Abstract 1775: Therapeutic reactivation of cell death in refractory hematologic cancers using a broad spectrum BIM BH3 death helix

Abstract

Abstract BCL-2 family proteins are essential regulators of cellular life and death and, when deregulated, contribute to the development, maintenance, and chemoresistance of human cancer. Whereas multidomain anti-apoptotic proteins such as BCL-2 guard against apoptosis, the multidomain pro-apoptotic proteins BAX and BAK induce cell death through mitochondrial damage. The BH3-only members act as cellular antennae, poised to transmit signals of cellular injury to their multidomain counterparts, and deliver the death message via conserved alpha-helical BH3 domains. Interaction-based neutralization or genetic deletion of BCL-2 family death proteins is a common mechanism employed by cancer cells to mount an apoptotic blockade against chemotherapy and radiation treatments. The design of next-generation therapeutics based on the molecular architecture of the BIM BH3 helix offers the unique advantage of recapitulating BIM's natural capacity to directly target the full complement of anti- and pro-apoptotic BCL-2 proteins. By inserting a hydrocarbon staple into BIM BH3, we developed a Stabilized Alpha-Helix of BCL-2 domain (SAHB) peptide capable of targeting the three modes of BCL-2 family-mediated apoptotic blockade, achieving (1) anti-apoptotic inhibition, (2) pro-apoptotic direct activation, and (3) BIM BH3 replacement. We demonstrate that BIM SAHB is helical and targets BCL-2 family proteins and their complexes with high affinity in vitro and in cells. BIM SAHB, but not a binding interface mutant, induces dose-responsive apoptosis of resistant hematologic cancer cells as measured by viability, annexin V binding, and caspase 3/7 activation assays. Importantly, we demonstrate by immunoprecipitation that reactivation of the death program correlates with MCL-1/BAK dissociation and BAX activation. Non-malignant mouse embryonic and human fibroblasts demonstrate relative resistance to BIM SAHB, indicative of a therapeutic window for treatment. To evaluate the therapeutic potential of BIM SAHB in vivo, we examined the effect of pharmacologic BIM BH3 replacement in the B-cell lymphoproliferative disease of Bim−/− bone marrow-reconstituted mice. Strikingly, animals treated with BIM SAHB displayed significant TUNEL positivity within the aberrant lymphoid infiltrates, whereas no effect was observed in mice treated with vehicle or the mutant control SAHB. In addition, the surrounding parenchymal tissue was unaffected by BIM SAHB treatment, highlighting the selectivity of action and therapeutic window in vivo. Thus, we find that broad and multimodal targeting of the BCL-2 family pathway by pharmacologic replacement of the BIM BH3 helix can overcome formidable pathologic barriers to cell death in vitro and in vivo, and represents a promising strategy for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2011-1775