BH3 mimetic drugs cooperate with Temozolomide, JQ1 and inducers of ferroptosis in killing glioblastoma multiforme cells

Abstract

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer, with treatment options often constrained due to inherent resistance of malignant cells to conventional therapy. We investigated the impact of triggering programmed cell death (PCD) by using BH3 mimetic drugs in human GBM cell lines. We demonstrate that co-targeting the pro-survival proteins BCL-XL and MCL-1 was more potent at killing six GBM cell lines compared to conventional therapy with Temozolomide or the bromodomain inhibitor JQ1 in vitro. Enhanced cell killing was observed in U251 and SNB-19 cells in response to dual treatment with TMZ or JQ1 combined with a BCL-XL inhibitor, compared to single agent treatment. This was reflected in abundant cleavage/activation of caspase-3 and cleavage of PARP1, markers of apoptosis. U251 and SNB-19 cells were more readily killed by a combination of BH3 mimetics targeting BCL-XL and MCL-1 as opposed to dual treatment with the BCL-2 inhibitor Venetoclax and a BCL-XL inhibitor. The combined loss of BAX and BAK, the essential executioners of intrinsic apoptosis, rendered U251 and SNB-19 cells refractory to any of the drug combinations tested, demonstrating that apoptosis is responsible for their killing. In an orthotopic mouse model of GBM, we demonstrate that the BCL-XL inhibitor A1331852 can penetrate the brain, with A1331852 detected in both tumour and healthy brain regions. We also investigated the impact of combining small molecule inducers of ferroptosis, erastin and RSL3, with BH3 mimetic drugs. We found that a BCL-XL or an MCL-1 inhibitor potently cooperates with inducers of ferroptosis in killing U251 cells. Overall, these findings demonstrate the potential of dual targeting of distinct PCD signalling pathways in GBM and may guide the utility of BCL-XL inhibitors and inducers of ferroptosis with standard of care treatment for improved therapies for GBM.

Fig. 1. BH3 mimetic drugs targeting MCL-1 and BCL-XL kill GBM cells more potently compared to TMZ or JQ1 monotherapy.

Viability of the human GBM cell lines, SNB-19, SNB-75, U251, SF-268, SF-295 and SF-539, after treatment with the indicated concentrations of a TMZ, b JQ1 or c dual treatment with S63845 (MCL-1 inhibitor) plus A1331852 (BCL-XL inhibitor). ATP levels were determined after 5 days of continuous treatment using the CellTiter-Glo® assay with data presented relative to DMSO (vehicle control) treated cells. n = 3 independent experiments, data are presented as mean ± S.D. One-way ANOVA with the Dunnett’s multiple comparisons tests relative to DMSO vehicle treated cells. *P  <  0.05, **P < ,0.01, ***P  <  0.001, ****P  <  0.0001.

Fig. 2. Combination treatments with BH3 mimetic drugs targeting MCL-1, BCL-XL or BCL-2 causes increased apoptosis of GBM cells compared to single agent treatment.

a, b U251 and SNB-19 cells were treated as indicated with BH3 mimetic drugs as single agents or combinations of BH3 mimetic drugs at 1 µM each for 5 days. Cell viability was determined by staining with Annexin V plus DAPI followed by flow cytometric analysis. n = 3 independent experiments, data are presented as mean ± s.d. One-way ANOVA with the Dunnett multiple comparison test relative to DMSO (vehicle control) treated cells. c, d U251 or SNB-19 cells were treated with the indicated drug combinations for 16, 24 or 48 h. Cell viability was determined by staining with Annexin V plus DAPI followed by flow cytometric analysis. n = 3 independent experiments, data are presented as mean ± s.d. One-way ANOVA with the Tukey’s multiple comparisons tests relative to DMSO (vehicle control) treated cells or as indicated between treatment groups. *P  <  0.05, ***P  <  0.001, ****P  <  0.0001.

Fig. 3. Combinations of BH3 mimetic drugs with TMZ or JQ1 cause increased killing of GBM cells compared to TMZ or JQ1 monotherapy.

a U251 cells were treated for 5 days with 1 µM A1331852 (BCL-XL inhibitor), 1 µM S63845 (MCL-1inhibitor), 1 µM Venetoclax (BCL-2 inhibitor); 50 µM TMZ; 1 µM JQ1 as single agents or with the indicated drug combinations. Cell viability was determined by staining with Annexin V plus DAPI followed by flow cytometric analysis. n = 3 independent experiments, data are presented as mean ± s.d. One-way ANOVA with the Tukey’s multiple comparisons tests relative to DMSO (vehicle control) treated cells or as indicated. *P  <  0.05, **P < ,0.01, ***P  <  0.001, ****P  <  0.0001. b Effect of A1331852, TMZ and JQ1 as single agents or combination treatments as indicated on cell cycle progression. U251 and SNB-19 cells were treated for 48 h with 1 µM A1331852, 1 µM JQ1 or 50 µM TMZ. DMSO was used as vehicle control. Cells were fixed in 70% ethanol followed by DAPI staining and flow cytometric analysis. Percentages of cells in the sub-G1 (dying), G1, S and G2/M phases are indicated. Histograms shown are representative of results from three independent experiments.

Fig. 4. Cleavage of caspase-3 and PARP1, markers of apoptosis, is seen in U251 and SNB-19 cells after treatment with TMZ or JQ1 plus BH3 mimetic drugs.

a, c Western blot analysis of lysates from U251 or SNB-19 cells that had been treated with 1 µM A1331852 (BCL-XL inhibitor), 1 µM Venetoclax/ABT-199 (BCL-2 inhibitor) or 1 µM S63845 (MCL-1 inhibitor) for 16 h as single agents or the indicated drug combinations. b, d Western blot analysis of lysates from U251 or SNB-19 cells that had been treated for 4 days with 1 µM A1331852, 1 µM S63845, 1 µM Venetoclax/ABT-199, 50 µM TMZ or 1 µM JQ1. Membranes were probed for cleaved (activated) caspase-3 (CC3; p19, 17 fragments indicated) and cleaved PARP1. * indicates cleaved PARP1 fragment. Probing for β-actin was used as a loading control.

Fig. 5. U251 and SNB-19 cells lacking both BAX and BAK, the essential effectors of apoptosis, are profoundly resistant to killing by combination treatments with TMZ or JQ1 with BH3 mimetic drugs.

a Western blot analysis of lysates from U251 or SNB-19 cells that had been left untreated or treated with 1 µg/mL doxycycline for 5 days to induce expression of Bax and Bak sgRNAs. Blots were probed for BAX, BAK and β-actin, the latter used as a loading control. b, c, d U251 or SNB-19 cells were left untreated (control) or treated with 1 µg/mL doxycycline for 5 days to induce sgRNAs for Bax and Bak. b Cells were then treated for 48 h with 1 µM A1331852 (BCL-XL inhibitor), 1 µM S63845 (MCL-1 inhibitor) or 1 µM Venetoclax/ABT-199 (BCL-2 inhibitor) with the indicated combinations. Shown are representative dot plots following staining with Annexin V plus DAPI followed by flow cytometric analysis. c, d Cells were treated for 5 days with 1 µM A1331852 (BCL-XL inhibitor), 1 µM S63845 (MCL-1 inhibitor), 1 µM Venetoclax/ABT-199 (BCL-2 inhibitor), 50 µM TMZ, 1 µM JQ1 as single agents or with the indicated drug combinations. Cell viability was determined by staining with Annexin V plus DAPI followed by flow cytometric analysis. n = 3 independent experiments; data are presented as mean ± s.d. Two-way ANOVA with the Bonferroni’s multiple comparisons tests. *P  <  0.05, **P < 0.01, ***P  <  0.001, ****P  <  0.0001. e Western blot analysis of lysates from BAX/BAK DKO U251 cells that had been treated for 16 h with 1 µM A1331852, 1 µM Venetoclax/ABT-199 or 1 µM S63845 as single agents or with the combinations of agents indicated. f Western blot analysis of lysates from BAX/BAK DKO U251 cells that had been treated for 5 days with 1 µM A1331852, 1 µM S63845, 1 µM Venetoclax/ABT-199, 50 µM TMZ or 1 µM JQ1. Membranes were probed for cleaved (activated) caspase-3 (CC3; p19, 17 fragments indicated) and cleaved PARP1. * indicates cleaved PARP1 fragment. Probing for β-actin was used as a loading control. Lysates from parental U251 cells that had been treated with 1 µM A1331852 plus 1 µM S63845 were used as a positive control for cleaved caspase-3 and cleaved PARP1.

Fig. 6. A1331852 can penetrate the brain in an orthotopic mouse model of GBM.

a NOD/SCID/γc^-/- mice were intra-cranially injected with 5×10⁴ U251-mCh-Luc cells using a stereotactic frame. Successful engraftment and growth of tumour cells was detected on day 7 by bioluminescent imaging using the IVIS imaging system. b Platelet counts in wt NOD/SCID/γc^-/- mice intra-cranially injected with U251-mCh-Luc cells and then administered with vehicle (as a control) or 50 mg/kg body weight A1331852 (BCL-XL inhibitor) daily for 5 consecutive days. Blood was collected via cardiac puncture and shown are platelet counts 5 days post treatment. n = 5 wt NOD/SCID/γc^-/- mice administered with vehicle (as a control) and n = 4 wt NOD/SCID/γc^-/- mice administered with 50 mg/kg body weight A1331852 (BCL-XL inhibitor). Data are presented as mean ± s.d. student’s unpaired t-test ****P  <  0.0001. c Concentration of A1331852 in plasma, brain (healthy region or tumour region), spleen, and liver in tumour bearing mice following a 5-day treatment with this BH3 mimetic drug. Data are presented as mean ± s.d. n = 4 per tissue analysed.

Fig. 7. BH3 mimetic drugs cooperate with inducers of ferroptosis in the killing of GBM cells.

a, b U251 cells were treated for 24 h with the indicated concentrations of erastin (a) or RSL3 (b) (both inducers of ferroptosis) ± 1 μM liproxstatin-1 or 50 μM deferoxamine (both inhibitors of ferroptosis). Cell viability was determined by using the MTT assay and data are expressed relative to vehicle control treated cells. Data are presented as means ± s.e.m; n = 9 from 3 independent experiments. One-way ANOVA with the Dunnet’s multiple comparisons test. ****P  <  0.0001. Data were fitted using a non-linear regression curve. c, d U251 cells were treated for 24 h with the indicated concentrations of S63845 (MCL-1 inhibitor) plus A1331852 (BCL-XL inhibitor) ± the indicated concentrations of erastin (c) or RSL3 (d). Cell survival was determined as described above. Data are presented as means ± s.e.m, n = 9 from 3 independent experiments. Two-way ANOVA with the Tukey’s multiple comparisons tests relative to S63845 and A1331852 treated cells at the indicated concentrations; *P  <  0.05, **P < ,0.01, ***P  <  0.001, ****P  <  0.0001. Data were fitted using a non-linear regression curve. e, f Parental U251 cells and g, h BAX/BAK DKO U251 cells were treated for 24 h with vehicle control, 0.2, 0.4 or 0.8 µM S63845 plus 0.2, 0.4 or 0.8 µM A1331852 ± 1 μM liproxstatin-1, ± 2.5 μM erastin (e, g) or 100 nM RSL3 (f, h). Cell survival was determined as described above. Data are presented as means ± s.e.m, n = 9 from 3 independent experiments. Two-way ANOVA with the Tukey’s multiple comparisons tests relative to control or as indicated between treatment groups, *P  <  0.05, **P < ,0.01, ***P  <  0.001, ****P  <  0.0001.