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Apoptosis in leukemia: From molecular pathways to targeted therapies
Best Practice & Research Clinical Haematology, 1, 21, pages 5 - 11
Knowledge about apoptosis pathways developed in the last few years is already being translated into novel targeted therapies. Two illustrative approaches to this process are the development of small molecule BCL2 inhibitors and X-linked inhibitors of apoptosis proteins (XIAP) inhibitors. This work demonstrates both the development of new agents based on molecular mechanisms and how these agents further our understanding of the biology of apoptosis.
Key words: apoptosis, caspase, inhibitors, BCL2, XIAP, X-linked inhibitors of apoptosis protein, targeted therapies.
Fundamental knowledge about the molecular mechanisms of apoptosis is already being translated into novel therapies. Two illustrative approaches to this process are the development of BCL2 and XIAP inhibitors. These inhibitors include small molecules that block protein-protein interactions and antisense DNA oligonucleotides that inhibit the transcription of their target. This review uses the development of BCL2 and XIAP inhibitors to highlight how basic understandings of the apoptosis pathway can be translated into the clinic. Moreover, it demonstrates how, in an iterative process, the results of clinical trials inform us about the biology of the target and the apoptosis pathway.
Apoptosis is a morphologic process characterized by cell shrinkage, membrane blebbing and nuclear condensation. Molecularly, apoptosis is a complex series of events with multiple positive and negative feedback loops and integration into other critical intracellular pathways including cell cycle progression and phospho-signaling pathways. Classically, apoptosis culminates in the activation of caspases, cysteine proteases that cleave critical intracellular proteins and thereby induce the final stages of cell death. Multiple pathways lead to caspase activation and several of these pathways are already being targeted for therapeutic intervention. One of these pathways is the receptor-mediated or extrinsic pathway that is activated when ligands such as FAS and TRAIL bind to death receptors on the cell surface causing these receptors to oligomerize. Oligomerization of death receptors leads to binding of adaptor proteins such as FADD that in turn permits the binding, dimerization and activation of upstream caspases such as caspases 8. Once activated, caspase 8 is released into the cytoplasm where it cleaves and activates downstream effector caspases, such as caspase 3 leading to cell death and apoptosis. The mitochondrial or intrinsic pathway of caspase activation is initiated by damage to the mitochondria that results in the release a number of proteins into the cytoplasm including cytochrome c. When released into the cytoplasm, cytochrome c complexes with Apaf-1 and this complex recruits upstream caspases, such as caspase 9. Upon recruitment, caspase-9 dimerizes, activates and in turn cleaves and activates downstream effector caspase 3 causing cell death and apoptosis1 and 2 ( Figure 1 )
A host of proteins regulate the apoptosis pathway positively and negatively. Among these proteins is the BCL2 family of anti-apoptotic proteins. This family of proteins blocks the mitochondrial pathway by stabilizing the mitochondrial membrane. These proteins also form inactivating heterodimers with pro-apoptotic proteins such as Bax and Bak. This latter function has been directly targeted with small molecule inhibitors that prevent BCL2 and its homologues from binding and inhibiting the pro-apoptotic proteins Bax and Bak. 3
Another regulator of the caspase activation pathway is XIAP. XIAP is a member of the Inhibitor of Apoptosis Protein family of anti-apoptotic proteins and is a potent inhibitor of active caspases 9 and 3. By blocking active caspase 3, XIAP inhibits the downstream portion of the caspase cascade and thus blocks apoptosis triggered by multiple caspase activation pathways.4 and 5 Like BCL2, both small molecule and antisense oligonucleotides have been developed to block the activity of XIAP.
The preclinical and clinical activity of BCL2 and XIAP inhibitors will be reviewed here to provide examples of how translational research is impacting clinical care.
Inhibition of the BCL2 family of anti-apoptotic proteins
BCL2 antisense oligonucleotides
Both antisense and small molecule approaches have been used to target BCL2 and its family members. Antisense oligonucleotides directed at BCL2 act at the level of BCL2 mRNA. Specifically, these antisense oligonucleotides bind BCL2 mRNA, resulting in the formation of sense-antisense heterodimers. These heterodimers recruit endogenous RNAase H enzymes that cleave and degrade the native sense mRNA leaving the anti-sense molecule intact. The antisense oligonucleotide is then released back into the cytosol where it is capable of inhibiting additional native BCL2 mRNA. Thus, by degrading BCL2 mRNA, translation is impaired and BCL2 protein levels decrease. In preclinical studies, BCL2 antisense oligonucleotides induced apoptosis, sensitized malignant cells to chemotherapy, and delayed tumor growth in xenografts.6 and 7 Based on these studies, the clinical grade antisense BCL2 inhibitor oblimersen sodium (Genasense®), was advanced into clinical trials. Several phase 3 studies have already been conducted including a pivotal phase 3 trial of oblimersen sodium in combination with chemotherapy in patients with CLL. 8 In this trial, patients with relapsed or refractory CLL were randomized to receive fludarabine and cyclophosphamide salvage chemotherapy with or without oblimersen sodium. The addition of oblimersen sodium to standard salvage chemotherapy improved the rate of complete response (CR) and nodular partial response (PR) from 7 to 17%. Despite this improvement in CR, there was no change in overall survival or time to progression for the study population as a whole. Interestingly, in subgroup analysis, a survival advantage was noted in the fludarabine-sensitive patients but not in the subgroup that was fludarabine-refractory. To date, the FDA has not approved oblimersen sodium for the treatment of CLL, largely due to the lack of survival advantage in the entire cohort.
Thus, while the preclinical efficacy of BCL2 antisense was dramatic, the success in the clinical setting has been more modest. The limited clinical efficacy of BCL2 antisense has raised questions about the value of an antisense approach to inhibiting BCL2 and has also has raised concern about antisense strategies in general as a therapeutic modality. A number of reasons may explain why BCL2 antisense did not produce more significant clinical benefit. First, BCL2 antisense oligonucleotides specifically target BCL2 but not its homologues. Therefore, redundancy among the BCL2 family members may negate any potential impact of inhibiting this single family member. If true, then pan-BCL2 inhibitors may be required to achieve clinical efficacy. Second, the amount of target knockdown required to inhibit BCL2's function in patients is unknown, but the∼20% target knockdown that was observed in previous studies may not be adequate. Finally, the lack of significant clinical benefit in the reported phase 3 trials may simply reflect the inclusion of patients with highly refractory disease. In the phase 3 study in CLL, the CR rate was increased to 17%. Thus, most patients still did not achieve CR and would not be expected to have a survival advantage. Potentially, studies including patients with less chemoresistant diseases may be required to demonstrate greater rates of CR with a resultant survival benefit from BCL2 antisense oligonucleotides.
Small-molecule BCL2 inhibitors
Given the modest efficacy of BCL2 antisense oligonucleotides, interest in developing small molecule BCL2 inhibitors has increased. The pro-apoptotic proteins Bax and Bak bind BCL2 in a pocket formed by BCL2's BH1-BH2-BH3 domains. Peptides or small molecules that bind this groove in the BCL2 protein can block the interactions between BCL2 and Bax/Bak. As such, the function of BCL2 is inhibited and the pro-apoptotic function of Bax and Bak is left unchecked.
A number of different approaches have been pursued to develop small molecule BCL2 inhibitors, including work by GeminX Biotechnologies and Abbott Laboratories ( Table 1 ). GeminX Biotechnologies used a high throughput screening assay to identify inhibitors of BCL2 protein-protein interactions. From this screen, they identified Obatoclax, a pan-BCL2 inhibitor that binds and inhibits the activity of BCL2, Bcl-xl, MCL1 and A1.9, 10, and 11 Obatoclax is delivered intravenously and is currently being evaluated in clinical trials. For example, in a phase 1 study of Obatoclax in patients with refractory hematologic malignancies including AML, patients received up to 40 mg/m2 of Obatoclax by continous infusion over 24 hours every 2 weeks. At the highest dose level, dose-limiting toxicities included QTc prolongation. Other non-dose limiting toxicities included mild neurologic side effects such as euphoric mood, somnolence, and dizziness. Apoptosis was observed in 10/14 patients as evidenced by increased levels of oligonucleosomal DNA, a marker of DNA fragmentation. 12
|Identified||High thoughput screen||Rational drug design|
|Specificity||Pan-Bcl family||Preferential BCL2 inhibitor|
|Development status||Phase 2||Phase 1|
A contrasting approach to developing a small molecule BCL2 inhibitor was employed by Abbott Laboratories who developed a preferential BCL2 inhibitor. Using rational drug design, they synthesized molecules that docked into BH1-BH2-BH3 pocket of BCL2. With medicinal chemistry, the initial compounds were modified to select agents such as ABT-737 and ABT-263 that preferentially bound and inhibited BCL2 over MCL1 and A1. 13 The Abbott BCL2 inhibitor ABT-263 is an oral formulation that has recently entered phase 1 clinical trials in patients with refractory malignancies including leukemias ( www.abbott.com ). Both Obatoclax and the Abbott BCL2 inhibitors demonstrated potent anti-tumor activity when evaluated in preclinical studies.9, 11, and 13 Thus, the results from clinical trials will be particularly informative as to which BCL2 inhibition strategy is most safe and effective.
XIAP is 1 of at least 8 members of the IAP family and a potent inhibitor of caspases 3, and 9 (reviewed in Schimmer et al 2006). By inhibiting the downstream effector caspases, XIAP blocks multiple apoptosis signals. Structurally, XIAP contains 3 BIR (Baculovirus IAP Repeat Domains) and a RING finger. The function of XIAP's BIR 1 domain is unknown, while its BIR 2 domain with its N-terminal linker binds and inhibits caspases 3 and 7, and the BIR 3 domain binds and inhibits active caspases 9. XIAP's RING finger contains an E3 ligase that ubiquitinates several proteins including caspase-3, but the physiological importance of this domain is uncertain. Overexpression of XIAP in malignant cell lines induces to chemoresistance14, 15, and 16, and in some studies of patients with malignancies including AML, increased XIAP expression is associated with a poor outcome.17 and 18 Given these observations and the biological role of XIAP in apoptosis, there was interest in developing XIAP inhibitors. Like BCL2, both antisense and small molecule inhibitors have been developed to block XIAP.
XIAP antisense oligonucleotides
AEG35156 is an antisense oligonucleotide directed against XIAP that is currently being developed by Aegera Therapeutics (Montreal, Canada). In the initial phase 1 study, patients with refractory tumors received escalating doses of AEG35156 by continuous infusion over 7 days. Increased liver enzymes and thrombocytopenia were dose limiting toxicities. Knockdown of XAP levels were observed in 2 patients. 19 Encouraged by these results, phase 1 and 2 studies of AEG35156 as a bolus infusion over 2 hours have been initiated, including a phase 1/2 trial of XIAP antisense in combination with reinduction chemotherapy for relapsed and refractory AML ( www.aegera.com ). Like BCL2 antisense oligonucleotides, it is unknown whether sufficient target knockdown can be achieved to inhibit XIAP's function. Interestingly, a small amount of XIAP knockdown may be sufficient to induce apoptosis, because positive feedback loops in the caspase activation pathway, allow a small amount of active caspase-3 to amplify the apoptosis signal and induce cell death. It is also unknown whether the presence of other IAP family members will negate the effects of specifically blocking XIAP.
Small molecule XIAP inhibitors
Small molecule inhibitors of XIAP that bind and inhibit the BIR 3 and BIR 2 domains this target are also being developed. These molecules were identified through rationale drug design and high throughput screening.20, 21, 22, 23, 24, and 25 As an example of such an approach, our group developed an enzymatic high throughput screen to identify small molecules that derepressed XIAP-mediated inhibition of recombinant caspase 3. Through a screen of a million-compound combinatorial library and the subsequent deconvolution steps, we identified several small molecule XIAP inhibitors including the polyphenylurea inhibitors that bound and blocked the activity of XIAP's BIR 2 domain.25 and 26 These compounds induced apoptosis in malignant cells over normal cells through a mechanism consistent with inhibition of XIAP.15 and 26
Subsequently, we used these molecules as tools to study the role of XIAP in AML and normal hematopoietic cells. 27 In this study, we demonstrated that our polyphenylurea XIAP inhibitors preferentially induced apoptosis in AML cell lines and primary patient samples preferentially over normal hematopoietic cells. The study also revealed that the activity of the XIAP inhibitor in the AML patient samples was related to levels of XIAP protein expression. Our XIAP inhibitors induced cell death most potently in AML samples with high levels of XIAP, but the compounds were less active in samples with low to absent levels of XIAP. These results suggest that that there may a specific subgroup of AML patients where XIAP levels are increased and contribute to disease pathogenesis. In these patients, inhibiting XIAP may be useful therapeutically.
Small molecule XIAP inhibitors are being developed by several groups and are advancing through preclinical evaluation towards clinical trial. In the interim, they serve as useful biological tools to understand the role of the IAP proteins in normal and malignant cells.
Summary and conclusions
Through basic investigations of the mechanisms of apoptosis, drugable targets such as BCL2 and XIAP have been identified. For both of these targets, antisense oligonucleotide and small molecule inhibitors have been developed. These inhibitors have already entered clinical trial or are at an advanced stage of development. The process of discovering and developing these compounds highlights how fundamental science can be translated into new therapies for malignancies including AML. Perhaps less widely recognized, however, is that the clinical and preclinical data provide important information about the biology of the apoptosis pathway. Thus, in a positive feedback loop, understanding the biology of apoptosis leads to new targeted therapies, and in the course of using these new therapies, our understanding of the biology of apoptosis improves.
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☆ This work was supported by the Canadian Institutes of Health Research (CIHR), and the Canadian Cancer Society. A.D.S. is the recipient of a CIHR Clinician Scientist Award.
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