Inducing Synthetic Lethality in A3B-expressing Cancer Cells through WEE1-inhibition
Introduction
The cytosine deaminase, APOBEC3B (A3B), is over-expressed in a variety of cancers and is a driver of cancer mutation, contributing to the development of therapeutic resistance. A3B deaminates single-stranded DNA cytosines to uracils, which are mostly repaired through the base excision repair (BER) pathway. However, errors during BER can lead to DNA double-strand breaks that must be repaired to maintain the integrity of the genome. WEE1 is a kinase involved in the DNA damage response as a mediator of the cell cycle. When DNA damage is detected, WEE1 becomes activated and phosphorylates CDK1. Phosphorylation of CDK1 inhibits its activity and halts the cell cycle at the G2/M checkpoint, allowing DNA damage repair mechanisms to act before the cell undertakes mitosis. We hypothesize that inhibiting WEE1 will cause the DNA damage that results from A3B activity to accumulate, resulting in increased chromosomal instability and causing a synthetic lethal effect in cancer cells with high A3B-expression and activity.
Methods
To investigate the potential synthetic lethal combination of A3B expression and WEE1-inhibition, two JHOC5 clones, clone 5 and clone 7, were selected for high A3B expression and activity. These clones were engineered to lack A3B (A3B-KO) or express a control CRISPR lacZ construct (A3B-WT). A3B-knockout was confirmed by immunoblots, DNA deaminase activity assays, and Sanger sequencing. The drugs examined in this study were AZD1775, a WEE1-inhibitor, and ZNL 02-096, a WEE1-degrader. The effectiveness of the drugs at inhibiting or degrading WEE1 was assessed by immunoblotting with antibodies for WEE1, phospho-CDK1, and CDK1. Cell viability following drug treatment was assessed with CellTiter Glo assays and colony-formation assays. Drug-induced DNA damage was assessed with immunofluorescence assays using an antibody for γ-H2AX.
Results
Treatment with AZD1775 and ZNL 02-096 reduced the concentrations of p-CDK1 in A3B-KO and A3B-WT JHOC5 cells in a dose-dependent manner. ZNL 02-096 also reduced the concentration of WEE1. CellTiter Glo assays of cell viability showed that cell viability was reduced by AZD1775 and ZNL 02-096 in both the A3B-KO and A3B-WT JHOC5 cells of both clone. In clone 5, but not clone 7, the EC50 values of both drugs were significantly lower in the A3B-WT cells compared to that observed in A3B-KO cells, suggesting a synthetic lethal effect. Colony-formation assays with AZD1775-treatment did not show significant differences in cell viability between the A3B-KO and A3B-WT cells of either clone. Colony-forming assays with clone 5, but not clone 7, cells showed a significantly lower EC50 value when A3B-WT cells were treated with ZNL 02-096 compared to A3B-KO cells. In both clones, immunofluorescence assays showed significantly higher γ-H2AX staining in A3B-WT cells treated with AZD1775, compared to A3B-KO cells. In clone 5 but not clone 7 cells, A3B-WT cells showed significantly higher γ-H2AX staining than A3B-KO cells.
Conclusion
WEE1-inhibition may form a synthetic lethal pair with A3B-expression that results in increased DNA damage and reduced cell viability in JHOC5 cells. However, this effect may be influenced by additional, cell-specific factors.