Phosphoproteome Analysis Reveals Differential Mode of Action of Sorafenib in Wildtype and Mutated FLT3 Acute Myeloid Leukemia (AML) Cells

Abstract

Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD⁺ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD^- and FLT3-ITD⁺ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 μm, whereas FLT3-ITD⁺ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD^- cells. However, we identified two FLT3-ITD^- cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD^- and FLT3-ITD⁺ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib.

Fig. 1.

Sensitivity to sorafenib is not depended on FLT3 status. AML cell lines MV4-11, MONO-MAC-1 and SKM-1 were treated with 0.01–10 μm sorafenib for 72 h. MV4-11 (FLT3 ITD positive) and MONO-MAC-1 (FLT3 wild-type) are more sensitive to sorafenib than SKM-1 cells. The proliferation (A) and metabolic activity (B) was significantly inhibited on MV4-11 and MONO-MAC-1 cells from 0.1–10 μm sorafenib. Apoptotic (Annexin V-FITC⁺ and propidium iodide⁻) (C) and necrotic cells (Annexin V FITC⁺ and propidium iodide⁺) (D) were determined by flow cytometry. Highest amount of apoptosis rates were only observed in MV4-11 cells after treatment with sorafenib. Necrosis rates were increased with 5 and 10 μm sorafenib in MV4-11 and SKM-1. Results are displayed as the mean ± S.D. of three independent experiments. Symbols (*: MV4-11, #: MONO-MAC-1 and +: SKM-1) represents a statistically significance difference between sorafenib and DMSO treated cells with a p value <0.05.

Fig. 2.

Effects of sorafenib on cell morphology. MV4-11 and MONO-MAC-1 cells were treated for up to 72 h with DMSO or sorafenib (0.2 μm) and stained using Pappenheim method to assess cellular morphology. Cytomorphology was altered in both cell lines. In MONO-MAC-1 cells an extensive cytoplasmic vacuolization could be observed whereas in MV4-11 cells, sorafenib induced more shrinkage characteristics with cell fragmentation into apoptotic bodies.

Fig. 3.

Workflow for phosphoproteome analysis. The AML cell lines MV4-11, MONO-MAC-1, and SKM-1 were SILAC-labeled, treated with DMSO, 0.01 μm sorafenib, and 0.2 μm sorafenib for two hours. Each experiment was performed in three replicates with a complete label switch according to the depicted experimental design. The pooled lysates of each experiment were subjected to a global, quantitative phosphoproteomics workflow (see Methods for more details). In total, 30,812 phosphorylation sites were identified in this study.

Fig. 4.

Effect of sorafenib on the phosphoproteome. The plots show the effect of treatment with 0.01 μm (top) and 0.2 μm (bottom) sorafenib on the phosphoproteome of MV4-11 (left), MONO-MAC-1 (middle), and SKM-1 (right). Each dot represents a phosphorylation site with its respective log-10 ratio between the sorafenib and DMSO treated cells and the standard deviation of the log-ratios across the three replicates. Significantly regulated phosphorylation sites are colored red (based on MeanRank test with 0.05 FDR).

Fig. 5.

mTOR and MEK/ERK pathways. The significantly regulated phosphorylation sites for MV4-11 (A) and MONO-MAC-1 (B) treated with 0.2 μm sorafenib are mapped to the mTOR and MEK/ERK pathway downstream of the receptor tyrosine kinases FLT3 and RET. Each bubble represents a significantly regulated phosphosite; the ratio between DMSO and sorafenib treated cells is color coded (red: upregulated, blue: downregulated). White bubbles contain the number of detected but nonregulated sites (at 0.05 FDR).

Fig. 6.

Activation of mTOR pathway and sorafenib's kinase profile. (A) Baseline phosphorylation (log 10) of proteins involved in the mTOR pathway. Each dot represents the log 10 intensity in each replicate DMSO experiment; the horizontal bars correspond to the respective mean. Lines with asterisks denote pairs for which the baseline phosphorylation is significantly different in MV4-11 and MONO-MAC-1 (t test, p < 0.05). (B) Kinase profile of sorafenib in MV4-11 and MONO-MAC-1. The horizontal bars represent the kinases identified as binders and their respective dissociation constants.

Fig. 7.

Effects of linivanib and vandetanib on MV4-11 and MONO-MAC-1 cells. MV4-11 and MONO-MAC-1 cells were treated for up to 72 h with DMSO, sorafenib (0.01 μm), linifanib (0.01 μm) or vandetanib (0.01 μm) to assess cell proliferation (A) and metabolic activity (B). In both cell lines a significant decrease of metabolic activity were induced with sorafenib and linivanib. Inhibition with RET kinase inhibitor vandetanib has no significant effects. Results are displayed as the mean ± S.D. of three independent experiments (* significant treatment effects versus DMSO treated cells).