BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer

Abstract

Metastatic BRAF^V600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAF^V600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAF^V600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAF^V600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-X_L, and that combining encorafenib with a BCL-X_L inhibitor significantly enhances apoptosis in BRAF^V600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-X_L inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-X_L inhibition, we also examined the effect of combining encorafenib with the BCL-X_L -targeting PROTAC DT2216, and the novel BCL-2/BCL-X_L inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAF^V600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-X_L inhibition significantly enhances apoptosis in pre-clinical models of BRAF^V600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

Fig. 1. BRAF inhibitors induce predominantly cytostatic effects in BRAF^V600E CRC cell lines.

A, B BRAF^V600E CRC cell lines were treated with vemurafenib (Vem, 5 μM) or encorafenib (Enc, 100 nM) for 72 h and stained with propidium iodide (PI) and analysed by FACS to determine (A) apoptosis induction (percentage of cells with sub-diploid DNA content), and (B) percentage of cells in S-phase. Values shown are mean ± SEM from a representative experiment performed in technical triplicate. Similar results were obtained in two additional independent experiments. Groups were compared using an unpaired Student’s t-test using Welch’s correction; *(p ≤ 0.05), **(p ≤ 0.01) and ***(p ≤ 0.001). C BRAF^V600E CRC cell lines were treated with encorafenib (100 nM) plus cetuximab (10 μg/mL) (Enc+Cet) or irinotecan (10 μM)(Irino) for 72 h and apoptosis levels determined by PI staining as above. Values shown are mean ± SEM from a representative experiment performed in technical triplicate. Similar results were obtained in 2 additional independent experiments. Differences were compared using one-way ANOVA with Tukey’s multiple comparison testing; ns (not significant), *(p ≤ 0.05), **(p ≤ 0.01) and ****(p ≤ 0.0001). D, E Western blot analysis for BIM_S/L/EL, MCL-1 and BCL-X_L in BRAF^V600E CRC cell lines treated with (D) encorafenib (100 nM) or vemurafenib (5 μM) or (E) encorafenib (100 nM) plus cetuximab (10 μg/mL) for 6 h. β-tubulin was used as a loading control.

Fig. 2. CRCs express high basal expression of BCL-X_L and MCL-1.

A Basal mRNA expression levels of pro-survival genes in human CRCs. Data obtained from the COAD cohort profiled by the cancer genome atlas (TCGA)(n = 266). B Basal mRNA expression of pro-survival genes in CRC cell lines. mRNA expression was extracted from n = 58 CRC cell lines profiled by RNA-seq analysis as previously reported [29]. C Relative mRNA expression of pro-survival genes in primary BRAF^WT versus BRAF^V600E CRCs. Data obtained from the TCGA COAD cohort (WT, n = 236, BRAF-mutant, n = 30). D Relative mRNA expression of pro-survival genes in BRAF^WT versus BRAF^V600E CRC cell lines. mRNA expression was extracted from n = 47 BRAF^WT and n = 11 BRAF^V600E CRC cell lines profiled by RNA-seq analysis by [29].

Fig. 3. Effect of combining BRAF inhibitors with BH3 mimetics on apoptosis in BRAF^V600E CRC cell lines.

BRAF^V600E CRC cell lines were treated with encorafenib (100 nM) alone and in combination with either (A) the MCL-1 inhibitor S63845 (1 μM), (B) the BCL-X_L inhibitor A-1331852 (10 nM) or (C) the BCL-2 inhibitor ABT-199 (1 μM) for 72 h and apoptosis induction determined by PI staining and FACS analysis. Values shown are mean ± SEM from a representative experiment performed in technical triplicates. Differences between groups were compared using one-way ANOVA with Tukey’s multiple comparison testing; ns (not significant), *(p ≤ 0.05), ***(p ≤ 0.001) and ****(p ≤ 0.0001).

Fig. 4. BRAF + BCL-X_L inhibitor induced apoptosis requires BIM induction.

A Validation of CRISPR-mediated deletion of BIM in COLO 201 cells. Cells were maintained in 1 μg/mL of doxycycline (+DOX) to induce BIM deletion and subsequently treated with encorafenib (100 nM) for 6 h to induce BIM expression. BIM expression [extra-long (BIM_EL), Long (BIM_L) and short (BIM_S) forms] was assessed by western blot with β-tubulin used as a loading control. B Control and BIM-deleted COLO 201 cells were treated with encorafenib (100 nM) and A-1331852 (10 nM) alone and in combination for 72 h and apoptosis determined by PI staining and FACS analysis. Values shown are mean ± SEM from a representative experiment performed in technical triplicate. Similar results were obtained in a second independent experiment. One-Way ANOVA, with Tukey’s multiple comparison testing; ****(p≤0.0001). C Western blot analysis of cleaved caspase 3 (Cl. Caspase 3) induction following treatment of control and BIM-deleted COLO 201 cells with encorafenib (100 nM) plus A-1331852 (10 nM) for 12 h. β-tubulin was used as a loading control.

Fig. 5. Effect of combining the BCL-X_L -targeting PROTAC, DT2216, with encorafenib in BRAF^V600E CRC cells.

A Western blot analysis of BCL-X_L expression following treatment of BRAF^V600E CRC cells with DT2216 (10, 100 and 1000 nM) for 24 h. β-tubulin was used as a loading control. B BRAF^V600E CRC cells were treated with encorafenib (100 nM) and DT2216 (10, 100 or 1000 nM) alone and in combination for 72 h and apoptosis determined by PI staining and FACS analysis. Values shown are mean ± SEM from 3 independent experiments, except for LIM2405 (values shown are mean ± SEM from 2 independent experiments). Differences were compared using one-way ANOVA with Tukey’s multiple comparison testing; ns (not significant), *(p ≤ 0.05), **(p ≤ 0.01), ***(p ≤ 0.001), and ****(p ≤ 0.0001).

Fig. 6. Anti-tumour effects of encorafenib plus AZD4320/AZD0466 on BRAF^V600E CRC cells in vitro and in vivo.

A COLO 201 cells were treated with encorafenib (100 nM) and AZD4320 (500 nM) alone and in combination for 72 h in vitro and apoptosis determined by PI staining and FACS analysis. Values shown are mean ± SEM from 3 independent experiments. Differences were compared using one-way ANOVA with Tukey’s multiple comparison testing; ****(p ≤ 0.0001). B Effect of encorafenib and AZD0466 on the growth of COLO 201 xenografts. NOD scid gamma mice (n = 8 per group) were subcutaneously injected with 2 × 10⁶ COLO 201 cells into the left and right flanks (n = 16 tumours per group except the combination treatment group where n = 14). Mice were then randomised to receive either vehicle control, encorafenib (20 mg/kg, b.i.d, og), AZD0466 (103 mg/kg, once weekly, I.V) or the combination, for 22 days. Tumour volume was determined by caliper measurements every second day and normalised to the volume at day 1 of treatment. Values shown are mean ± SEM. (C) Weight (g) and (D) representative images of excised tumours at experimental endpoint. Hash (#) depicts one less mouse (two less tumours) for the combination treatment group. E Relative change in mouse body weight from treatment commencement. Values shown are mean ± SEM. F Representative images of immunohistochemical staining of BIM in resected tumours and corresponding annotation performed using HALO software depicting areas of low (yellow), moderate (orange) and strong (red) BIM staining. G Quantification of BIM staining (percentage of positively stained tumour cells) in resected tumours. Values shown are mean ± SEM. Differences were compared using one-way ANOVA with Tukey’s multiple comparison testing; *(p≤0.05), **(p≤0.01), ***(p≤0.001) and ****(p≤0.0001).