BCL-XL is an actionable target for treatment of malignant pleural mesothelioma

Abstract

Despite having one of the lowest survival rates of all cancers, there have been no new approved treatments for malignant pleural mesothelioma (MPM) in over a decade. Standard-of-care treatment relies on Cisplatin plus Pemetrexed chemotherapy. Here, we tested a suite of BH3-mimetic drugs targeting BCL-2 pro-survival proteins of the intrinsic apoptotic pathway. We found BCL-XL is the dominant pro-survival protein in a panel of cell lines in vitro, though potent, synergistic cell killing occurred with MCL-1 co-targeting. This correlates with high-level expression of BCL-XL and MCL-1 in cell lines and a large cohort of patient tumour samples. BCL-XL inhibition combined with Cisplatin also enhanced cell killing. In vivo BCL-XL inhibition was as effective as Cisplatin, and the combination enhanced tumour growth control and survival. Genetic ablation of MCL-1 also enhanced the effects of BCL-XL inhibitors, in vivo. Combined, these data provide a compelling rationale for the clinical investigation of BH3-mimetics targeting BCL-XL in MPM.

Fig. 1. Expression of BCL-2 family members in MPM cell lines and their sensitivity to BH3-mimetics.

A Cell lysates from established, and patient-derived MPM and other solid tumour cell lines were analysed by Western blot and probed for pro-survival or pro-apoptotic BAK/BAX and BH3-only proteins. Blots were re-probed for GAPDH or β-actin as a loading control. * Indicates non-specific band. B Relative mRNA levels for a wider set of BCL-2 genes were determined for a subset of cell lines by qRT-PCR, and generally reflect the protein expression pattern. Heatmap summarises this data. C Single agent activity of BH3-mimetics in MPM cell lines as determined by CellTiter-Glo viability assays after 72 h treatment. Drugs targeting BCL-XL were most effective. Data represent mean ± SEM (n = 3).

Fig. 2. Drug combination studies with ABT-263.

Co-treatment of MPM cells with ABT-263 and either A S63845 or B Cisplatin enhances responses over ABT-263 alone. Cell viability was determined using CellTiter-Glo assays after 72 h treatment. Data represent mean ± SEM (n = 3).

Fig. 3. BH3-mimetic combinations reduce cell viability by inducing apoptosis.

MSTO-211H cells were treated with ABT-263 or A-1331852 for 72 h in the presence or absence of A S63845 or B Cisplatin and apoptosis induction monitored by FACS using Annexin V/propidium iodide staining. C Apoptosis induction was confirmed for drugs as single agents (left panel) or in combination (right panel), by treating cells in the presence of the pan-caspase inhibitor Q-VD-OPh. Data represent mean ± SEM (n = 3) *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 (unpaired Students t-test).

Fig. 4. BCL-XL inhibition results in tumour growth control in MPM xenografts.

A MSTO-211H xenograft tumour volumes measured during and following treatment with A-1331852 (25 mg/kg, 14 days by oral gavage) and Cisplatin (4 mg/kg, once on day 1 intraperitoneal injection), the combination, or appropriate controls. B Tumour masses at Day 19 (i.e. 1 day after the final dose of A-1331852 or vehicle administration). Each point is the mass of an individual tumour (photographed) with the bar indicating the mean ± SEM (n = 3) significance determined by Student’s t-test (unpaired). C Kaplan–Meier survival curves with log-rank analysis of mice treated with A-1331852, Cisplatin and combinations of both, with relevant vehicle controls. Survival endpoint was when tumours reached 1000 mm³ as dictated by the ethics approval associated with this experiment. Significance determined by Log-rank (Mantel–Cox test). D Immunohistochemistry analysis of tumours for cleaved Caspase-3 and Ki67. Values represent the mean % positively stained cells for each antibody in five different fields of view. Data are mean ± SEM (n = 3), significance determined by Student’s t-test (unpaired). E Responses of MSTO-211H cells to Cisplatin determined by Western blot. Cell lysates were prepared 72 h after treatment with indicated concentrations of Cisplatin and Western blots probed with antibodies to the indicated BCL-2 family proteins. Western blots were reprobed with antbody to GAPDH as a loading control. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

Fig. 5. MCL-1 deletion increases responses to BCL-XL in vivo.

A MSTO-211H cells (with Cas9/MCL-1 sgRNA) tumour volumes measured during and following treatment with A-1331852 (25 mg/kg, 14 days by oral gavage), feeding doxycycline-containing chow, or appropriate controls. B Tumour masses at Day 19 (i.e. 1 day after the final dose of A-1331852 or vehicle administration). Each point is the mass of an individual tumour (photographed) with the bar indicating the mean ± SEM (n = 3). Significance determined by Student’s t-test (unpaired). C Kaplan–Meier survival curves with log-rank analysis of mice treated with A-1331852, doxycycline and combinations of both, with relevant vehicle controls. Survival endpoint was when tumours reached 1000 mm³ as dictated by the ethics approval associated with this experiment. Significance determined by Log-rank (Mantel–Cox test). D Immunohistochemistry analysis of tumours for cleaved Caspase-3 and Ki67. Values represent the mean % postively stained cells for each antibody in five different fields of view. Data are mean ± SEM (n = 3), significance determined by Student’s t-test (unpaired). ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

Fig. 6. BCL-2 protein expression in MPM patient samples.

A Quantitation of the percentage of patient tumour samples which have high or low expression of each BCL-2 family pro-survival protein (BCL-XL, MCL-1, and BCL-2) and pro-apoptotic protein (BAK and BAX) based on stratified H-scores. B Kaplan–Meier overall survival curves and correlation with expression of BCL-XL, MCL-1, BCL-2. Significance in Kaplan–Meier survival analysis was performed using the Mantel–Cox test.