysates of 32D cells expressing FLT3-wild-type (wt) that were left untreated (lane 1) or were stimulated by FLT3-ligand (FL) for durations indicated (lanes 2 and 3) were subjected to immunoblotting with antibodies against pBTK, BTK, pERK, and actin. (F) Tetrazolium salt (XTT)-based proliferation analysis of 32D cells expressing FLT3-ITD and GFP, or the respective control cells expressing GFP, that were treated with either DMSO or quizartinib/ibrutinib at the final concentrations indicated. Results (from 3 independent experiments; mean ± SD) are shown for cells that had been treated for 3 days. (G-H) XTT-based proliferation analysis of (G) Ba/F3 cells expressing either GFP or FLT3-ITD in combination with GFP and (H) murine myeloid progenitor cells expressing either HOXA9 and MEIS1 in combination with BFP or HOXA9 and MEIS1 in combination with both BFP and FLT3-ITD that have been treated with ibrutinib in DMSO or with DMSO control at the final concentrations indicated. Results are representative for 3 independent experiments and are shown for cells that had been treated for 3 days; mean ± SD are shown for technical replicates. (I) Colony formation assays of 32D cells expressing FLT3-ITD and GFP or the respective control cells in the presence of DMSO or ibrutinib (upper panel) or upon BTK knockdown (lower panel). Results (mean ± SD) are from 4 independent experiments. *P < .05 between the different treatment groups (upper panel: ibrutinib compared with DMSO; lower panel: BTK KD compared with nsp) using Student t test.

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