Confounding off-target effects of BH3 mimetics at commonly used concentrations: MIM1, UMI-77, and A-1210477

Abstract

Targeting anti-apoptotic BCL2 family proteins has become an attractive therapeutic strategy for many cancers, and the BCL2-selective inhibitor ABT-199 (venetoclax) has obtained clinical success. However, MCL1 can promote drug resistance and overall cancer cell survival. Thus, there is a critical need to develop an effective drug that antagonizes MCL1. However, most putative MCL1 inhibitors have been misclassified as they fail to directly inhibit MCL1 in cells, but rather induce the pro-apoptotic protein NOXA. We have investigated three putative MCL1 inhibitors: MIM1, UMI-77, and A-1210477. All three compounds were developed in cell-free assays and then found to be cytotoxic, and hence assumed to directly target MCL1 in cells. Here, we investigated whether these compounds directly inhibit MCL1 or inhibit MCL1 indirectly through the induction of NOXA. Both MIM1- and UMI-77-induced NOXA through the unfolded protein response pathway, and sensitized leukemia cells to ABT-199; this cytotoxicity was dependent on NOXA suggesting that these compounds do not directly target MCL1. A-1210477 was the only compound that did not induce NOXA, but it still sensitized cells to ABT-199. A-1210477 induced accumulation of MCL1 protein consistent with it binding and preventing MCL1 degradation. However, at concentrations used in several prior studies, A-1210477 also induced cytochrome c release, caspase activation, and apoptosis in a BAX/BAK-independent manner. Furthermore, the release of cytochrome c occurred without loss of mitochondrial membrane potential. This apoptosis was extremely rapid, sometimes occurring within 0.5–1 h. Hence, we have identified a novel mechanism of apoptosis that circumvents the known mechanisms of cytochrome c release. It remains to be determined whether these unexpected mechanisms of action of putative BH3 mimetics will have therapeutic potential.

AT101 [(-)-Gossypol] Selectively Inhibits MCL1 and Sensitizes Carcinoma to BH3 Mimetics by Inducing and Stabilizing NOXA

“…While many of the putative BH3 mimetics were shown to induce NOXA in leukemia and lymphoma cell lines, it was unknown whether these compounds would induce NOXA in carcinoma cell lines, or which compound is the most potent. We chose three mimetics whose mechanisms of NOXA induction were established by our laboratory: AT101, S1 and UMI-77 [ 18 , 20 , 25 ]. In addition, we have shown that microtubule-disrupting agents can also induce NOXA, and used BNC105 as a representative agent [ 26 ].…”

“…AT101 alone induced little apoptosis in any of the cell lines despite induction of NOXA ( Figure 1 A). We and others have shown that NOXA-inducing compounds can sensitize cancer cells to other compounds, such as inhibitors of BCL2 and BCL-XL [ 18 , 33 , 34 ]. We sought to determine whether AT101 could sensitize carcinoma cells to other BH3 mimetics.…”

“…NOXA is an effective, natural antagonist of MCL1 that can both kill MCL1-dependent cells and synergize with other compounds. Many putative BH3 mimetics were misclassified as direct MCL1 inhibitors, yet they were still able to sensitize cancer cells to other compounds because of their ability to induce NOXA, revealing their therapeutic potential [ 17 , 18 ]. Here, we show that AT101, a previously misclassified BH3 mimetic enantiomer of gossypol, induces NOXA in multiple carcinoma cell lines and also sensitizes in a NOXA-dependent manner.…”

“…Many of the reported BH3 mimetics designed to antagonize MCL1 were found instead to induce NOXA through the unfolded-protein response (UPR) pathway [ 17 , 18 ]. In addition, apoptotic events caused by these compounds, whether through single-agent activity or through combination with another BH3 mimetic, were determined to be NOXA dependent.…”

Silibinin declines blue light-induced apoptosis and inflammation through MEK/ERK/CREB of retinal ganglion cells

“…When apoptosis is triggered, Bax moves to the mitochondrial outer membrane to promote the opening of the permeability transition (PT) pore. Sequentially, cytochrome C will be released to the cytoplasm and further activates the caspase cascade to induce apoptosis [13]. Here through western blotting, it was demonstrated that silibinin treatment would reduce the expression of Bax, cleaved caspase-3 and cleaved caspase-9, while it upregulated the expression of Bcl2 (Figure 2).…”