Abstract 2723: The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia

Abstract

Abstract Different Checkpoint kinase inhibitors (Chk-i) have been developed to increase the cytotoxic effect of genotoxic agents inhibiting the key elements of the DNA damage response (DDR) pathways. Our group has already showed the efficacy of this class of compounds in single agent in different in vitro/ex vivo/in vivo studies for the treatment of acute lymphoblastic leukemia (ALL). The aim of the study was to evaluate the efficacy of a Chk1/Chk2 inhibitor in combination with the topoisomerase II inhibitor doxorubicin for the treatment of ALL. Firstly we evaluate the efficacy of doxorubicin on human B (NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathways. Cells were treated with doxorubicin (0.25-2.5 uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. The induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was also evaluated in all the cell lines. Due to the inhibitory effect of the compound on the topoisomerase II enzyme and due to the activation of the cell cycle checkpoint, cells were arrested in G2/M phase. Then the effectiveness of the Chk-i as a chemo-sensitizer agent was evaluated. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk-i (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a synergistic effect in term of reduction of the cell viability and induction of apoptosis. The effect of the combination was also analyzed using western blot looking for specific marker of activation of the DDR pathway showing the same synergistic effect. Moreover the effect of the combination on cell cycle profile was evaluated using a double staining Pi/Anti-phospho-Histone H3 ser10 (marker of mitosis). Cell lines were pre-treated for 18 hours with doxorubicin and then with the Chk-i for different time points (1, 2, 3, 6 and 9 hours). The treatment with Chk-i removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells positive for the mitotic marker p-HH3 (ser10) and increasing the percentage of cells in sub-G1 phase. In our opinion the combination between the Chk1 inhibitor,LY2606368, and the topoisomerase II inhibitor, doxorubicin, could be a promising strategy for the treatment of B/T-ALL. Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Andrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Enrico Derenzini, Anna Ferrari, Valentina Robustelli, Viviana Guadagnuolo, Cristina Papayannidis, Maria Chiara Abbenante, Sandro Grilli, Giovanni Martinelli. The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2723.