Abstract
Introduction: The gene encoding anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in a large subset of all neuroblastoma patients and plays an important oncogenic role. Previous studies with BCL-2 inhibitor ABT263 showed favorable antitumor activity for BCL-2-dependent neuroblastoma cells, but unfortunately the clinical use of ABT263 was associated with dose-limiting thrombocytopenia due to the concomitant inhibition of anti-apoptotic protein BCL-extra large (BCL-XL). Therefore, the more specific BCL-2 inhibitor ABT199 was developed. In this study, we explored the preclinical therapeutic potential of ABT199 for neuroblastoma treatment and strategies to prevent ABT199 resistance.
Experimental design: IC50 and LC50 values of ABT199 were determined for 24 neuroblastoma cell lines to study predictive biomarkers for sensitivity to ABT199. In vitro target inhibition and apoptosis were studied in predictive biomarker-positive and -negative cell lines. The in vivo efficacy of ABT199 was subsequently investigated in a BCL-2-dependent neuroblastoma xenograft model and compared with the in vivo efficacy of ABT263. Next, in vitro and in vivo effects of ABT199 on other BCL-2 family members were studied by Western Blot analysis and Affymetrix mRNA profiling. Lastly, combined effects of ABT199 and MCL-1 knockdown using shRNA or inhibition with the selective inhibitor A-1210477 were studied in vitro.
Results: The sensitivity of neuroblastoma cell lines to ABT199 was best predicted by protein levels of BCL-2 and BCL-2-like protein 11 (BIM)/BCL-2 complex. ABT199 killed neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels more potently than low expressing cell lines with average LC50 values of 0.17 versus 15.46 μmol/L, respectively. In vitro effects on cell viability nicely correlated with target-specific effects on BIM displacement from BCL-2 and downstream effects on apoptotic markers cytochrome c and cleaved PARP. Target-specific effects and effects on apoptotic markers were dose-dependent, indicating that BIM displacement from BCL-2, cytochrome c release from the mitochondria and cleaved PARP can be utilized as pharmacodynamics biomarkers of ABT199 efficacy. Oral treatment of female NMRI homozygous (nu/nu) mice with KCNR neuroblastoma xenografts expressing high BCL-2 levels with 100 mg/kg/d ABT199 for three consecutive weeks resulted in significant tumor growth inhibition. However, similar treatment with ABT263 demonstrated superior antitumor activity over ABT199 (i.e., complete tumor regression). The superior in vivo activity of ABT263 was most probably the result of the simultaneous inhibition of multiple anti-apoptotic BCL-2 family proteins by ABT263, since maximum BIM displacement from BCL-2 was observed after ABT199 treatment. ABT199 furthermore induced a strong apoptotic response, as was shown by the remarkable increase in cleaved caspase-3 levels.
We showed in vitro and in vivo that neuroblastoma cells might survive ABT199 treatment due to NOXA-mediated upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and sequestration of released BIM by MCL-1. In vitro knockdown of MCL-1 sensitized high BCL-2-expressing neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Results were confirmed with the selective MCL-1 inhibitor A-1210477.
Conclusions: Taken together, the results presented in this study strongly suggest that children with neuroblastoma tumors expressing high levels of BCL-2 and BIM/BCL-2 complex might benefit from combined treatment with ABT199 and inhibitors of MCL-1.
Citation Format: Laurel T. Bate-Eya, Ilona J.M. den Hartog, Ida van der Ploeg, Linda Schild, Jan Koster, Rogier Versteeg, Huib N. Caron, Jan J. Molenaar, M. Emmy M. Dolman. MCL-1 inhibition improves ABT199 efficacy for BCL-2-dependent neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B09.
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