NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death

PMAIP1/NOXA gene amplification increases DLBCL vulnerability to BCL2 inhibitors. (A) Representative FISH staining in U-2932 and Ri-1 cells demonstrating an increase BCL2 (red) and PMAIP1/NOXA (green) copy numbers. Centrosome of chromosome 18 is shown in light blue. (Magnification: 63×.) (B) NOXA genetic silencing attenuates S55746 activity in Ri-1 DLBCL cells. Ri-1 cells were transfected with 1 μM scramble or PMAIP1/NOXA siRNA and incubated with increasing concentrations of S55746 (0.1, 0.25, and 0.5 μM). (C) Cell viability was assessed for cells in B by MTS assay after 48 h. Error bars represent SEM of triplicate experiments. Differences between groups were calculated with the Student t test. ***P = 0.005, ****P < 0.0001. (D) NOXA ectopic overexpression enhanced the efficacy of S55746 in HBL-1 DLBCL cells. (E) Cell viability was assessed for cells in D by MTS assay after 48 h. Viability data were normalized to effect of NOXA overexpression alone. Error bars represent SEM of triplicate experiments. *P < 0.05, ***P < 0.0005.

BCL2 inhibitors deplete MCL1 in DLBCL cells harboring NOXA gene amplification by a caspase-dependent mechanism. (A) DLBCL cells Ri-1 and U-2932 harboring NOXA genetic amplification were incubated with 0.25 μM of S55746 or venetoclax (ABT199) for 72 h. Neither drug had an effect on BCL2 protein levels, but both drugs depleted MCL1 protein. Within the time frame and drug concentrations, BCL2 inhibitors also increased NOXA protein levels in these cell lines. (B) S55746-induced MCL1 depletion was prevented by the pan-caspase inhibitor Z-VAD-fmk. Ri-1 cells were incubated with S55746 (0.05 μM) and increasing concentrations of ZVAD-fmk for 24 h, before protein levels were determined by Western blotting. In the absence of caspase inhibition, S55746 cleaved caspase 3 and PARP and depleted MCL1. These events were reversed by the pan-caspase inhibitor ZVAD-fmk.

BCL2 inhibition increases MCL1 protein abundance in a BIM-dependent manner. (A) Western blot analysis showing the effect of BCL2 inhibitors S-55746 and ABT-199 with two doses (0.25 μM and 1 μM for 24 h) on MCL-1, BCL2, and Bim protein expression in Bim-positive cell lines (SU-DHL-4 and HBL-1) vs. Bim-negative cell lines (Mino and Jeko-1). (B) SU-DHL-4 cells were treated with 0.1 DMSO, 0.5 μM S-55746, 0.5 μM ABT199, 5 μM A-1210477, and 5 μM UMI-77, respectively, for 24 h. The interaction of Bim and MCL-1 was determined by immunoprecipitation (IP) and analyzed by Western blot analysis.

S55746 synergizes with panobinostat in vitro and in vivo in DLBCL. (A) Western blot showing increase in NOXA protein levels and decrease in MCL1, after treatment with the combination of panobinostat and S55746 for 24 h. (B) Heat maps showing the effect of the combination of S55746 and panobinostat on cell viability. Percentage of cell viability is depicted in a colorimetric scale from red (high) to green (low) normalized to DMSO (control). Values are the mean ± SD of three separate determinations. Cells were incubated with increasing concentrations of S55746 and panobinostat for 24 h and cell viability was determined by Celltiter-Glo assay. (C) NSG mice (n = 8 per treatment group) were injected with DLBCL PDX and with either vehicle, panobinostat(5 mg/kg five times weekly), UMI-77 (60 mg/kg every other day), S55746 (75 mg/kg five times weekly), or the two drugs together for 3 wk and observed until death after the end of the treatment. Differences among groups were calculated with the ANOVA with Dunnett’s test. *P = 0.04, **P = 0.003, ***P = 0.0007, ****P < 0.0001.