CHEMICAL BIOLOGY

Programmed Cell Death as a Therapeutic Approach

 

Kurt Deshayes, Genentech, Inc., Department of Protein Engineering, South San Francisco, California

doi: 10.1002/9780470048672.wecb472

 

Although turnover rate varies greatly with cell type, the survival of multicellular organisms requires the constant renewal of healthy cells and the removal of pathogen-infected or damaged cells. A critical component of homeostasis is the proper regulation of programmed cell death or apoptosis. Both oversensitivity of crucial cells and resistance of malfunctioning cells to apoptotic stimuli threaten survival. A lack of death response permits mutant cells to survive normal death stimuli and potentially proliferate. Conversely, excess apoptosis removes cells required for organism survival. Restoring homeostasis by controlling apoptosis is a potentially powerful technique for alleviating disease.

Biologic Background

Programmed cell death allows the removal of overpopulated cells, pathogen-infected cells, malformed cells, or cells that have sustained genetic damage (1). Several disease states are linked either to a lack of apoptotic response or to unwanted cell death (2) (see Table 1).

 

Table 1. Diseases Associated with Programmed Cell Deatha

 

Diseases Associated with

Diseases Associated with

Downregulated Apoptosis

Upregulated Apoptosis

1.

Cancer

1.

 

 

A. Hormone-dependent

tumors

Breast cancer

Prostrate cancer

Ovarian cancer

B. Follicular lymphomas

Neurodegenerative disorders

 

 

A. Alzheimer’s disease

 

 

B. Parkinson’s disease

 

 

C. Cerebellular degeneration

 

 

D. Amyotrophic lateral sclerosis

E. Retinitis pigmentosa

C. P53 mutant carcinomas

2.

AIDS

2.

Autoimmune diseases

3.

Ischemic injury

 

A.

Lupus

 

A. Stroke

 

B.

Glomerulonephetis

 

B. Myocardial infarction

3.

Viral Infectons

 

C. Reperfusion injury

A.

Herpesviruses

4.

Toxin-induced liver disease

 

B.

Poxviruses

5.

Aplastic anemia

 

C.

Adenoviruses

 

 

aAdapted from Reference 2.

 

When activated by cellular stress (e.g., hypoxia, DNA damage, or nutrient withdrawal), programmed cell suicide is initiated by the intrinsic apoptotic pathway (Fig. 1). A key step, the release of cytochrome c through changes in the mitochondria outer membrane permeabilization, is regulated by the Bcl-2 protein family (3). Cytochrome c in combination with the Apaf-1 protein catalyzes the processing of Caspase-9 (cysteinyl, aspartate-specific protease) within the apoptosome (4). Caspase-9 initiates hydrolysis of zymogen procaspase-3 and procaspase-7, which, when activated, lead to the degradation of cellular targets and eventual cellular suicide (5). Caspase activity is regulated by Inhibitor of apoptosis (IAP) proteins, which are negative (prosurvival) apoptosis regulators (6). Conversely, the IAP proteins are antagonized by a second mitochondrial activator of caspases (Smac) protein and released from the mitrochondria alongside cytochrome c, which serves as a positive (proapoptotic) apoptosis regulator (7).

Another important mechanism for promoting programmed cell death is the binding of ligands to the death receptors, which occurs in the extrinsic pathway (8) (Fig. 1). The death receptors recruit and activate caspase-8, which in turn regulates effector caspase-3 and caspase-7. Caspase-8 processes the Bcl-2 family member Bid, which collaborates with other members of the Bcl-2 family to induce cytochrome c release from the mitochondria and thereby activates the downstream intrinsic pathway (9).

Changes in the balance between the proapoptotic and prosurvival signals modulate the response to apoptotic stimuli. Cancer cells are a prominent example of how overexpression of prosurvival factors leads to a harmful repression of programmed cell death. In contrast, neurodegenerative disorders (Parkinson’s and Alzheimer’s diseases) have been suggested to result from excess cell death in slowly regenerating neurons, which leads to a loss of function (2).

Presumably, targeting the apoptotic pathway can be a means to treat cancer by antagonizing the prosurvival components or by agonizing the proapoptotic components. Recent positive results with agents that target the apoptosis pathway have increased interest in cancer therapies directed at this pathway. It is quite possible that antagonizing selected proteins within the apoptosis pathways may not be sufficient always for broadly applicable therapy. However, the connectivity between the pathways can be exploited by the judicious application of multiple agents that act in concert, perhaps synergistically, on the different components of the pathway to give broad therapeutic benefit.

When thinking about proapoptotic therapies, agents can be designed that trigger a cascade of enzymes, such that once the proteins have been activated the process will continue without the help of the therapeutic agent. This is strikingly different from a therapy that relies on enzyme inhibition for their therapeutic effect. The latter often requires chronic dosing to maintain continued enzyme inhibition. Apoptosis-inducing reagents may require only doses sufficient to overcome initial apoptotic roadblocks. Furthermore, fast-growing solid tumors often are hypoxic and nutrient-starved, conditions that would lead a normal cell into apoptosis. To avoid programmed cell death, cancer cells keep apoptosis in check through the overexpression of prosurvival factors. The studies discussed in the following sections suggest that cancer cells are primed for programmed cell death and, therefore, are more sensitive to toward the induction of apoptosis.

 

Figure 1. The extrinsic apoptotic pathway is triggered when death receptors are engaged by their cognate ligands, which results in recruitment of the adaptor protein FADD and the apical caspase, caspase-8. This recruitment leads to the activation of caspase-8 and subsequent activation of the effector caspase-3 and caspase-7. The intrinsic apoptotic pathway is triggered by stimuli, such as irradiation, chemotherapeutic agents, or growth factor withdrawal. Activation of proapoptotic BH3-only members of the Bcl-2 family by the p53 pathway neutralizes the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, which leads to the disruption of the mitochondrial membrane potential and to the release of cytochrome c and Smac into the cytoplasm. These events result in Apaf-1 -mediated activation of caspase-9 and subsequent activation of the effector caspase-3 and caspase-7 and then culminate in cell suicide. XIAP is the last line of defense against cellular suicide and acts by inhibiting caspases. Other IAP proteins (ML-IAP, cIAP1, and cIAP2) do not inhibit caspases directly but sequester the proapoptotic molecule Smac, which prevents it from blocking the action of XIAP. Smac mimetics bind to IAP proteins and block their inhibitory activity by antagonizing the critical IAP-caspase and IAP-Smac interactions.

 

Targeting the Bcl-2 Family of Proteins

It was first observed in type Type 2 B-cell lymphoma that the translocation of the Bcl-2 gene leads to radical overexpression of Bcl-2 (10). The overproduction of Bcl-2 has been shown to be transforming (10). Members of the Bcl-2 protein family contain as many as four characteristically helical Bcl-2 homology motifs (BH1-BH4). Bcl-2 proteins are divided into classes, as prosurvival proteins (Bcl-2, Bcl-xL, Mcl-1, A-1, and Bcl-w), proapoptotic proteins (Bak and Bax), and proapoptotic proteins that contain only the BH3 motif (Bim, Bid, Puma, and Noxa) (3, 11). The manner in which the different Bcl-2 family members interact and the mechanisms by which cytochrome c release is initiated are unclear, but the central role of the Bcl-2 family in apoptosis, and the therapeutic potential of regulating these proteins, is well established.

Changes in the ratio of prosurvival to proapoptotic Bcl-2 proteins regulate cellular response to apoptotic stimuli. For example, decreasing the production of proapoptotic proteins will sensitize cells to apoptosis (12). Genta, Inc. (Berkeley Heights, NJ) is investigating this method as a potential cancer therapy. More specifically, antisense oligonucleotides that correspond to the first six codons of the Bcl-2 RNA are used to stop production of the prosurvival Bcl-2 proteins (Genasense; Genta, Inc.). Promising results have been observed in lymphoma patients, and phase III clinical trials are underway (13). However, in another clinical trial, Genasense did not show sufficient efficacy in the treatment of malignant melanoma to receive FDA approval (14).

Another potential therapeutic approach is to mimic the proapoptotic BH3 only proteins with small molecules that bind to the prosurvival members of the Bcl-2 family and promote apoptosis. This interaction involves a large hydrophobic interface, which is a challenging target for a small-molecule antagonist. Nonetheless, progress in the development Bcl-2 antagonists has been reported. For example, a natural product, Gossypol, isolated from cottenseed oil, was shown to have spermicidal activity, and tested originally as a potential male contraceptive (15). Gossypol is believed to bind to the prosurvival Bcl-2 proteins (16) (see Fig. 2). The cytotoxic effects of Gossypol have been redirected as a potential anticancer therapeutic. Ascenta (San Diego, CA) has moved the R enantiomer of Gossypol, AT-101, into phase II human clinical trials. In these trials, the pan-specific Bcl-2 inhibitor has been combined with Rituxan (Genentech, Inc., South San Francisco, CA) in the treatment of relapsed CLL (chronic lymphocytic leukemia) (17). Recent results with MEFs (mouse embryo fibroblasts) that express neither Bax nor Bak show that Gossypol can kill cells independent of the proapoptotic Bcl-2 family proteins, which suggests that another mechanism is responsible for Gossypol-induced cell killing (18).

Work on small-molecule BH3 mimetics has been reported by groups from Abbott Labs (Abbott Park, IL) (19), the Hamilton group at Yale (New Haven, CT) (20), GeminX (Montreal, Quebec, Canada) (21), and University of Michigan (Ann Arbor, MI) (22) (see Fig. 2). All of these agents are believed to antagonize the prosurvival Bcl-2 members by mimicking the action of the BH3 domain. The Abbott small molecule (ABT-737) was developed using the SAR (structure activity relationship) by NMR (nuclear magnetic resonance) method of Dr. Stephen Fesik (19). This molecule binds with subnanomolar potency to members of the prosurvival Bcl-2 family, Bcl-2, Bcl-xL, and Bcl-w, but does not bind to the other widely expressed member of the family, Mcl-1. In vitro and in vivo studies show ABT-737 has excellent efficacy against cell lines that do not express Mcl-1 (some lymphomas and small-cell lung cancer) but limited potency against cancers that highly express Mcl-1 (18). However, when combined with an agent that specifically antagonizes Mcl-1, good efficacy is seen in cell lines where ABT-737 does not demonstrate single agent activity (18). These results raise issues about which Bcl-2 family members must be targeted to achieve broad efficacy and suggest that therapeutic efficacy against diverse cancers may require a pan-specific Bcl-2 family antagonist. Data from GeminX (21) indicate that broad inhibition of the Bcl-2 proteins may be viable clinically because its pan-specific Bcl-2 family antagonist, Obatoclax (GeminX), demonstrated activity against hematological tumors in a phase I clinical study.

 

Figure 2. Small-molecule antagonists of prosurvival Bcl-2 proteins: (a) ABT-737 (19); (b) GeminX (21); (c) Yale (20); (d) Gossypol (16); and (e) the University of Michigan (22).

 

Targeting the Inhibitor of Apoptosis Proteins

IAP proteins prevent cell death through interactions between their BIR (baculoviral) IAP repeat domains and the proteases that are critical for the initiation and execution of apoptosis, caspase-3, caspase-7, and/or caspase-9 (23). X-chromosome linked IAP (XIAP) is a ubiquitously expressed IAP protein and a potent inhibitor of caspases that plays a critical role in resistance to chemotherapeutic agents and other proapoptotic stimuli. Although no direct genetic mutation defines XIAP as an oncogene, XIAP overexpression is common in cancer and has been linked to poor patient prognosis (24). In another class of IAP proteins, c-IAP1 and c-IAP2 are unique among IAP proteins for their ability to interact with TRAF1 and 2 (tumor necrosis factor receptor-associated factors 1 and 2) (25, 26). Importantly, c-IAP1 and c-IAP2 also are targets of genetic amplification, which correlates with resistance to chemotherapy and radiotherapy (27). The (melanoma inhibitor of apoptosis protein) (ML-IAP) is upregulated in melanomas but not expressed in most normal adult tissues (28).

Changes in IAP expression modulate apoptotic response to cellular stress. For example, an antisense oligonucleotide developed by Aegera Therapeutics, Inc. (Montreal, Quebec, Canada), AEG35156, reduces expression of XIAP. Treatment with AEG3516 increases apoptosis in studies with myeloid leukemia cells, and the antisense approach currently is in phase I/II clinical trials for refractory AML (acute myeloid leukemia) in combination with chemotherapy (29). The strategy of up-regulating IAP action to inhibit apoptosis has been validated by transfection of a gene-encoding XIAP in a rat glaucoma model (30).

The Smac protein promotes apoptosis by neutralizing the IAP proteins through interactions of the Smac N-terminal region with the BIR domains of the IAP (31, 32). The interaction between Smac and IAP proteins disrupts the IAP inhibition of caspases, which allows caspase activation and eventual cell suicide. In vivo and in vitro studies reveal that cell killing via either the intrinsic or the extrinsic pathways can be enhanced by Smac mimetics (33-36). Collectively, these results suggest that Smac mimetics that bind to the BIR domains of the IAPs extricate the caspases in a manner analogous to Smac.

Significantly, interactions between Smac and the BIR3 domain of XIAP are localized to the four N-terminal residues of mature processed Smac (31, 32), suggest that a small-molecule mimetic might be sensible. The immediate challenge in developing a small-molecule Smac mimetic is rescaffolding the key binding determinants of the tetrapeptide onto a platform that exhibits drug-like properties. This rescaffolding has become an active area of research with several groups producing nonpeptide Smac mimics. Examples of small-molecule Smac mimetics under preclinical investigation are shown in Fig. 3, with significant progress reported by groups from Abbott (37), University of Michigan (38), Genentech (39), Novartis (Basel, Switzerland) (40), Princeton University (Princeton, NJ) (41), and Texas Southwestern Medical Center (Dallas, TX) (25). Based on these reports, it seems that an effective Smac mimetic will require a free amino group and a small hydrophobic amino acid at the N-terminus, a proline derivative in the third position, and an aromatic group at the fourth position. The studies mentioned above demonstrate Smac mimetics are effectors of both the extrinsic and the intrinsic apoptosis pathways.

 

 

Figure 3. Small-molecule IAP antagonists from (a) Abbott (37); (b) Novartis (40); (c) Texas Southwestern (25); (d) the University of Michigan (38); and (e) Genentech (39).

Targeting the P53 Pathway

The transcription factor p53 responds to cellular stress by promoting the production of growth suppressors and proapoptotic factors that lead to cell cycle arrest, senescence, or apoptosis (42). Activation of the p53 pathway is known to target more than 16 genes that regulate apoptosis (43). Significantly, when detecting DNA damage, p53 initiates the expression of proapoptotic BH3-only proteins Puma (p53-upregulated modulator of apoptosis) and Noxa (43) (see the “Targeting the Bcl-2 Family of Proteins” section above). Approximately 50% of cancers show mutations or deletions in the p53 gene, which silences a critical mechanism for limiting the propagation of damaged cells. Most alterations that lead to loss of p53 function consist of single amino acid mutations that destabilize the protein core (44). Function has been rescued in these mutants by binding of peptides derived from the C-terminal domain (44) or small molecules (45). Activity of C-terminal peptides is believed to be at least partially caused by antagonizing the binding of the p53 C-terminus to the DNA-binding motif of p53. The small-molecule activators developed by Pfizer (New York, NY) shown in Fig. 4, CP-31398 and CP-257042, restore p53 function by stabilizing the protein core (45). The potency of these molecules requires a hydrophobic group within a fixed distance from an ionizable group, which suggests a specific interaction with the protein. Studies with CP-31398 show inhibition of tumor growth in melanoma and colon cancer xenograft models.

Regulation of p53 activity is complex, including numerous posttranslational modifications. One such modification is ubiquitination by the E3 ligase MDM2 (murine double minute 2); the interaction between MDM2 and p53 has been identified as a potential target for therapeutic intervention. MDM2 binds to p53 in the transcription activation domain, blocking p53 function while simultaneously targeting p53 for ubiquitination and degradation. Cancers with functional p53 often silence p53 by the overexpression of MDM2 (46). Studies that use either blocking antibodies and peptides or reduction of MDM2 levels employing antisense oligonucleotides demonstrate that disruption of the p53-MDM2 interaction activates the p53 pathway, which blocks proliferation and promotes apoptosis (47, 48). Examination of a crystal structure of a p53-derived peptide bound to MDM2 reveals that three amino acids (Phe19, Trp23, and Leu26) project deep into a hydrophobic-binding pocket on the MDM2 surface (46, 49). The localized nature of the interface suggests MDM2 as a potential small-molecule target. The Hamilton group used an approach analogous to the Bcl-2 work mentioned above to obtain a helix mimic that binds to MDM2 (50). By screening for molecules that only show activity in cells with wild-type p53, researchers at the Karonlinska Institutet (Stockholm, Sweden) discovered a small molecule they named RITA, which shows in vivo p53-dependent antitumor activity (51) (see Fig. 4). A major advance in this area is the work reported by the Hoffman-La Roche team (Nutley, NJ) in their development of a series of potent MDM2 antagonists they named the nutlins (Fig. 4). This class of MDM2-binding molecules was identified initially in a high-throughput screen and followed by extensive structural optimization that yielded potency in the 100-300-nM range (52).

Studies using an osteosarcoma SJSA-1 xenograft tumor model demonstrated that a 20-day course of treatment with racemic nutlin-3 resulted in a 90% inhibition of tumor growth as compared with vehicle treatment. Resolution of the racemate to give the active nutlin-3 enantiomer increased potency two fold, and studies with enantiomerically pure compound evidenced 100% growth inhibition in the SJSA-1 model as well as in an LnCaP prostrate xenograft model. Both models showed significant tumor regression, which suggests that the disruption of the MDM2-p53 interaction is antiproliferative and proapoptotic and that antagonizing MDM2 suppression of p53 is a promising therapeutic approach for cancers that retain wild-type p53 function (49).

 

 

Figure 4. Small molecules that stabilize mutant p53, (a) CP-31398 and (b) CP-257042 (45). Small molecule antagonists of MDM2 binding, (c) RITA (51); (d) Nutlin-1; (e) Nutlin-2; and (f) Nutlin-3 (52).

Targeting the Death Receptors

One of the most promising advances in apoptosis-cancer therapies is the activation of the extrinsic pathway through the death receptors (53). This is an extremely active area of biology research, but with little potential for a small-molecule approach. An attractive feature of the extrinsic pathway is that its unique activation mechanism provides an avenue for promoting cell death in cancers that resist current therapies (8). Another feature of the extrinsic pathway is that many cancer cells highly express death receptors but do not express the decoy receptors that are found on normal cells, which increases the susceptibility of cancer cells to death receptor activation (54). As mentioned earlier, the potential for agents that target multiple components of apoptosis holds promise for effective treatment of resistant cancers. An exciting example of this potential is illustrated in a study in which a ligand that activates death receptors, Apo2L/TRAIL, was combined with a Smac-derived peptide (36); complete regression was observed in a mouse glioma model, one of the most difficult cancers to treat and one for which no current viable therapy exists. This result suggests that activation of the extrinsic pathway in combination with apoptosis-sensitizing agents is an effective strategy for treating cancers resistant to conventional therapies.

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Further Reading

Weinberg R. The Biology of Cancer. 2006. Garland Science, London, United Kingdom.

 

See Also

Cell Death: Apoptosis and Necrosis

Chemical Tools to Dissect the Apoptotic Pathways

The Apoptosome and Caspase Activation, Chemistry of

DNA Damage

Gene Therapy and Cell Therapy

Mitochondria: Topics in Chemical Biology