Fig. 3
Talazoparib enhances INO and γ-calicheamicin cytotoxicity in ALL cell lines in terms of reduction of cell viability, induction of apoptosis and DNA damage. A Heatmaps showing the effect on cell viability of subtoxic concentrations of INO in combination with talazoparib for 72 h in ALL cells. In the heatmaps, the color scale represents the values of mean normalized cell viability (% of cell viability relative to control) of at least three independent experiments. The white number inside each square of the heatmap represents the combination index (CI) value. C.I < 1 means synergism; C.I = 1–1.3 means additivity; C.I > 1.3 means antagonist; B two-dimensional ZIP synergy map of ALL cell lines treated with increasing concentrations of INO and talazoparib for 72 h. Zero Interaction potency (ZIP) score is expressed as δ value. The black cross represents the highest values of ZIP score for each combination. C Reduction of cell viability of ALL cell lines treated with γ-calicheamicin (RS4;11 = 25 pM; REH = 160 pM; SUP-B15 = 3 pM; KOPN-8 = 20 pM) in combination with talazoparib (RS4;11 = 1 μM; REH = 5 μM; SUP-B15 = 0.2 μM; KOPN-8 = 1 μM), olaparib (RS4;11 = 10 μM; REH = 50 μM; SUP-B15 = 3 μM; KOPN-8 = 54 μM) or veliparib (RS4;11 = 70 μM; REH = 100 μM; SUP-B15 = 50 μM; KOPN-8 = 98 μM) for 24 h. Histograms represent the mean and standard deviation of normalized cell viability of three independent experiments. D Histograms represent the percentage of Annexin V + cells of CD22-positive ALL cell lines treated with INO (SUP-B15 = 4.9 ng/mL, RS4;11 = 14.7 ng/mL, KOPN-8 = 44.2 ng/mL and REH = 14.7 ng/mL) and talazoparib (SUP-B15 = 37.0 nM, RS4;11 = 333.3 nM, KOPN8 = 111.1 nM and REH = 1000 nM) for 48 h (results of at least three independent experiments). E Histograms represent the percentage of cells in different cell-cycle phases after 18, 24 and 48 h of treatment with INO alone or in combination with talazoparib. ALL cell lines were treated with INO (SUP-B15 = 4.9 ng/mL, RS4;11 = 14.7 ng/mL, KOPN-8 = 44.2 ng/mL and REH = 14.7 ng/mL) and/or talazoparib (SUP-B15 = 37.0 nM, RS4;11 = 333.3 nM, KOPN8 = 111.1 nM and REH = 1000 nM). Histograms represent mean ± standard deviation of at least three independent experiments. F Histograms represent the variation of G2/M and S phase cells between INO in combination with talazoparib and cells treated with INO alone. Positive fold-change (FC) indicates increased values while negative FC indicates decreased values. G Immunoblot analysis of ALL cells treated with INO (SUP-B15 = 4.9 ng/mL; RS4;11 = 14.7 ng/mL; KOPN8 = 44.27 ng/mL; REH = 14.7 ng/mL,) and talazoparib (SUP-B15 = 37.0 nM; RS4;11 = 333.3 nM; KOPN8 = 111.1 nM; REH = 1000 nM) for 24 h. β-actin was used for loading normalization. The numbers above the bands represent the relative quantification of band intensity. On the right, histograms represent the average signal obtained from relative band quantification of at least three independent experiments. H Histograms showing the percentage of pH2AX+/pMPM2+ cells in ALL cell lines treated with subtoxic concentrations of INO for 48 h and with talazoparib (IC50 values after 24 h of treatment) for additional 6 h. I Comet assay in SUP-B15 cell lines treated with γ-calicheamicin (IC50 value) in combination with talazoparib (IC50 value) for 18 h. Scatter-plots with histograms show the variation of Tail Area, Tail Length and Tail moment between controls and treatments. J Clonogenic results of RS4;11, REH, SUP-B15 and KOPN-8 cells treated with sub-toxic concentrations of γ-calicheamicin (SUP-B15 = 0.07 pM; REH = 2 pM; RS4;11 = 0.0013 pM; KOPN-8 = 0.2 pM) and talazoparib (SUP-B15 = 0.00019 μM; REH = 0.055 μM; RS4;11 = 0.0016 μM; KOPN-8 = 0.0055 μM) for 10–14 days. K Clonogenic results of RS4;11, REH, SUP-B15 and KOPN-8 cells treated with sub-toxic concentrations of INO (SUP-B15 = 4.8 ng/mL; RS4;11 = 17.4 ng/mL ng/mL; KOPN8 = 48 ng/mL; REH = 4.8 ng/mL) and talazoparib (SUP-B15 = 0.00019 μM; REH = 0.055 μM; RS4;11 = 0.0016 μM; KOPN-8 = 0.0055 μM) for 10–14 days