Background
Methods
Drugs and cell lines
Primary leukemic cells and treatment
Immunohistochemistry analysis
Cell viability and cell proliferation assay
Light and fluorescence microscopy analyses
Flow cytometry
Immunoblotting
Quantitative PCR and gene expression
Gene expression profile
Statistical analysis
Results
Wee1 transcript is highly expressed in ALL primary samples
AZD-1775 reduces cell viability and induces apoptosis in ALL cell lines
AZD-1775 changes the cell cycle profile and activates the DNA damage response pathway in ALL cell lines
AZD-1775 reduces the cell viability of primary leukemic samples
AZD-1775 sensitizes leukemia cells to clofarabine and other genotoxic agents
24 h | 48 h | 72 h | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Clofarabine (nM) | Clofarabine (nM) | Clofarabine (nM) | |||||||||
Cell lines | 2.5 | 5 | 10 | 2.5 | 5 | 10 | 2.5 | 5 | 10 | ||
REH | AZD-1775 (nM) | 6.9 | 0.70 | 0.80 | 0.95 | 0.61 | 0.88 | 0.99 | 0.78 | 1.07 | 1.12 |
20.6 | 0.80 | 0.70 | 0.94 | 0.85 | 0.95 | 1.06 | 0.78 | 1.02 | 1.15 | ||
61.7 | 1.32 | 0.70 | 0.91 | 1.61 | 1.38 | 1.05 | 0.90 | 1.10 | 1.09 | ||
185.2 | 1.64 | 0.70 | 0.82 | 2.56 | 2.02 | 0.92 | 1.26 | 1.09 | 0.81 | ||
555.6 | 0.89 | 0.46 | 0.65 | 1.00 | 0.82 | 0.55 | 0.43 | 0.24 | 0.29 | ||
1666.7 | 1.10 | 0.43 | 0.76 | 0.33 | 0.34 | 0.34 | 0.62 | 0.64 | 0.68 | ||
5000.0 | 2.23 | 1.11 | 0.77 | 1.00 | 0.26 | 1.01 | 1.81 | 1.83 | 1.88 | ||
NALM-6 | AZD-1775 (nM) | 6.9 | 0.24 | 0.45 | 0.55 | 0.60 | 0.46 | 0.75 | 0.73 | 1.06 | 1.15 |
20.6 | 0.20 | 0.30 | 0.49 | 0.49 | 0.56 | 0.96 | 0.76 | 1.02 | 1.17 | ||
61.7 | 0.26 | 0.22 | 0.39 | 0.66 | 0.76 | 0.93 | 0.84 | 1.08 | 1.14 | ||
185.2 | 0.45 | 0.34 | 0.33 | 1.99 | 1.31 | 0.80 | 1.24 | 1.16 | 0.97 | ||
555.6 | 0.63 | 0.39 | 0.27 | 1.38 | 0.93 | 0.66 | 0.85 | 0.66 | 0.57 | ||
1666.7 | 0.55 | 0.36 | 0.33 | 9.87 | 0.84 | 1.04 | 0.20 | 0.26 | 0.37 | ||
5000.0 | 0.84 | 0.64 | 0.71 | 2.05 | 1.98 | 2.19 | 0.49 | 0.55 | 0.66 | ||
NALM-19 | AZD-1775 (nM) | 6.9 | 0.46 | 0.54 | 0.85 | 0.52 | 0.59 | 0.81 | 1.02 | 1.20 | 1.09 |
20.6 | 0.45 | 0.43 | 0.60 | 0.35 | 0.54 | 0.68 | 1.00 | 1.18 | 1.08 | ||
61.7 | 0.56 | 0.39 | 0.62 | 0.38 | 0.41 | 0.43 | 0.91 | 1.01 | 0.92 | ||
185.2 | 0.73 | 0.32 | 0.44 | 0.49 | 0.35 | 0.20 | 0.91 | 0.79 | 0.73 | ||
555.6 | 0.60 | 0.21 | 0.32 | 0.20 | 0.14 | 0.18 | 0.53 | 0.50 | 0.74 | ||
1666.7 | 0.68 | 0.27 | 0.41 | 0.21 | 0.15 | 0.27 | 0.69 | 0.75 | 1.10 | ||
5000.0 | 1.39 | 0.76 | 0.69 | 0.56 | 0.53 | 0.70 | 1.75 | 1.79 | 2.43 | ||
RPMI-8402 | AZD-1775 (nM) | 6.9 | 0.21 | 0.26 | 0.50 | 0.23 | 0.36 | 1.39 | 8.20 | 1.23 | 1.46 |
20.6 | 0.14 | 0.24 | 0.63 | 0.34 | 0.42 | 11.91 | 0.69 | 0.78 | 1.43 | ||
61.7 | 0.22 | 0.21 | 0.62 | 0.77 | 0.53 | 3.17 | 0.55 | 0.83 | 1.26 | ||
185.2 | 0.41 | 0.26 | 0.81 | 1.48 | 1.43 | 2.37 | 0.66 | 0.80 | 0.73 | ||
555.6 | 0.57 | 0.36 | 0.54 | 0.95 | 0.61 | 0.48 | 0.43 | 0.41 | 0.08 | ||
1666.7 | 0.60 | 0.38 | 0.32 | 0.55 | 0.33 | 0.21 | 0.28 | 0.10 | 0.12 | ||
5000.0 | 0.37 | 0.26 | 0.05 | 0.09 | 0.02 | 0.04 | 0.22 | 0.24 | 0.26 | ||
Cell lines | 5 | 10 | 20 | 5 | 10 | 20 | 5 | 10 | 20 | ||
CCRF-CEM | AZD-1775 (nM) | 6.9 | 0.01 | 0.02 | 0.23 | 0.74 | 1.13 | 1.15 | 1.01 | 1.61 | 1.06 |
20.6 | 0.01 | 0.01 | 0.08 | 0.92 | 2.29 | 1.30 | 0.43 | 2.41 | 1.02 | ||
61.7 | 0.01 | 0.01 | 0.02 | 0.64 | 1.07 | 0.70 | 4.20 | 4.81 | 0.98 | ||
185.2 | 0.01 | 0.01 | 0.02 | 1.13 | 0.78 | 0.29 | 0.91 | 0.83 | 0.76 | ||
555.6 | 0.01 | 0.01 | 0.01 | 0.20 | 0.19 | 0.22 | 0.39 | 0.51 | 0.72 | ||
1666.7 | 0.05 | 0.06 | 0.09 | 0.44 | 0.42 | 0.45 | 0.80 | 0.94 | 1.08 | ||
5000.0 | 0.42 | 0.45 | 0.30 | 1.40 | 1.22 | 0.96 | 2.02 | 2.11 | 1.87 | ||
MOLT-4 | AZD-1775 (nM) | 6.9 | 0.00 | 0.09 | 0.25 | 1.62 | 8.99 | 8.15 | 1.00 | 1.03 | 1.27 |
20.6 | 0.11 | 0.15 | 0.32 | 2.26 | 0.85 | 10.07 | 0.80 | 1.14 | 1.02 | ||
61.7 | 0.32 | 0.24 | 0.40 | 0.69 | 0.75 | 0.55 | 0.95 | 1.19 | 0.62 | ||
185.2 | 0.57 | 0.27 | 0.23 | 1.24 | 0.94 | 0.43 | 0.81 | 0.53 | 0.26 | ||
555.6 | 0.31 | 0.16 | 0.18 | 0.88 | 0.61 | 0.40 | 0.00 | 0.11 | 0.12 | ||
1666.7 | 0.40 | 0.28 | 0.30 | 1.31 | 1.42 | 0.42 | 0.31 | 0.31 | 0.32 | ||
5000.0 | 1.61 | 1.76 | 1.66 | 6.06 | 5.49 | 5.24 | 0.92 | 2.04 | 0.93 |
AZD-1775 sensitizes leukemic cells to the tyrosine kinase inhibitors
24 h | 48 h | |||||||
---|---|---|---|---|---|---|---|---|
BOS (nM) | BOS (nM) | |||||||
Cell line | 10 | 25 | 50 | 10 | 25 | 50 | ||
BV-173 | AZD-1775 (nM) | 6.9 | 0.95 | 1.11 | 0.99 | 1.45 | 1.03 | 1.27 |
20.6 | 1.03 | 1.21 | 1.01 | 1.59 | 0.98 | 1.29 | ||
61.7 | 0.73 | 0.86 | 0.62 | 0.88 | 0.72 | 1.08 | ||
185.2 | 0.71 | 0.77 | 0.71 | 0.59 | 0.65 | 1.01 | ||
555.6 | 1.26 | 0.75 | 0.61 | 0.85 | 0.78 | 1.03 | ||
1666.7 | 1.14 | 0.92 | 0.81 | 1.08 | 1.08 | 1.24 | ||
5000.0 | 1.82 | 1.71 | 1.58 | 2.44 | 1.86 | 2.02 | ||
Cell line | 500 | 1000 | 2000 | 500 | 1000 | 2000 | ||
SUP-B15 | AZD-1775 (nM) | 6.9 | 0.52 | 0.96 | 1.23 | 0.59 | 0.84 | 1.23 |
20.6 | 0.64 | 0.72 | 1.11 | 0.61 | 0.99 | 0.98 | ||
61.7 | 0.7 | 1.36 | 0.91 | 0.73 | 0.83 | 0.89 | ||
185.2 | 1.05 | 1.51 | 0.79 | 0.79 | 0.88 | 0.79 | ||
555.6 | 1.07 | 1.3 | 1.14 | 1.18 | 1.22 | 1 | ||
1666.7 | 0.44 | 0.54 | 0.46 | 0.5 | 0.55 | 0.54 | ||
5000.0 | 0.51 | 0.49 | 0.4 | 0.41 | 0.41 | 0.31 |
24 h | 48 h | 72 h | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Bosutinib isomer (nM) | Bosutinib isomer (nM) | Bosutinib isomer (nM) | |||||||||
Cell line | 500 | 1000 | 2000 | 500 | 1000 | 2000 | 500 | 1000 | 2000 | ||
NALM-6 | AZD-1775 (nM) | 6.9 | 0.57 | 0.69 | 0.79 | 0.62 | 1.00 | 1.21 | 0.59 | 0.79 | 1.14 |
20.6 | 0.33 | 0.48 | 0.64 | 0.65 | 0.81 | 1.07 | 0.47 | 0.68 | 0.99 | ||
61.7 | 0.35 | 0.39 | 0.65 | 0.67 | 0.65 | 0.97 | 0.68 | 0.73 | 0.93 | ||
185.2 | 0.48 | 0.41 | 0.41 | 0.91 | 0.65 | 0.68 | 0.89 | 0.73 | 0.63 | ||
555.6 | 0.63 | 0.43 | 0.36 | 0.54 | 0.45 | 0.47 | 0.41 | 0.40 | 0.48 | ||
1666.7 | 0.67 | 0.51 | 0.41 | 0.65 | 0.66 | 0.68 | 0.53 | 0.59 | 0.66 | ||
5000.0 | 1.65 | 1.43 | 1.21 | 1.58 | 1.61 | 1.62 | 1.42 | 1.43 | 1.52 | ||
Cell lines | 250 | 500 | 1000 | 250 | 500 | 1000 | 250 | 500 | 1000 | ||
NALM-19 | AZD-1775 (nM) | 6.9 | 0.43 | 0.75 | 1.01 | 1.99 | 2.94 | 0.20 | 0.19 | 0.28 | 0.35 |
20.6 | 0.38 | 0.47 | 0.63 | 1.53 | 1.15 | 0.12 | 0.24 | 0.29 | 0.28 | ||
61.7 | 0.19 | 0.27 | 0.23 | 0.20 | 0.12 | 0.09 | 0.44 | 0.48 | 0.31 | ||
185.2 | 0.27 | 0.24 | 0.24 | 0.42 | 0.23 | 0.15 | 0.67 | 0.48 | 0.26 | ||
555.6 | 0.38 | 0.31 | 0.28 | 0.41 | 0.31 | 0.25 | 0.29 | 0.28 | 0.21 | ||
1666.7 | 0.76 | 0.68 | 0.69 | 0.82 | 0.72 | 0.69 | 0.47 | 0.44 | 0.38 | ||
5000.0 | 1.78 | 1.95 | 1.77 | 2.04 | 2.09 | 1.93 | 1.19 | 1.30 | 1.13 | ||
MOLT-4 | AZD-1775 (nM) | 6.9 | 1.23 | 0.93 | 1.11 | 0.99 | 1.03 | 1.28 | 0.67 | 1.22 | 1.26 |
20.6 | 0.78 | 0.61 | 0.78 | 0.85 | 0.89 | 1.07 | 0.74 | 0.89 | 1.07 | ||
61.7 | 0.77 | 0.53 | 0.43 | 0.62 | 0.63 | 0.71 | 0.91 | 0.85 | 0.77 | ||
185.2 | 0.53 | 0.40 | 0.23 | 0.39 | 0.35 | 0.34 | 0.35 | 0.37 | 0.53 | ||
555.6 | 0.22 | 0.25 | 0.21 | 0.34 | 0.40 | 0.50 | 0.35 | 0.43 | 0.67 | ||
1666.7 | 0.40 | 0.60 | 0.65 | 0.95 | 1.12 | 1.14 | 0.92 | 0.96 | 1.17 | ||
5000.0 | 2.46 | 2.25 | 3.02 | 3.06 | 3.26 | 3.49 | 3.54 | 3.30 | 3.52 | ||
Cell line | 2000 | 5000 | 10000 | 2000 | 5000 | 10000 | 2000 | 5000 | 10000 | ||
CCRF-CEM | AZD-1775 (nM) | 6.9 | 0.75 | 0.78 | 0.84 | 0.75 | 0.78 | 0.84 | 1.06 | 0.65 | 0.39 |
20.6 | 0.38 | 0.62 | 0.79 | 0.38 | 0.62 | 0.79 | 1.21 | 0.57 | 0.52 | ||
61.7 | 0.27 | 0.54 | 0.74 | 0.27 | 0.54 | 0.74 | 0.53 | 0.49 | 0.52 | ||
185.2 | 0.26 | 0.46 | 0.58 | 0.26 | 0.46 | 0.58 | 0.28 | 0.43 | 0.49 | ||
555.6 | 0.25 | 0.65 | 0.53 | 0.25 | 0.65 | 0.53 | 0.35 | 0.49 | 0.37 | ||
1666.7 | 0.84 | 0.93 | 0.81 | 0.84 | 0.93 | 0.81 | 0.66 | 0.79 | 0.46 | ||
5000.0 | 3.13 | 2.76 | 0.99 | 3.13 | 2.76 | 0.99 | 2.08 | 1.97 | 0.88 |
24 h | 48 h | |||||||
---|---|---|---|---|---|---|---|---|
Ponatinb (nM) | Ponatinb (nM) | |||||||
Cell line | 25 | 50 | 100 | 25 | 50 | 100 | ||
BV-173 | AZD-1775 (nM) | 6.9 | 0.39 | 0.57 | 1.16 | 0.29 | 0.31 | 0.67 |
20.6 | 0.34 | 0.59 | 1.04 | 0.33 | 0.34 | 0.50 | ||
61.7 | 0.33 | 0.55 | 0.99 | 0.16 | 0.17 | 0.56 | ||
185.2 | 0.31 | 0.49 | 0.90 | 0.06 | 0.11 | 0.48 | ||
555.6 | 0.47 | 0.53 | 0.83 | 0.02 | 0.07 | 0.20 | ||
1666.7 | 0.53 | 0.58 | 0.86 | 0.01 | 0.01 | 0.12 | ||
5000.0 | 1.23 | 1.33 | 1.38 | 0.06 | 0.07 | 0.15 | ||
Imatinib (nM) | Imatinib (nM) | |||||||
Cell line | 250 | 500 | 1000 | 250 | 500 | 1000 | ||
BV-173 | AZD-1775 (nM) | 6.9 | 0.39 | 0.41 | 0.62 | 0.41 | 0.03 | 0.02 |
20.6 | 0.58 | 0.43 | 0.69 | 0.43 | 0.04 | 0.03 | ||
61.7 | 0.32 | 0.33 | 0.39 | 0.18 | 0.06 | 0.05 | ||
185.2 | 0.23 | 0.31 | 0.47 | 0.15 | 0.07 | 0.05 | ||
555.6 | 0.50 | 0.44 | 0.48 | 0.11 | 0.08 | 0.07 | ||
1666.7 | 0.57 | 0.50 | 0.53 | 0.07 | 0.20 | 0.17 | ||
5000.0 | 1.05 | 1.18 | 1.21 | 0.61 | 1.19 | 1.56 |