Multiple BH3 Mimetics Antagonize Antiapoptotic MCL1 Protein by Inducing the Endoplasmic Reticulum Stress Response and Up-regulating BH3-only Protein NOXA

Abstract

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X L , or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation

“…In contrast, dasatinib, a tyrosine kinase inhibitor, induced a dose-dependent phosphorylation of Mcl-1 (Fig. 4E) and obatoclax, a pan-Bcl-2 inhibitor, up-regulated Noxa expression (not shown), which are consistent with previous studies (24,34). Interestingly, a time-and dose-dependent decrease in the protein expression of BimEL was also observed following Mcl-1 degradation in cells treated with maritoclax (Fig.…”

“…This mode of action is different from that of obatoclax, which is predicted to bind at the p1 and p2 binding sites of Mcl-1 (37) and does not destabilize Mcl-1 (34). While obatoclax up-regulates Noxa (34), maritoclax has no effect on Noxa protein levels (Fig.…”

pRb/E2F-1-mediated caspase-dependent induction of Noxa amplifies the apoptotic effects of the Bcl-2/Bcl-xL inhibitor ABT-737

“…Transcriptional upregulation of Noxa was previously described in response to treatment with BH3-mimetics. 24,42 As Noxa potently binds to Mcl-1, 6 its ability to promote ABT-737 induction of cell death generally relies on its capacity to inhibit Mcl-1 that is not targeted by ABT-737. [13][14][15][16][17] Silencing of Mcl-1 increased cell death rates in ABT-737-treated U251 cells (without affecting Noxa expression in untreated or ABT-737-treated cells, Supplementary Figures S3A and S3B).…”

Gossypol Increases Expression of the Pro-apoptotic BH3-only Protein NOXA through a Novel Mechanism Involving Phospholipase A2, Cytoplasmic Calcium, and Endoplasmic Reticulum Stress

“…We reported previously that several putative BH3 mimetics, including gossypol, activate the integrated stress response (ISR) rather than directly inhibiting the anti-apoptotic BCL2 proteins (25). The ISR involves phosphorylation of eIF2␣ followed by the induction of ATF4, ATF3, and ultimately the BH3-only protein NOXA, which in turn can inhibit the BCL2 family members MCL1 and BFL1.…”