Abstract 4920: Intratumoral levels of BAX-BAK heterodimer as a specific on-target pharmacodynamic biomarker of drug action by novel BH3 mimetics

Abstract

Abstract Background: Promising small-molecule inhibitors of pro-survival Bcl-2 family proteins (BH3 mimetics) are currently under clinical development. Bcl-2, Bcl-xL and Mcl-1 proteins sequester BAK and BAX, which are released by BH3 mimetics, allowing them to activate and form homo- or heterodimers at the mitochondrial membrane. Such oligomerization results in mitochondrial membrane porations, release of cytochrome c and activation of executioner caspases leading to apoptosis. Therefore, measurement of BAK-BAX heterodimers on the mitochondrial membrane is considered an on-target pharmacodynamic biomarker of drug action by BH3 mimetics. We describe development and validation of a quantitative BAX-BAK heterodimer assay as well as fitness-for-purpose for proof of mechanism studies during clinical trials. Method: The BAX-BAK heterodimer sandwich immunoassay was developed on the Luminex® platform using monoclonal antibodies specific to active BAX and BAK proteins. A recombinant fusion protein containing BAX and BAK domains, connected by a 20-amino acid linker, was used as a calibrator to mimic oligomer formed in situ by the noncovalent association of BAX and BAK proteins. Preparation of the mitochondrial fraction from core needle biopsies followed Standard Procedures described previously for a 15-biomarker apoptosis multiplex (Srivastava et al., Clin Can Res 2016). BH3 mimetics, navitoclax (NSC-759659) and an Mcl-1 inhibitor NSC-798846 (aka S63845 by Kotschy et al., Nature 2016) were used to demonstrate effectiveness of the PD assay for monitoring pharmacodynamic response. Results: The BAX-BAK heterodimer sandwich assay demonstrated satisfactory precision (CV<19%) and accuracy (mean analytical recoveries of 95%), with a minimum sample load of 4-μg protein/well. Using clinically validated sample collection and processing procedures, the assay revealed 10-45 fold increases in BAX-BAK heterodimers in multiple hematologic cancer cell lines (AMO-1, MV411, THP-1) treated for 4-6 hours with 100-1,000 nM NSC-798846 and NSC-759659. Increased levels of BAX-BAK correlated with 10-12 fold increases in cleaved caspase-3 levels measured with the apoptosis multiplex. The BAX-BAK biomarker assay has been extensively evaluated in murine tumor xenograft models and is ready for clinical use to evaluate core needle tumor biopsies. Conclusion: We have developed and validated an assay for BAX-BAK heterodimers as a biomarker of target engagement by BH3 mimetics. This assay is suitable for use in preclinical and clinical studies not only to confirm target engagement but also to optimize dose and schedule of BH3 mimetics as single agents or in combination regimens. The BAX-BAK biomarker enables pharmacodynamic assessment of investigational BH3 mimetics for clinical proof-of-mechanism studies and early clinical development. Funded by NCI Contract No HHSN261200800001E. Citation Format: Apurva K. Srivastava, Jeevan P. Govindharajulu, Dominic Esposito, Melinda G. Hollingshead, James H. Doroshow, Ralph E. Parchment. Intratumoral levels of BAX-BAK heterodimer as a specific on-target pharmacodynamic biomarker of drug action by novel BH3 mimetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4920.