Figure 2.
Oncogenic FLT3 signaling triggered by very short FLT3 insertions and their sensitivity to FLT3 inhibitors. (A) A schematic representation of the FLT3 receptor and its different domains is shown. These include the N-terminal (N) and C-terminal (C) regions, immunoglobulin-like (Ig-like) domains, transmembrane (TM) domain, JM domain, and TKD. For a more focused view, an enlarged representation of the aa domain 576 to 597 within the JM domain is provided. This highlights the aa modifications introduced by the 4 FLT3-VSIs (referred to as F, L, Y, and R) selected for modeling. (B) Cells were starved of serum and/or cytokines for 6 hours. Protein extracts were subjected to western blotting with the indicated antibodies. (C) TF-1 and Ba/F3 cells were cultured with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), respectively. Cell viability was monitored by trypan blue exclusion assay over a period of 8 days (n = 3). (D) TF-1 or Ba/F3 cells expressing FLT3-VSIs or a classic FLT3-ITD mutations were incubated with either vehicle (−) or 3nM (+) quizartinib (QUIZ) for 48 hours. Western blots were performed using the indicated antibodies. (E-F) TF-1 or Ba/F3 cells transduced with the empty vector were cultured with 10% fetal bovine serum and GM-CSF or IL3, respectively, whereas cells expressing FLT3 variants were cultured without cytokines. Cells were treated with either vehicle (0.1% dimethyl sulfoxide) or various concentrations of QUIZ, gilteritinib (GILT), midostaurin (MIDO), or ponatinib (PONA) for 48 hours. Cell viability was measured using the luminescence-based ATPlite assay (n = 3 biological replicates, each including 3 technical replicates). (E) Cell viability assays performed on Ba/F3 and TF-1 cells exposed to QUIZ were plotted using the log(inhibitor) vs response—variable slope (4 parameters) model implemented in GraphPad (Prism, v9.5.0). (F) Half maximal inhibitory concentrations (IC50) for each TKI and FLT3 variant type were determined in Ba/F3 and TF-1 cell lines using GraphPad. Vertical bars indicate standard deviations. Statistical significance levels are indicated as ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.

Oncogenic FLT3 signaling triggered by very short FLT3 insertions and their sensitivity to FLT3 inhibitors. (A) A schematic representation of the FLT3 receptor and its different domains is shown. These include the N-terminal (N) and C-terminal (C) regions, immunoglobulin-like (Ig-like) domains, transmembrane (TM) domain, JM domain, and TKD. For a more focused view, an enlarged representation of the aa domain 576 to 597 within the JM domain is provided. This highlights the aa modifications introduced by the 4 FLT3-VSIs (referred to as F, L, Y, and R) selected for modeling. (B) Cells were starved of serum and/or cytokines for 6 hours. Protein extracts were subjected to western blotting with the indicated antibodies. (C) TF-1 and Ba/F3 cells were cultured with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), respectively. Cell viability was monitored by trypan blue exclusion assay over a period of 8 days (n = 3). (D) TF-1 or Ba/F3 cells expressing FLT3-VSIs or a classic FLT3-ITD mutations were incubated with either vehicle (−) or 3nM (+) quizartinib (QUIZ) for 48 hours. Western blots were performed using the indicated antibodies. (E-F) TF-1 or Ba/F3 cells transduced with the empty vector were cultured with 10% fetal bovine serum and GM-CSF or IL3, respectively, whereas cells expressing FLT3 variants were cultured without cytokines. Cells were treated with either vehicle (0.1% dimethyl sulfoxide) or various concentrations of QUIZ, gilteritinib (GILT), midostaurin (MIDO), or ponatinib (PONA) for 48 hours. Cell viability was measured using the luminescence-based ATPlite assay (n = 3 biological replicates, each including 3 technical replicates). (E) Cell viability assays performed on Ba/F3 and TF-1 cells exposed to QUIZ were plotted using the log(inhibitor) vs response—variable slope (4 parameters) model implemented in GraphPad (Prism, v9.5.0). (F) Half maximal inhibitory concentrations (IC50) for each TKI and FLT3 variant type were determined in Ba/F3 and TF-1 cell lines using GraphPad. Vertical bars indicate standard deviations. Statistical significance levels are indicated as ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.

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