Enhance the Efficacy of Doxorubicin Destabilizing the G2/M Checkpoint with a CHK1/CHK2 Inhibitor Pre-Treatment on Acute Lymphoblastic Leukemia

The anthracycline antibiotic, doxorubicin, inhibits the topoisomerase II enzyme resulting in the abrogation of DNA synthesis and in the induction of DNA damages. This compound is normally used in several chemotherapy schedules for the treatment of leukemia. From a biological point of view, it has been reported that doxorubicin-treated cells arrest in G2/M phase as a consequence of the induction of DNA damages and of the induction of the DNA damage response (DDR) pathway. The activation of the G2/M checkpoint is regulated by three kinases: the cell-cycle checkpoint kinase 1 (CHK1) and 2 (CHK2) and WEE1. The activity of these kinases, which act as important tumor-suppressors on normal cells, can protect tumor cells from genotoxic agents. During the last years several studies have showed the efficacy of selective DDR inhibitors in single agent or in combination with DNA damaging agents. On these bases, the aim of the study was to evaluate the effects of a selective CHK1/CHK2 inhibitor (hereafter CHKi) in combination with doxorubicin (hereafter Doxo) on a panel of B and T acute lymphoblastic leukemia (ALL) cell lines (NALM-6, NALM19, RPMI-8402, MOLT-4 and CCRF-CEM). To this purpose we firstly evaluate the effect of Doxo in single agent on B-/T-ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathway. Cells were treated for 24/48 hours with increasing concentration of Doxo (0.25, 0.5 and 1 uM) showing a dose-dependent reduction of the cell viability, induction of apoptosis and increment of cells arrested in S or G2/M phases of the cell cycle. No differences in term of sensitivity to Doxo were seen between ALL cell lines with wildtype or mutated TP53. Then we evaluated the chemosensitizer efficacy of the CHKi against Doxo on the panel cell lines. To this purpose cells were treated simultaneously with increasing concentration of Doxo in combination with the CHKi for 24/48 hours and then the combination index of the two compounds was evaluated using Compusy Software. Only an additive effect in term of reduction of the cell viability was seen when the two compounds were added simultaneously. Based on these results we performed different schedules of combinations firstly treating with Doxo and then with CHKi or vice versa, defining the most effective schedule when cells were treated firstly with the CHKi (1 hour) and then with Doxo (24 hours). In this experimental setting the strongest effect was seen on MOLT-4 (TP53 mutated) cell line in which the pre-treatment with the CHKi impaired the G2/M checkpoint induced, later, by Doxo. Immunoblotting analyses showed that, in MOLT-4, the effect on the cell viability was associated with an increment of DNA damage (gH2AX) which was significantly higher in the combination. Interestingly on MOLT-4 the treatment with Doxo in single agent activated the cell cycle inhibitor p21 but not in the sample treated with the combination. On NALM-6 cell line (TP53 wildtype), the treatment with Doxo alone or in combination with CHKi activated p21. This data was associated with the poor effectiveness of the combination on NALM-6 cell line (poor reduction of cell viability and of induction of gH2AX). Although further experiments should be performed, first of all to investigate the role of p21 on the G2/M checkpoint, in our opinion the combination between Doxo and CHKi could be a promising strategy for the treatment of ALL. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, HARMONY project.