Mutations in the TP53 gene are usually associated with very poor prognosis in hematological malignancies, including strong resistance to therapy. The concept of synthetic lethality based on the inhibition of Chk1 kinase represents a promising approach for the potential elimination of these highly aggressive tumor cells. After this inhibition, the blockade of all three major cell cycle checkpoints may be presumed - the G1/S (owing to TP53 mutation), and S and G2/M (both through the Chk1 inhibition). This should result in uncontrolled cell proliferation followed by mitotic catastrophe and cell death.
The possibility of this concept was tested in 17 B-cell leukemia (n=8) or B-cell lymphoma (n=9) permanent cell lines, with eleven of them harboring TP53 mutation and/or deletion.
The cell lines were cultured in 96-well plates in quadruplicates (50 000 cells per well) with the half of plate pre-treated (2 h) by Chk1 inhibitor (SCH900776) (200 nmol/l). Subsequently, three nucleoside analogs - fludarabine, cytarabine and gemcitabine - were applied in four different concentrations for 72 h. The range of concentrations was optimized for each cell line and each drug separately in order to enable a monitoring of potential sensitization effect of Chk1 inhibition. Fludarabine was administered between concentrations 20-0125 μg/ml, cytarabine between 1.6 μg/ml−0.4 ng/ml, and gemcitabine between 25-0.25 ng/ml. The viability with and without Chk1 inhibition was assessed using WST-1 agent (Cell proliferation reagent; Roche).
In general, the cell lines having TP53 defects were much more resistant to the cytostatics than wt-TP53 cells. The Chk1 inhibitor on its own exhibited none or only negligible effect on cell viability (≥ 90% in comparison with untreated control). The effect of Chk1 inhibition for the sensitization to the tested drugs was evaluated if this sensitization was apparent in at least two bordering concentrations of the cytostatics. Using this approach, a significant sensitization effect (p<0.01; ANOVA test) was noted in 36% of mut-TP53 and none wt-TP53 cell lines for fludarabine, 45% of mut-TP53 and 17% of wt-TP53 cell lines for cytarabine, and in 82% of mut-TP53 and 33% of wt-TP5 3 cell lines for gemcitabine. In total, 9 out of 11 cell lines harboring TP53 defect were prone to clear sensitization for at least one of the drugs using the Chk1 inhibition. In analyzed cell lines, this inhibition resulted at the molecular level in enhanced phosphorylation of H2AX after 48 h treatment in comparison with cells treated with fludarabine on its own, pointing to a more extensive DNA damage.
Our results show that inhibition of Chk1 kinase can be rational way how to eliminate highly aggressive and resistant leukemia and lymphoma cells harboring TP53 mutations. The work also shows a utility of SCH900776 molecule – which is now being tested also in clinical trials - for effective Chk1 inhibition. The work was supported by the project FNUSA-ICRC (CZ.1.05/1.1.00/02.0123), MUNI/A/0784/2011, and CZ.1.07/2.4.00/17.0042.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.