2-Methoxy antimycin reveals a unique mechanism for Bcl-x(L) inhibition

Overexpression of Bcl-x(L) in multiple cancers correlates with resistance to chemotherapy and radiation therapy, and provides a rationale for development of small-molecule Bcl-x(L) inhibitors. Based on knockout studies, nonneoplastic cells also require Bcl-x(L) survival functions, particularly when challenged with cytotoxic agents. We analyze the selective cytotoxicity of one Bcl-x(L) inhibitor, 2-methoxy antimycin A, toward cells with excess exogenous Bcl-x(L) in isogenic cell line pairs. This selectivity, characteristic of a gain-of-function mechanism, is not shared by other known Bcl-x(L) inhibitors, including BH3I-2, HA14-1, ABT-737, gossypol, or the stapled BH3 helical peptide SAHB-BID. We show that Bcl-x(L) overexpression induces a shift in energy metabolism from oxidative phosphorylation to glycolysis. Treatment with 2-methoxy antimycin A acutely reverses the metabolic effects of Bcl-x(L), causing mitochondrial hyperpolarization and a progressive increase in mitochondrial NAD(P)H. We identify an additional small-molecule Bcl-x(L) inhibitor, NSC 310343, establishing a class of Bcl-x(L) inhibitors with gain-of-function activity. In contrast to other Bcl-x(L) inhibitors, combining gain-of-function Bcl-x(L) inhibitors with a standard inducer of apoptosis, staurosporine, enhances selective cytotoxicity toward Bcl-x(L)-overexpressing cells. These results provide an example of the intersection of bioenergetic metabolism and Bcl-x(L) functions and suggest a metabolic basis for the gain-of-function mechanism of Bcl-x(L) inhibitors.