Bcl-2 turns deadly

Abstract

Small-molecule inhibitors of anti-apoptotic Bcl-2 proteins and BH3 mimetic peptides are promising anticancer agents. A recent study identifies a Nur77-based peptide that converts anti-apoptotic Bcl-2 proteins into proapoptotic molecules, providing another potential cancer therapeutic strategy.

Bcl-2 family proteins are frequently deregulated in tumors and can confer resistance to therapeutic agents. Although their mechanisms of action are still under investigation, Bcl-2 family proteins are key regulators of mitochondrial cell death pathways. In damaged cells, three subsets of Bcl-2 family proteins interact to determine cell fate. The anti-death Bcl-2 family members (Bcl-2, Bcl-x_L) compete with specific members of the pro-death BH3-only subset (Bid, Bim, Bad) to prevent oligomerization of the membrane-permeabilizing family members that damage mitochondria and kill cells (Bax, Bak). A wide variety of therapeutic strategies have been developed to target Bcl-2 family proteins, including BH3 mimetic peptides derived from the BH3 domains of Bim and of caspase-cleaved Bid (tBid) that bind and activate Bax to kill cells¹. The small-molecule inhibitor ABT-737 was designed to mimic the BH3 domain of Bad by binding in a cleft on the front of Bcl-2 and Bcl-x_L and inactivating their anti-apoptotic function². However, because N-terminal truncation of Bcl-2 and Bcl-x_L by caspase cleavage can convert these anti-apoptotic factors into potent pro-death molecules^3,4, it is theoretically possible to exploit this event as a therapeutic strategy. In a recent report, Kolluri et al.⁵ found that a short peptide corresponding to the nuclear orphan receptor Nur77 binds to and converts Bcl-2 into a pro-death molecule. This finding opens the door for the development of small molecules with similar actions for the treatment of cancer.

Nur77 and related orphan nuclear receptors are implicated in the elimination of autoreactive T cells, brain development and other processes⁶. Following up on their earlier report that Nur77 induces apoptosis by binding and converting Bcl-2 into a pro-apoptotic factor, Kolluri et al.⁵ found that a nine-amino-acid peptide (NuBCP-9) that corresponds to a region of Nur77 required for interaction with Bcl-2 is also pro-apoptotic. NuBCP-9 and its enantiomer bind the N-terminal BH4 domain and adjacent unstructured loop domain of Bcl-2, an important regulatory region where phosphorylation and caspase cleavage occurs. The authors conclude that NuBCP-9 works through a Bcl-2—dependent mechanism to induce cell death by showing that Bcl-2 knockout fibroblasts (bcl-2^-/- MEFs) are more resistant to NuBCP-9—induced apoptosis. Fitting with this model, but contrary to the norm, Bcl-2 overexpression enhances NuBCP-9—induced cell death in T-cell leukemia-derived cells. Importantly, injection of NuBCP-9 peptide suppresses growth and induces apoptosis in tumor cells xenografted in mice.

How does the NuBCP-9 peptide convert Bcl-2 into a pro-death factor? Kolluri et al.⁵ show that direct binding of NuBCP-9 to Bcl-2 induces conformational changes in the Bcl-2 protein (Fig. 1), detected by shifts in biophysical properties and by exposure of an antibody epitope in the BH3 domain of Bcl-2. Exposure of the BH3 helix in tBid and Bax is critical for their pro-apoptotic function, and this mechanism appears to be conserved during the conversion of Bcl-2 to its pro-apoptotic conformation. The authors provide evidence that NuBCP-9—converted Bcl-2 does not adopt the membrane-permeabilizing function of Bax, and does not mimic the BH3-only proteins Bid or Bim, which can directly activate Bax. Instead, converted Bcl-2 mimics a different group of BH3-only proteins (for example, Bad) that bind to and inactivate the anti-death protein Bcl-x_L, thereby releasing the brakes on tBid activation of Bax to kill cells¹. These experimental systems are complex, however, so other possible mechanisms should be explored.

Figure 1.

Small molecules convert anti-death factors into pro-death factors. A peptide derived from Nur77 (designated NuBCP-9) converts anti-death Bcl-2 and Bcl-B into pro-death factors that activate Bax- and Bak-dependent cell death¹⁰. NuBCP-9 binds the anti-apoptotic conformation of Bcl-2 in the N-terminal loop domain and induces a conformational change. In the latter conformation, the BH3 domain is exposed and Bcl-2 promotes apoptosis.

The results of Kolluri et al.⁵ suggest a mechanism in which a major conformational change that likely involves both the N and C termini of Bcl-2 serves as a molecular switch between anti-death and pro-death activities. The ability of NuBCP-9 to flip this switch is likely to be distinct from the mechanism of the BH3 mimetic ABT-737 now in clinical trials. While ABT-737 kills tumor cells by binding and inactivating Bcl-2 and Bcl-x_L, it also unexpectedly protects neurons from hypoxia-induced synaptic decline and from increased mitochondrial permeability, which implies that ABT-737 also inactivates the pro-death function of Bcl-x_L and perhaps also Bcl-2 (ref. ⁷). In contrast, the Nur77 mimetic peptide NuBCP-9 inhibits only the anti-death conformation to promote the switch to a pro-death form of Bcl-2, potentially enhancing its antitumor effects.

Does Bcl-2 conversion to a pro-death factor also occur during physiological processes, and does Nur77 mediate the conversion of Bcl-2 to a pro-death factor in animals? Interestingly, Thompson and Winoto⁶ recently reported that Nur77 translocation to mitochondria and conversion of Bcl-2 into a BH3-exposed killer protein occurs during elimination of autoreactive T lymphocytes using two different mouse models. These observations imply a physiological role for Nur77-induced pro-death activity of Bcl-2, though a causal role for Bcl-2 in negative selection has not yet been established. Because the Nur77 mimetic NuBCP-9 does not target Bcl-x_L, it will avoid the complicating side effects of thrombocytopenia associated with ABT-737, but future studies will be required to determine whether NuBCP-9 causes immunosuppression.

Another important question is whether this death conversion of Bcl-2 can be harnessed to treat cancer. The particularly potent effects of NuBCP-9 against Bcl-B provide a reasonable target, as Bcl-B was reported recently to be elevated in several human cancers, where it is thought to confer resistance to chemotherapeutic drugs and irradiation therapy⁸. Efforts are now underway to identify small molecules that mimic the effects of the Nur77 peptide NuBCP-9 (ref. ⁹).