FIGURE 4.

CX-4945 potentiates Venetoclax induced cytotoxicity. (A) Histograms depicting the percentage of Annexin V positive Jeko-1, Granta-519 and Rec-1 cells after 24 h treatment with DMSO (not higher than 0.1% v/v; white columns), CX-4945 (1 μM for Jeko-1 and Granta-519, 0.5 μM for Rec-1; grey columns), VEN (20 nM for Jeko-1, 1.25 nM for Granta-519 and 50 nM for Rec-1; black columns) or their combination (striped columns). Data are expressed as ratio over DMSO treated cells, mean ± SD of n = 10 (Jeko-1), n = 9 (Granta-519) and n = 6 (Rec-1) independent experiments. * indicates p < 0.05 compared to DMSO treated cells; # indicates p < 0.05 compared to both CX-4945 and VEN only treated cells. (B) Histogram showing the percentage of Annexin V positive B cells derived from one MCL patient after 24 h treatment with DMSO (not higher than 0.1% v/v; white column), CX-4945 (1 μM and 2.5 μM; grey columns), VEN (10 nM; black column) or their combination (striped columns). Data are expressed as ratio over DMSO treated cells. (C) Histogram showing the percentage of Annexin V positive healthy B cells treated for 24 h with DMSO (not higher 0.1% v/v; white column), CX-4945 (1 μM and 2.5 μM; grey columns), VEN (5 and 10 nM; black columns) or their combination (striped and dotted colums). Data are normalized on DMSO treated cells and are expressed as the mean ± SD of n = 3 independent experiments. (D) Representative WB showing the expression levels of uncleaved and cleaved PARP in Jeko-1 and Rec-1 cells treated as in (A). β-actin was used as loading control. Experiments were repeated at least three times for each cell line. (E) Representative WB showing the expression levels of Mcl-1 in Jeko-1, Granta-519 and Rec-1 cells treated as in (A). β-actin was used as loading control. Experiments were repeated at least three times for each cell line. (F) Dose response curves of Jeko-1 and Rec-1 cells incubated for 72 h with increasing concentrations of CX4945 (green curves), VEN (red curves) or their combination (purple curves). For Jeko-1, IC₅₀ of the single treatments were 2.30 μM for CX4945 and 6.68 μM for VEN. IC₅₀ for CX4945 used in combination with VEN was 0.74 μM, while IC₅₀ for VEN used in association with CX-4945 was 2.13 μM. The calculated CI was 0.64. For Rec-1, IC₅₀ of the single treatments were 0.76 μM for CX-4945 and 4.0 μM for VEN. IC₅₀ for CX-4945 used in combination with VEN was 0.23 μM, while IC₅₀ for VEN used in association with CX-4945 was 1.22 μM. The calculated CI was 0.60. Data are presented as averaged percentage over control of three independent experiments for each cell line.