The BH3 domain of BCL-2 family proteins is a critical interaction motif that mediates the crosstalk among anti- and pro-death members. We have applied hydrocarbon stapling to restore alpha-helical structure to synthetic BH3 domain peptides, recapitulating the architecture observed in the native context and/or when bound to their physiologic targets. By generating Stabilized Alpha-Helices of BCL-2 domains (SAHBs) modeled after the BID and BIM BH3 domains, we previously detected direct, quantifiable, and functional binding interactions with both anti- and pro-apoptotic BCL-2 family proteins. Like BIM BH3, PUMA BH3 is well known to broadly engage the variety of anti-apoptotic targets. However, the ability of PUMA BH3 to directly bind and activate BAX remains unresolved. Here, we report the synthesis of PUMA SAHB, a structurally-stabilized PUMA BH3 helix that, in addition to targeting the complete spectrum of anti-apoptotic proteins, directly binds to BAX. Structural analysis revealed that PUMA SAHB engages the α1/α6 trigger site of BAX in a manner analogous to the BIM BH3 helix. PUMA SAHB binding triggers functional BAX activation as reflected by dose-responsive and sequence-dependent BAX-mediated liposomal and mitochondrial release. Importantly, biotin-PUMA SAHB and Flag-PUMA protein pull-downs from cellular lysates confirm binding to the corresponding native multidomain anti- and pro-apoptotic targets. As PUMA has been implicated in driving apoptosis of a variety of neuronal cell types, we explored the therapeutic potential of PUMA SAHB activity in the context of neuroblastoma. Treatment of a variety of neuroblastoma cell lines, including SH SY5Y, Kelly and IMR5, induced dose-responsive caspase 3/7 activation and cell death. To confirm the on-target effects of PUMA SAHB, genetically modified cells lacking BAX and BAX were similarly treated, but no caspase 3/7 activation or viability effects were observed. Thus, we find that a stapled PUMA BH3 helix is a dual anti-apoptotic inhibitor and pro-apoptotic direct activator that may serve as an effective pharmacologic trigger of apoptosis in cancers of neural origin.

This work was supported by NIH grant R01 CA050239 to L.D.W. and an NSF Graduate Research Fellowship to A.L.E.

Citation Format: Amanda L. Edwards, Evripidis Gavathiotis, James L. LaBelle, Craig R. Braun, Kwadwo Opoku-Nsiah, Gregory H. Bird, Loren D. Walensky. Multimodal activation of apoptosis by a hydrocarbon-stapled PUMA BH3 helix. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4611. doi:10.1158/1538-7445.AM2013-4611