Direct Activation of BAX by BTSA1 Overcomes Apoptosis Resistance in Acute Myeloid Leukemia

Abstract

SUMMARY The BCL-2 family protein BAX is a central mediator of apoptosis. Overexpression of anti-apoptotic BCL-2 proteins contributes to tumor development and resistance to therapy by suppressing BAX and its activators. We report the discovery of BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site and induces conformational changes to BAX leading to BAX-mediated apoptosis. BTSA1-induced BAX activation effectively promotes apoptosis in leukemia cell lines and patient samples while sparing healthy cells. BAX expression levels and cytosolic conformation regulate sensitivity to BTSA1. BTSA1 potently suppressed human acute myeloid leukemia (AML) xenografts and increased host survival without toxicity. This study provides proof-of-concept for direct BAX activation as a treatment strategy in AML.

Overcoming resistance to mitochondrial apoptosis by BZML‐induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells

“…Our results showed that cytosolic cytochrome c levels increased while the mitochondrial cytochrome c levels decreased in BZML‐treated A549 and A549/Taxol cells. Additionally, mitochondrial outer membrane permeabilization can be affected by the Bcl‐2 family proteins . We found that BZML could time dependently up‐regulate the expression of Bax and down‐regulate the expression of Bcl‐2 in A549 and A549/Taxol cells (Figure C).…”

“…12 Our results showed that cytosolic cytochrome c levels increased while the mi- permeabilization can be affected by the Bcl-2 family proteins. 34,35 We found that BZML could time dependently up-regulate the expression of Bax and down-regulate the expression of Bcl-2 in A549 and A549/Taxol cells ( Figure 3C). Importantly, caspase-9…”

Bax mitochondrial residency is more critical than Bax oligomerization for apoptosis

“…One of these drugs, Venetoclax, is specific for heterodimers containing Bcl-2 and has been approved by the FDA to treat a certain form of chronic lymphocytic leukemia (Cang et al, 2015). In addition, compounds directly activating Bax can also promote MOMP and apoptosis (Reyna et al, 2017). MOMP has therefore been validated as a key process promoting cell death.…”

Liposomal Permeabilization Assay to Study the Functional Interactions of the BCL-2 Family

“…Additionally, the liposome permeabilization assay has been adapted as a screening tool to identify and characterize peptides, small molecules or antibodies that specifically bind to BCL-2 family members and modulate their function [7–9]. For instance, a small molecule that directly induces the activation of the BAX trigger site was characterized using this permeabilization assay [10]. Additionally, the inhibitory potential of MCL-1 small molecules binders was determined with this biochemical technique [11].…”

“…To quantify membrane permeabilization, a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and a cationic quencher (DPX: p -xylene-bis-pyridinium bromide) are incorporated within the liposomes [6]. As the liposome is permeabilized by activated BAX [10] or BAK [15], ANTS and DPX diffuse apart, and an increase in fluorescence is detected using a fluorescence plate reader. Here we will discuss in more detail the experimental procedures to measure tBID-induced BAX-mediated permeabilization of liposomes.…”