Article Figures & Data
Figures
- Figure 1.
GS87 is a highly specific and potent GSK3 inhibitor that induces AML cell differentiation. A, molecular structure of GS87. B, GS87 is a potent GSK3 inhibitor. In vitro kinase assay for GSK3α and GSK3β. C, GS87 leads to extensive AML differentiation as measured by respiratory burst capacity. OCI, HL-60, and NB4 cells were treated with GS87 (30 μmol/L), Li (10 mmol/L), or SB (20 μmol/L) for 72 hours and differentiation capacity was measured using the NBT reduction assay. D, GS87 leads to more effective AML differentiation than other clinically used GSK3 inhibitors. HL-60 cells were treated with GS87 (30 μmol/L), tideglusib (30 μmol/L), or LY-2090314 (30 μmol/L) for 72 hours and differentiation was measured by the NBT assay. **, P < 0.01. NBT, nitrotetrazolium blue.
- Figure 2.
GS87 promotes monocytic differentiation of AML cells. A–C, GS87 leads to extensive acute myeloid leukemia (AML) differentiation as measured by immunophenotyping. AML cell lines were treated with GS87 (30 μmol/L or as indicated), Li (10 mmol/L), or SB (20 μmol/L) for 72 hours and cell surface expression of CD11b and CD14 (HL-60 cells) were measured by flow cytometry. **, P < 0.01. D, treatment with GS87 induces morphologic changes consistent with monocytic differentiation in HL-60 cells compared with untreated cells. Cells were stained using Wright–Giemsa stain. Images were taken using the built-in EVOS XL core camera and acquisition software using a 100× oil objective. The arrows indicate representative cells with marked nuclear condensation or vacuoles. The boxed cells are shown expanded to better illustrate the morphology. E, GS87 leads to AML differentiation in primary patient leukemic cells. Primary patient leukemia cells treated with GS87, Li, or SB showed the highest increase in the cell surface expression of CD11b after GS87 treatment.
- Figure 3.
GS87 inhibits AML cell proliferation and causes irreversible growth inhibition. A, GS87 impairs AML proliferation. Equal numbers of HL-60, U937, THP-1, and NB4 cells were treated with GS87 (30 μmol/L), Li (10 mmol/L), or SB (20 μmol/L) for 72 hours and the MTT assay was performed. The MTT signal was normalized to the vehicle-treated control. B, GS87 modulates the cell cycle of AML cells. HL-60 cells were treated with GS87, Li, or SB for 24 hours and assessed for cell-cycle status. Representative flow results are shown and the histogram is an average of three independent studies. **, P < 0.01; NS, no significant difference. C, GS87 inhibits colony formation of AML cells but not normal marrow mononuclear cells. OCI, HL-60, and NB4 cells and normal marrow mononuclear cells were treated with GS87 for 72 hours, after which drug was washed away and equal numbers of viable cells were cultured in semisolid soft agar medium for 7 to 10 days. Colony formation of cells was normalized to a vehicle-treated control.
- Figure 4.
GS87 effectively modulates GSK3-dependent proteins. A, GS87-induced differentiation is independent of β-catenin. OCI cells with stable knockdown of β-catenin showed similar differentiation as compared with cells transfected with empty vector after treatment with GS87 for 3 days as measured by NBT reduction. **, P < 0.01. B and C, GS87 modulates GSK3 target proteins important in cell proliferation and differentiation more effectively than Li and SB. HL-60 cells were treated as indicated for 2 and 4 days (B) or 4 days (C) and assessed by Western blot analysis for key AML growth and differentiation proteins that can be modulated by GSK3. NBT, nitrotetrazolium blue.
- Figure 5.
GS87-induced AML differentiation is dependent on MAPK signaling. A, comparison of GSK3 inhibitor modulation of gene expression in AML cells. HL-60 cells were treated for 48 hours and the expression of the indicated genes was assessed. Venn diagram of significantly regulated genes (left); validation of microarray results by real-time PCR (right). B, multiple components of the MAPK signaling cascade are activated by GS87 treatment. HL-60 cells were treated with GS87, Li, or SB and the activation of p38, ERK, and JNK were analyzed by Western blot analysis at various time points using phospho-specific antibodies. C, chemical inhibition of ERK (PD08959 10 μmol/L) abrogates the differentiation capacity of GS87 in HL-60 cells. **, P < 0.01.
- Figure 6.
GS87 demonstrates efficacy in a circulating AML mouse model system. A, mice treated with GS87 showed longer survival compared with those treated with vehicle or the standard AML therapeutic cytarabine in a mouse model of human primary AML. B, bone marrow blast percentage in mice with primary human AML sacrificed because of moribund state or at study endpoint of 90 days showed significantly higher percentage of blasts in the vehicle and cytarabine groups compared with the GS87 group. Note: the GS87 mice tested were the surviving mice at the end of the study period. C and D, mouse model of circulating HL-60 cells showing a reduced disease burden in the bone marrow (C) and evidence of AML differentiation (D) in the GS87-treated mice.
Tables
- Table 1.
List of upstream regulators related to MAPK signaling identified from pathway analysis of genes significantly modulated (>2-fold) after treatment with GS87 (30 μmol/L), Li (10 mmol/L), or SB (20 μmol/L) for 48 hours
Upstream regulator Activation fold change P MAPK pathway—GS87 SB203580 −5.84 5.43E−13 PD98059 −5.231 7.30E−26 U0126 −4.754 5.18E−15 SP600125 −3.885 1.92E−09 SB202190 −3.024 1.36E−10 MAPK10 2.197 1.37E−04 MAPK 2.287 6.33E−09 MAP2K1/2 2.365 6.66E−09 MAPK13 2.404 4.15E−04 MAPK12 2.418 2.17E−04 MAP3K7 2.494 1.23E−06 MAP2K3 2.760 1.10E−05 MAPK7 2.768 2.01E−05 MAPK3 3.056 1.45E−06 MAP2K1 3.092 4.83E−10 MAPK14 3.429 8.35E−04 MAP3K8 3.765 2.11E−06 ERK1/2 3.805 8.31E−11 ERK 3.961 8.06E−17 JNK 3.966 7.79E−07 MAPK pathway—SB U0126 −2.949 9.97E−07 LY294002 −2.681 6.23E−05 PD98059 −2.662 2.13E−05 SB203580 −2.153 2.32E−04 ERK 2.000 1.70E−05 MAPK pathway—Li JNK 2.221 4.88E−03 ERK 2.592 3.47E−04
Supplementary Data
- Supplementary Figure Legends - Supplementary Figure Legends
- Supplementary Figure 1 - GS87 does not significantly impact the viability of AML cells
- Supplementary Figure 2 - Representative flow histograms showing CD11b staining of AML cells after GS87 treatment
- Supplementary Figure 3 - ATRA leads to marked induction of CD11b in Acute Promyelocytic Cells
- Supplementary Figure 4 - GS87 does not lead to observable alterations in normal hematopoiesis
- Supplementary Figure 5 - Heatmap of Z-scores of genes that are significantly dysregulated
- Supplementary Figure 6 - Heatmap of Z-scores of genes that are significantly dysregulated
- Supplementary Table 1 - Primer sequences
- Supplementary Table 2 - Kinase profiling
- Supplementary Table 3 - Microarray differential expression
- Supplementary Table 4 - Microarray gene overlap
- Supplementary Table 5 - Microarray pathway analysis
Volume 15, Issue 7
