Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells

Abstract

Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B-/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B-/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.

Keywords: ATR; Acute lymphoblastic leukemia; CHK1; Cell cycle; Doxorubicin.

Fig. 1

Subtoxic concentrations of Dox activate the G2/M cell cycle checkpoints in ALL cell lines. A Histograms showing cell viability analysis of RPMI-8402, SUP-B15, and REH cells treated with increasing concentration of Dox (RPMI-8402 from 5 to 0.25 μM, dilution 1:2; SUP-B15 and REH from 1 to 0.05 μM, dilution 1:2) for 24 h (*p < 0.05; **p < 0.01; ***p < 0.001). B Cell cycle analyses of RPMI-8402, SUP-B15, and REH cells treated with increasing concentration of Dox for 24 h. In the graph, the bars represent the mean ± standard deviation of at least three independent experiments (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). C Representative cell cycle graphs of RPMI-8402, SUP-B15, and REH treated with or without subtoxic concentrations of Dox (RPMI-8402, 0.1 μM; SUP-B15 and REH, 0.05 μM) for 48 h. Cell cycle profile of control and Dox-treated cells are represented in white and gray, respectively

Fig. 2

The pharmacological inhibition of ATR kinase enhances Dox cytotoxicity in ALL cells. A Heatmaps showing the effect of subtoxic concentrations of Dox for 48 h in combination with VE-821 for additional 24 h on cell viability of RPMI-8402, SUP-B15, and REH cells. B Heatmaps showing the effect of subtoxic concentrations of Dox for 48 h in combination with VE-821 for additional 48 h of primary ALL leukemic cells (n = 3). In the heatmaps, colors scale represents the values of mean normalized cell viability (% of cell viability relative to control) and numbers are combination index (CI) values. C Histograms showing the percentage of cells across cell cycle phases and D the percentage of Annexin V⁺ cells after treatment with subtoxic concentration of Dox (RPMI-8402, 0.1 μM; SUP-B15 and REH, 0.05 μM) for 48 h and then with VE-821 (5 μM) for additional 24 h. E Histograms representing the absolute number of cells during treatment with Dox (RPMI-8402, 0.1 μM; SUP-B15 and REH, 0.05 μM) for 48 h and then with or without VE-821 (5 μM) for further 9 days. Bars in C–E represent the mean ± standard deviation of at least three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). The drug schedule is reported in the top left of A, B, and E

Fig. 3

The pharmacological inhibition of CHK1 kinase enhances Dox cytotoxicity in ALL cells. A Heatmaps showing the effect of subtoxic concentrations of Dox for 48 h in combination with VE-821 for additional 24 h on cell viability of RPMI-8402, SUP-B15, and REH cells. B Heatmaps showing the effect of subtoxic concentrations of Dox for 48 h in combination with VE-821 for additional 48 h of three primary ALL leukemic cells. In the heatmaps, colors scale represents the values of mean normalized cell viability (% of cell viability relative to control) and numbers are combination index values. C Histograms showing the percentage of cells across cell cycle phases and D the percentage of Annexin V⁺ cells after treatment with subtoxic concentration of Dox (RPMI-8402, 0.1 μM; SUP-B15 and REH, 0.05 μM) for 48 h and then with PX (RPMI-8402: 0.0075 μM; SUP-B15: 0.03 μM; REH:0.05 μM) for additional 24 h. E Histograms representing the absolute number of cells during treatment with Dox (RPMI-8402, 0.1 μM; SUP-B15 and REH, 0.05 μM) for 48 h and then with or without PX (RPMI-8402: 0.0075 μM; SUP-B15: 0.03 μM; REH: 0.05 μM) for further 9 days. Bars in C–E represent the mean ± standard deviation of at least three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). The drug schedule is reported in the top left of A, B, and E

Fig. 4

The inhibition of the ATR-CHK1 pathway compromises mitotic regulation in ALL cell lines. A Box and whiskers plots representing mitotic index analysis of RPMI-8402, SUP-B15, and REH cells treated with Dox (RPMI-8402; 0.1 μM; SUP-B15 and REH: 0.05 μM) for 48 h and then with VE-821 (5 μM) or B PX (RPMI-8402: 0.0075 μM; SUP-B15: 0.03 μM; REH: 0.05 μM) for additional 3 h. Mitotic indices have been calculated as follows: (average number of mitotic/average total number of cells) * 100. C Histograms representing the normalized number of tripolar spindles expressed as percentage of total cell count. In the graph, black and gray bars represent Dox + VE-821 and Dox + PX combinations, respectively. D Immunofluorescence analysis showing tripolar spindles in RPMI-8402, SUP-B15, and REH cells treated with Dox (RPMI-8402: 0.1 μM; SUP-B15 and REH: 0.05 μM) for 48 h and then with VE-821 (5 μM) for additional 3 h. In the picture, cells were stained with DAPI (blue), anti-tubulin antibody (green), and anti-pericentrin antibody (red). Scale bar: 5 μm. E DAPI labeled DNA showing lagging chromosomes in RPMI-8402, SUP-B15, and REH cells treated with Dox (RPMI-8402: 0.1 μM; SUP-B15 and REH: 0.05 μM) for 48 h and then with VE-821 (5 μM) or F PX (RPMI-8402: 0.0075 μM; SUP-B15: 0.03 μM; REH: 0.05 μM) for additional 3 h. In the pictures, lagging chromosomes are pointed by yellow arrows. Scale bar: 5 μm. G DAPI labeled DNA showing chromosomes integrity analysis of RPMI-8402, REH, and SUP-B15 cells treated with Dox and then with VE-821. In the figures, fragmented chromosomes are pointed by white arrows. Scale bar: 20 μm. In the figures, statistical significance was represented as asterisks (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 5

The pharmacological inhibition of the ATR-CHK1 kinases causes G2/M checkpoint override in ALL cell lines. A Relative mRNA expression of CCNB1, CDK1, CHK1, CHK2, ATM, and ATR genes in RPMI-8402 and SUP-B15 cells treated with Dox (0.1 and 0.05 μM, respectively) for 48 h and with PX (0.0075 and 0.03 μM, respectively) or B VE-821 (5 μM) for further 3 h. In the graph, bars represent the mRNA level normalized on the control sample. The mean ± standard deviation of three independent experiments is shown. C Representative western blots of RPMI-8402 and SUP-B15 cells treated with Dox for 48 h and with PX or VE-821 for further 3 h. β-Actin was used for loading normalization. D Histograms showing ratio of pATR/ATR, pATM/ATM, pCHK1/CHK1, pCHK2/CHK2, and pCDK1/CDK1 protein levels in RPMI-8402 (black) and SUP-B15 (gray) cells treated with Dox (0.1 and 0.05 μM, respectively) for 48 h and with PX (0.0075 and 0.03 μM, respectively) or E VE-821 (5 μM) for further 3 h. Protein expression ratio is reported as the mean ± standard deviation of at least three independent experiments. In the figures, statistical significance was indicated by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 6

Schematic representation of the effect of ATR-CHK1 inhibitors in Dox damaged cells. A Graphical representation of the hypothetical mechanism of response to Dox in leukemic cells as monotherapy or B in combination with ATR-CHK1 inhibitors