Targeted manipulation of apoptosis in cancer treatment

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1 Targeted manipulation of apoptosis in cancer treatment Justin A Call, S Gail Eckhardt, D Ross Camidge Lancet Oncol 2008; 9: Published Online August 28, 2008 DOI: /S (08) Developmental Therapeutics and Gastrointestinal Malignancies Programs (J A Call MD, Prof S G Eckhardt MD) and Developmental Therapeutics and Thoracic Malignancies Programs (D R Camidge MD), University of Colorado Comprehensive Cancer Center, Aurora, CO, USA Correspondence to: Dr D Ross Camidge, Developmental Therapeutics and Thoracic Malignancies Programs, University of Colorado Comprehensive Cancer Center, Aurora, CO 80045, USA ross.camidge@uchsc.edu Apoptosis is a fundamental process in the development and maintenance of multicellular organisms and its regulation is commonly disrupted in human cancers. In vitro and in vivo, effective treatment of cancer with radiotherapy or anticancer drugs (or both) is frequently associated with increased markers of apoptosis. However, clinical resistance to treatment is common in many tumours, particularly with increasing lines of therapy. Diminished ability to undergo apoptosis might cause extensive therapeutic cross-resistance in cancer cells. With increased understanding of the regulatory and effector molecules of apoptosis new drugs have been developed that might manipulate the apoptotic balance in cancer cells in favour of cell death. This Review summarises the rationale for direct manipulation of various elements of apoptosis and describes agents that are currently under investigation in early-phase clinical trials in many different cancer types. Introduction Apoptosis is a highly regulated natural process that can remove unwanted, redundant, or damaged cells from multicellular organisms in an orderly, non-inflammatory way. In a typical adult human being, billions of cells undergo apoptosis every day. 1 In cancer cells, the presence of multiple genetic aberrations and the overall cellular stresses of malignant transformation should be associated with substantial proapoptotic activity, and a high basal apoptotic rate is common in many cancers (figure 1). However, cancers persist and dysregulation of apoptosis (by emphasis of antiapoptotic signals or de-emphasis of proapoptotic signals) might be essential for the survival of many cancers. Apoptosis is characterised by activation of effector caspases and cleavage of proteins that are essential for cell viability, resulting in DNA fragmentation, chromatin condensation, cell shrinkage, and membrane blebbing (figure 2). Traditional cytotoxic agents and radiotherapy indirectly manipulate apoptosis by driving proapoptotic signalling through the induction and recognition of cell damage. However, many new agents offer more direct targeted manipulation of apoptosis, either through inhibition or downregulation of antiapoptosis activity, Figure 1: Apoptosis in vivo Histological section of small-cell lung cancer, showing high basal level of apoptosis. Apoptotic bodies are shown by arrows (haematoxylin and eosin stain, magnification 40). Courtesy of Wilbur Franklin, University of Colorado, CO, USA. or through stimulation or upregulation of apoptosis effectors or initiators. These agents are in clinical development for treatment of various cancers (figure 3). Intrinsic and extrinsic apoptotic pathways Apoptosis is mediated by intracellular cysteine proteases called caspases, which can be positioned as upstream initiators or downstream effectors of apoptosis. Caspases are produced as inactive zymogens and are activated by proteolytic processing (for effector caspases) or by aggregation (for initiator caspases), leading to a series of amplification cascades. There is no evidence to suggest that apoptosis can occur in the absence of caspases, although other mechanisms of cell death exist, including autophagy and necrosis. 2 Two main molecular pathways lead to activation of effector caspases and apoptosis: the intrinsic and extrinsic pathways (figure 3). 3 The intrinsic pathway depends on the release of mitochondrial-derived cytochrome C by the proapoptotic BCL2 subfamily members BAX and BAK. This pathway usually follows P53-dependent transduction of cellular stress and damage signals, although other damage-recognition pathways exist. 2 There are three subfamilies of BCL2 proteins, all of which have a role in the intrinsic pathway through inhibition or promotion of apoptosis. 4 The antiapoptotic BCL2 subfamily members (BCL2, BCLX, and MCL1) interact with BAX and BAK to inhibit the release of cytochrome C. 4 On release from the mitochondrial intermembrane space, cytochrome C interacts with apoptotic proteaseactivating factor 1 and recruits and activates caspase-9. The complex of apoptotic protease-activating factor 1, caspase-9, and cytochrome C is called the apoptosome, which maintains caspase-9 in an active conformation. 5 Caspase-9 mediates activation of the effector caspases 3, 6, and 7, and produces positive feedback in the extrinsic pathway through further activation of caspase-8 and caspase-10 via caspase-6. 5 BAX and BAK also promote apoptosis by stimulating the release of smac protein from mitochondria, which inactivates inhibitors of apoptosis proteins (IAPs: XIAP; IAP1; IAP2; and survivin). XIAP is a direct caspase Vol 9 October 2008

2 A D B E Figure 2: Time-course photomicrographs of apoptosis in vitro HN12 cells treated with TRAIL undergoing apoptosis, showing nuclear fragmentation, cell shrinkage, and membrane blebbing. A=baseline. B F= min after TRAIL exposure. Courtesy of Andrew Thorburn, University of Colorado, CO, USA. inhibitor, and several other IAPs might ubiquitinate caspases to target them for degradation in the proteasome. However, how many IAPs work remains unclear. 6 Survivin, the smallest of the IAPs and the one most differentially expressed between malignant and healthy tissue, has several functions in cells independent of its antiapoptotic role; the same might apply to other IAPs. 7 C F By contrast, the extrinsic pathway depends on ligandbased activation of cell-surface death receptors (tumour necrosis factor [TNF] receptor, FAS1, or receptors for TNFrelated apoptosis inducing ligand [TRAIL, also known as Apo2L]). Ligands such as TNF, Fas ligand, or TRAIL interact with their respective receptors, leading to direct or indirect recruitment of the adaptor molecule FAS-associated death domain protein (FADD). This process activates the apoptosis initiators caspase-8 and caspase-10 in the deathinducing signal complex, resulting in cleavage and activation of the effector caspases 3, 6, and 7. 3 Caspase-8 and caspase-10 activation can also promote intrinsic pathway activation, in a cell-type specific manner, by the cleavage of BID, leading to direct activation of BAX and BAK. 3 Crosstalk between the intrinsic and extrinsic pathways is substantial, and several different activation signals can converge to push a cell beyond a putative apoptotic threshold, beyond which cell death by apoptosis becomes inevitable. 8 Inhibition of antiapoptotic signalling as a treatment strategy Members of the antiapoptotic BCL2 subfamily (eg, BCL2 and BCLX) are upregulated in many cancers. 9,10 Moreover, Extrinsic pathway FAS ligand TNF Mapatumumab (TRAIL-R1) Lexatumumab (TRAIL-R2) Apomab (TRAIL-R2) AMG-655 (TRAIL-R2) rh TRAIL (TRAIL-R1, TRAIL-R2) Intrinsic pathway Cell damage FAS1 TNF-R1 TRAIL-R1 TRAIL-R2 Upregulation of death receptors P53-dependent transduction P53-independent transduction FADD recruitment BID BAX and BAK Apoptosis inhibition by BCL2, BCLX, MCL1 Activated caspase-8 and caspase-10 Apoptosome/APAF1 Mitochondrial cytochrome C Oblimersen SPC2996 AT-101 GX ABT-263 Common pathway Activated effector caspases (caspase-3, caspase-6, and caspase-7) Activated caspase-9 Mitochondrial smac protein IAPs (XIAP, IAP1, IAP2, survivin) AEG35156 LY GDC-0152 YM-155 Terameprocol Apoptosis Figure 3: Intrinsic and extrinsic apoptotic pathways Agents in clinical development that directly manipulate these pathways are shown in green (extrinsic-pathway agonists), blue (inhibitors or downregulators of antiapoptotic BCL-2 subfamily), and red (inhibitors or downregulators of antiapoptotic family of inhibitors of apoptosis [IAP]). Inhibitory actions are shown as bars; activation as solid arrows; and pathway crosstalk as dashed arrows. TRAIL=TNF-related apoptosis inducing ligand. TNF=tumour necrosis factor. FADD=Fas-associated death domain.. Vol 9 October

3 some IAPs, particularly survivin but also XIAP, IAP1, and IAP2, are overexpressed in clinical samples and cell lines from various tumours. 7 Increased levels of anti apoptotic molecules might be essential to keep cancer cells above the apoptotic threshold. Healthy cells should have substantially less basal proapoptotic drive than do cancer cells, and effective approaches to block anti apoptotic activity alone could be associated with a wide therapeutic margin. Preclinical observations have shown that downregulation of BCL2, XIAP, or survivin and upregulation of smac signalling can trigger cell death in various cancer cell lines and xenografts, and can increase sensitivity to cytotoxic chemotherapy or radiotherapy while having little effect on healthy cells. 7,11 13 However, any clear associations between levels of antiapoptotic molecules and clinical outcomes have been hard to define. The ratio of antiapoptotic molecules to pro apoptotic molecules might be more important than absolute amounts. 14 Target Clinical development Oblimersen BCL2 I Melanoma (combined with dacarbazine) Chronic lymphocytic leukaemia (combined with fludarabine and cyclophosphamide) Myeloma (combined with dexamethasone) Acute myeloid leukaemia (combined with cytarabine and daunorubicin) Non-small-cell lung cancer (combined with docetaxel) Small-cell lung cancer (combined with carboplatin and etoposide) Acute myeloid leukaemia (combined with gemtuzumab) Prostate cancer (combined with docetaxel) Non-Hodgkin lymphoma (combined with rituximab) Chronic myeloid leukaemia (combined with imatinib) Gastrointestinal stromal tumour (combined with imatinib) Liver cancer (combined with doxorubicin) Renal cancer (combined with interferon) Myeloma (combined with thalidomide and dexamethasone) Merkel-cell carcinoma Colorectal cancer (combined with FOLFOX or irinotecan) Breast cancer (combined with doxorubicin and docetaxel) Oesophageal cancer (combined with cisplatin and fluorouracil) Non-Hodgkin lymphoma (combined with RICE or R-CHOP) Small-cell lung cancer (combined with paclitaxel) Melanoma (combined with albumin-bound paclitaxel and temozolomide) Waldenström s macroglobulinaemia SPC2996 BCL2 Relapsed or refractory chronic lymphocytic leukaemia LY Survivin Prostate cancer (combined with docetaxel) Acute myeloid leukaemia Hepatocellular carcinoma AEG35156 XIAP Pancreatic cancer (combined with gemcitabine) Breast cancer (combined with paclitaxel) Non-small-cell lung cancer (combined with carboplatin and paclitaxel) Acute myeloid leukaemia (combined with cytarabine and idarubicin) Phase I Solid tumours (combined with docetaxel) FOLFOX=folinic acid, fluorouracil, and oxaliplatin. RICE=rituximab, ifosfamide, carboplatin, and etoposide. R-CHOP=rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. Table 1: Antisense agents in clinical development that act against antiapoptotic targets To date, three main therapeutic strategies have been developed to block antiapoptotic signalling. Strategies have been directed mainly at the intrinsic pathway and include use of antisense oligonucleotides and direct small-molecule inhibitors or mimetics (figure 3, tables 1 and 2). Furthermore, several novel drugs with broad mechanisms of action that might include inhibition or downregulation of antiapoptotic signalling are also under investigation (eg, proteasome inhibitors and inhibitors of heat-shock protein 90). Antisense oligonucleotides Clinical approaches to use of antisense oligonucleotides have not been straightforward. Antisense molecules are taken up by cells mainly via endocytosis, but only a fraction escapes the endosome to be effective in the cell. 15 Antisense oligonucleotides hybridise to specific mrna sequences through base-pairing and interfere with their function through several mechanisms mainly recruitment of RNase-H-mediated message degradation. 15 Most antisense molecules under clinical investigation are first-generation drugs that, despite backbone modifications to enhance resistance to endogenous nuclease degradation, need protracted intravenous or subcutaneous infusions to address their short half-life. 15 BCL2 Oblimersen, the furthest advanced antiapoptotic antisense oligo nucleo tide, is a first-generation molecule against BCL2 (table 1). Downregulation of BCL2 in peripheral-blood mononuclear cells and in lymph-node biopsies containing tumour was noted in several phase I studies after protracted venous infusions; however, effects were modest. 15,16 In two randomised phase III chemotherapy combination trials, continuous infusion of oblimersen was associated with a significant increase in objective response rate compared with chemotherapy alone. 17,18 Myelosuppression (especially thrombocytopenia) was also increased by the antisense administration. However, the primary endpoint of overall survival benefit was not reached for either oblimersen added into dacarbazine for patients with melanoma, or oblimersen added into fludarabine and cyclophosphamide for patients with chronic lymphocytic leukaemia. 17,18 A randomised phase III trial in patients with advanced-stage small-cell lung cancer also showed that the addition of oblimersen to carboplatin and etoposide did not significantly change either the objective response rate or overall survival. 19 Clinical trials of continuous-infusion oblimersen combined with other traditional cytotoxic agents are ongoing in many other cancer types (table 1). Preclinical data suggest that short, intermittent intravenous infusions improve intratumoral cellular loading, retention of intact oblimersen oligonucleotide, and antitumour activity. A dose-escalation study 20 showed that oblimersen given as a weekly 2-h intravenous Vol 9 October 2008

4 infusion is feasible and achieves maximum blood concentrations that are ten-times higher than continuous intravenous infusion. However, whether this dose schedule will enhance anticancer activity and patient outcomes remains unknown. SPC2996 is a BCL2 antisense molecule that was developed by use of so-called locked nucleic acid technology: a high-affinity, biologically stable RNA analogue in which the normally flexible ribose sugar ring is fixed in a rigid conformation to give high RNA-binding affinity and resistance to enzyme degradation. SPC2996 is under assessment in phase I and phase II trials in patients with chronic lymphocytic leukaemia (table 1). 21 No trial results have been reported to date. Survivin LY is a second-generation antisense oligonucleotide that downregulates survivin expression. It is nuclease-resistant and highly potent in preclinical studies. 22 A single-agent phase I study of intravenous LY has been completed, but the results have not been published. Further studies are planned in patients with relapsed or refractory acute myeloid leukaemia, hormone-refractory prostate cancer, and advanced hepatocellular carcinoma (table 1). XIAP AEG35156 is a second-generation antisense oligonucleotide against XIAP. Preliminary results 23 of a singleagent clinical trial that assessed 7 days continuous intravenous infusion suggest that thrombocytopenia and transaminitis are potential side-effects of treatment. Clinical activity was noted in two of 16 patients: one patient with breast cancer, and one patient with non- Hodgkin lymphoma (coincident with documented XIAP downregulation in the tumour). Early-phase clinical trials of AEG35156 in combination with cytotoxic drugs are ongoing in acute myeloid leukaemia and in solid tumours (table 1). 24 Direct small-molecule inhibitors or mimetics The antiapoptotic BCL2 subfamily members (ie, BCL2, BCLX, and MCL1) have a hydrophobic BH3-binding pocket that interacts with proapoptotic subfamily members. 25 Gossypol is an enantiomeric polyphenol originally derived from cottonseed and investigated since the 1970s as a contraceptive for men and treatment for endometriosis. 26 Gossypol binds to BCL2, BCLX, and MCL1 in the BH3-binding pocket. 25 The R-enantiomer is more potent preclinically in inhibiting proliferation and inducing apoptosis than is either the S-enantiomer or the racemic mixture (ie, that with equal R and S enantiomers). Oral racemic gossypol was assessed in the 1980s and 1990s, but R-gossypol is currently under development as an immediate-release oral tablet (AT-101, table 2). Preclinically, single-agent AT-101 induces apoptosis in various cancer cell lines, an effect that is increased when combined with radiotherapy or cytotoxic drugs such as docetaxel, topotecan, carboplatin, or etoposide. 11,27,28 A phase I/II study 29 of AT-101 combined with docetaxel and prednisone in nine men with hormone-refractory prostate cancer showed that treatment was tolerated well. Eight patients had a decreased concentration of prostate-specific antigen by 50% or more. Further phase I/II studies of AT-101 combined with various cytotoxic agents are under way for different cancer types (table 2). Obatoclax (GX15-070) binds at the BH3 groove of several BCL2 subfamily members and is cytotoxic when used alone against a range of cell lines. 30 Single-agent phase I studies of obatoclax in refractory chronic lymphocytic leukaemia and solid tumours have commenced. Toxic effects noted to date include peri-infusional drowsiness, euphoria, ataxia, and abdominal pain. 30,31 Single-agent phase II trials of obatoclax in various haematological tumours are under way, and phase I/II combination studies are ongoing in various solid cancers (table 2). Gossypol (AT101) Obatoclax (GX15-070) ABT-263 Target(s) BCL2, BCLX, MCL1 BCL2, BCLX, MCL1 BCL2, BCLX GDC-0152 XIAP, IAP1, IAP2 Terameprocol Survivin (EM-1421, M4N) Clinical development Non-Hodgkin lymphoma (combined with rituximab) Chronic lymphocytic leukaemia (combined with rituximab) Non-small-cell lung cancer (combined with docetaxel) Prostate cancer (combined with docetaxel) Glioblastoma multiforme Small-cell lung cancer (combined with topotecan or cisplatin and etoposide) Oesophageal cancer (combined with docetaxel, fluorouracil, and radiotherapy) Glioblastoma multiforme (combined with temozolomide and radiotherapy) Myelodysplastic syndrome Non-Hodgkin lymphoma Idiopathic myelofibrosis with myeloid metaplasia Hodgkin s lymphoma Small-cell lung cancer (combined with topotecan) Non-small-cell lung cancer (combined with docetaxel) Non-Hodgkin lymphoma (combined with bortezomib) Chronic lymphocytic leukaemia (combined with fludarabine and rituximab) Non-Hodgkin lymphoma Chronic lymphocytic leukaemia Small-cell lung cancer Phase I Solid tumours Cervical intraepithelial neoplasia High-grade glioma YM-155 Survivin Non-Hodgkin lymphoma Non-small-cell lung cancer Melanoma Prostate cancer Prostate cancer (combined with docetaxel and prednisone) *Treatments with broad mechanisms of action that might include inhibition or downregulation of antiapoptotic signalling are not listed (eg, proteasome inhibitors and inhibitors of heat-shock protein-90). Table 2: Small molecules and other agents that block antiapoptotic targets in clinical development* Vol 9 October

5 ABT-737 binds with high affinity to several members of the antiapoptotic BCL2 subfamily, including BCL2 and BCLX, but not MCL1. 32 Preclinically, MCL1 expression was identified as an important determinant of ABT-737 resistance. 32 ABT-263 is an orally bioavailable variant of ABT-737. ABT-263 has entered clinical testing as monotherapy in three phase I/II studies (in non-hodgkin lymphoma, in chronic lymphocytic leukaemia, and in solid tumours with a planned expanded cohort in smallcell lung cancer; table 2). 33 Up to now, objective responses have been reported in three patients with relapsed or refractory chronic lymphocytic leukaemia. 34 The proapoptotic function of smac relies on the presence of an N-terminal four aminoacid motif that binds in the so-called smac-binding groove of XIAP, blocking interaction with caspase Smac mimetics bind tightly in this groove on many IAPs, sensitising cancer cell lines to proapoptotic signals in vitro and in vivo. 36,37 A first-in-man phase I study of GDC-0152, a smac-mimetic (IAP antagonist), has started (table 2). Identification of a potential small-molecule binding site on survivin, distinct from the smac-binding groove, raises the possibility that specific small-molecule inhibitors of survivin could also enter clinical development (table 2). 38 Proteasome inhibitors The 26S proteasome regulates the cellular protein milieu through the degradation of ubiquinated proteins targeted for destruction. Inhibition of the proteasome might be directly proapoptotic (eg, through inhibition of the breakdown of proapoptotic molecules with a short half-life or through prevention of the breakdown of inhibitors of antiapoptotic molecules). Furthermore, proteasome inhibition might be indirectly apoptotic through stress responses triggered by the aberrant protein environ ment. Preclinically, small-molecule proteasome inhibitors such as bortezomib are active against many cancer cell lines. They reduce levels of nuclear factor-κb (a transcription factor upstream of BCL2 and BCLX) through preventing the degradation of its inhibitor I-κB, and they increase levels of FAS1, P53, and BAX, resulting in apoptosis. 39 Many chemotherapy drugs induce nuclear factor-κb and activate an antiapoptotic response, the inhibition of which might enhance treatment response. 39 Furthermore, bortezomib upregulates expression of TRAIL receptors, which might sensitise cancer cells to further apoptotic stimuli. 40 Although the downstream consequences of proteosome inibition might differ between cell types, preclinical data show that they can promote apoptosis either as monotherapy or when combined with traditional cytotoxics or extrinsic-pathway agonists across a large range of cancer-cell types. 39,40 However, although bortezomib is approved for use in multiple myeloma and mantle-cell lymphoma, extensive clinical testing in multiple cancer types including melanoma, sarcoma, neuro endocrine, colorectal, breast, and renal-cell carcinoma suggests it does not seem to have substantial activity alone against solid cancers. 39 chemo therapy combination studies are under way for various solid cancers, but results to date show little or no benefit from the addition of bortezomib. 39 Other approaches to inhibit antiapoptotic signalling Terameprocol (M4N, EM-1421) is a transcription inhibitor that reduces the levels of several proteins, including survivin, by binding to the promoter region of genes. 41 Terameprocol has entered clinical trials, including phase I/II trials of cervical intraepithelial neoplasia and highgrade glioma (table 2). 42 YM-155 was identified in a high-throughput screen for agents that suppressed survivin. Preclinically, it reduced survivin message and protein levels in a dose-dependent way, although its exact mechanism of action remains unclear. A phase I study 43 of 7 days continuous intravenous infusion of YM-155 every 3 weeks in patients with advanced cancer showed dose-limiting toxic effects of mucosal inflammation and neutropenia. Three of five patients with non-hodgkin lymphoma had a partial Response Evaluation Criteria in Solid Tumors (RECIST) response; two of nine patients with prostate cancer had a decrease in the levels of prostate-specific antigen; and one patient with non-small-cell lung cancer had a minor response. YM-155 showed modest activity when given alone as first-line treatment to 34 patients with metastatic melanoma: one patient had a complete response, one had a partial response, and 11 had stable disease. 44 A phase II trial of YM-155 alone in 33 patients with refractory non-small-cell lung cancer showed that 5% of patients had a response and 38% had a period of stable disease. 45 Preclinical studies have shown that YM-155 enhances the activity of docetaxel without increased toxic effects, and a phase I/II combination study in patients with hormonerefractory prostate cancer is under way (table 2). 13 Survivin is a client protein for the molecular chaperone heat-shock protein-90 (HSP-90). 46 General HSP-90 inhibitors such as 17-AAG and specific inhibitors of the survivin HSP-90 complex such as shepherdin are under investigation in early-phase clinical trials. 47 Moreover, immunotherapeutic approaches against antigenic peptide sequences of survivin are being assessed because of the differential expression of survivin in malignant cells compared with most healthy tissue. 48 Inhibition of antiapoptotic signalling: discussion The stress of malignant transformation and the damaging effects of traditional cytotoxic chemotherapy and radiotherapy generate proapoptotic signalling in cancer cells. For cancer cells to remain alive in this hostile environment, they must ignore these signals (eg, by selection of favourable P53 mutations), tolerate them Vol 9 October 2008

6 (eg, through upregulation of prosurvival signals such as BCL2 or IAPs), or do both. 4 Small-molecule inhibitors of specific antiapoptotic regulators offer substantial potential clinically. Antisense approaches have had suboptimum results to date, which might be due to drug delivery rather than target relevance. Determinants of sensitivity to most approaches that block antiapoptotic activity remain unclear, and identification of predictive molecular biomarkers are ongoing. For example, up to 50% of all cancers have aberrant P53, although not all mutations have the same functional consequences. 49 Whether tumour cells that are wildtype P53 rely on antiapoptotic mechanisms more than mutant P53 tumours, and will therefore be more sensitive to inhibition of antiapoptotic signalling in the clinic, remains to be seen. Recognition of the many interacting pathways that regulate apoptosis is an area of future investigation. Of particular focus will be combination strategies to block antiapoptotic signalling in conjunction with indirect or direct proapototic stimuli (eg, cytotoxics, radiotherapy, or extrinsic pathway agonists); and the optimum sequencing of these approaches. Direct proapoptotic stimulation as a treatment strategy Local administration of high-dose recombinant TNF-α is licensed in Europe for the treatment of peripheral sarcomas and melanomas through isolated limb perfusion. However, its use as a systemic anticancer agent has been ineffective because of severe toxic effects (including hypotension and organ failure) that are consistent with its role as a proinflammatory cytokine. 50 Low, persistent exposure to TNF-α seems to be more tolerable, but might cause cachexia and initiate carcinogenesis. 50 Currently, therapeutic anticancer death-receptor agonism is most actively being explored by approaches focused around TRAIL a naturally occurring protein that forms a soluble homotrimer and binds cell-surface complexes of TRAIL receptors TRAIL-R1 and TRAIL-R2 (also called death receptors 4 and 5). 51,52 A narrow population of immunological cells produce TRAIL, including some natural-killer cells, monocytes, dendritic cells, and T cells. Although fairly high levels of TRAIL-R1 and TRAIL-R2 cell-surface proteins are expressed in various tumours, weak expression is also found on a few healthy cells. 51,53,54 Increased expression of TRAIL-R1 and TRAIL-R2 seems to be an early step in carcinogenesis and might target aberrant cells for immune-mediated destruction. 53,54 Two main therapeutic approaches have been developed as direct proapoptotic strategies, involving the extrinsic pathway: TRAIL-receptor agonist antibodies; and recomb inant ligands. Figure 3 shows proapoptotic agents and table 3 shows their current clinical development. Proapoptotic agonist antibodies Several agonist (ie, stimulating) monoclonal antibodies that target TRAIL-R1 or TRAIL-R2 are being assessed in clinical trials. Mapatumumab (HGS-ETR1) is a human agonist antibody for TRAIL-R1 and is currently the only specific agonist against this receptor in clinical development (table 3). Preclinically, it induced apoptosis in various cancer cell lines and xenografts if some TRAIL-R1 was present. 55,56 However, no clear association between degree of TRAIL-R1 expression and antibody sensitivity has been recorded Similarly, the death-receptor target seems necessary, but not sufficient, for cell-line sensitivity to TRAIL-R2 agonist monoclonal antibodies. 55,56 In a single-agent phase I study 57 of mapatumumab, most patients who were given the highest dose (10 mg/kg) developed at least grade 1 or 2 transaminitis potentially a direct drug effect because hepatocytes might express functional TRAIL receptors. 3,57,58 Although most tumour samples available for testing in this all-comers phase I study stained positive for TRAIL-R1, no objective responses were noted. 57 Single-agent phase II studies of mapatumumab have been completed for patients with non- Hodgkin lymphoma, non-small-cell lung cancer, and colorectal cancer. One complete response and two partial Target Agonist antibodies Mapatumumab TRAIL-R1 (HGS-ETR1) Lexatumumab (HGS-ETR2) TRAIL-R2 Clinical development Myeloma (combined with bortezomib) Non-small-cell lung cancer (combined with carboplatin and paclitaxel) Non-Hodgkin lymphoma Colorectal cancer Chemotherapy combination studies under way Apomab TRAIL-R2 Non-small-cell lung cancer (combined with carboplatin, paclitaxel, and bevacizumab) Non-Hodgkin lymphoma (combined with rituximab) Sarcoma Colorectal cancer (combined with irinotecan and cetuximab) AMG-655 TRAIL-R2 Phase I or phase II Non-small-cell lung cancer (combined with pemetrexed or carboplatin and paclitaxel) LBY-135 TRAIL-R2 Phase I ongoing Recombinant ligands Recombinant human TRAIL TNFerade TRAIL-R1, TRAIL-R2 Tumour necrosis factor α APO010 FAS1 Phase I under way Non-small-cell lung cancer (combined with carboplatin, paclitaxel, and bevacizumab) Non-Hodgkin lymphoma (combined with rituximab) Pancreatic cancer (combined with fluorouracil and radiotherapy) Oesophageal cancer (combined with fluorouracil, cisplatin, and radiotherapy) Melanoma (combined with radiotherapy) Rectal cancer (combined with capecitabine and radiotherapy) Head and neck cancer (combined with fluorouracil, hydroxyurea, and radiotherapy, or with cetuximab and radiotherapy) Table 3: Extrinsic-pathway proapoptotic agents in clinical development Vol 9 October

7 responses were noted in 17 patients with follicular non- Hodgkin lymphoma, but no responses were seen in patients with lung or colorectal cancer studies exploring mapatumumab in combination with standard doses of gemcitabine and cisplatin, and carboplatin and paclitaxel, in patients with any tumour types in which these chemotherapy agents were considered appropriate by the investigator, have been completed. 62,63 Compared with historical controls, mapa tumumab did not seem to change the toxic effects of the standard chemotherapy or to interact with the chemo therapy pharmacokinetically. In the heavily pretreated mixed populations within these studies, objective reponses were modest. Randomised trial data will be needed to establish the true effect of mapatumumab on the efficacy and toxic effects of standard chemotherapy. Randomised phase II trials of mapatumumab in combination with bortezomib in myeloma, and with carboplatin and paclitaxel in non-small-cell lung cancer, are ongoing (table 3). Currently, at least three TRAIL-R2 agonist antibodies are in early clinical development: lexatumumab (HGS- ETR2), apomab, and AMG-655 (table 3) Phase I testing of lexatumumab showed liver disturbances at high doses and 10 mg/kg was selected as the recommended dose for phase II testing. No patient responses to lexatumumab given alone have been noted. A phase I/II combination study of lexatumumab with various common cytotoxic monotherapies or regimens (eg, gemcitabine, doxorubicin, pemetrexed, and FOLFIRI [irinotecan, fluorouracil, and folinic acid]) in tumours in which these chemotherapy agents were considered appropriate by the investigator, showed no evidence of pharmacokinetic interaction or increases in toxic effects compared with those expected from the chemotherapy alone. 67 Three of 41 patients had a partial RECIST response: two with colorectal cancer who received lexatumumab with folinic acid, fluorouracil, and irinotecan, and one with small-cell lung cancer who received lexatumumab with doxorubicin. 67 Apomab given every other week has been assessed. A maximum-tolerated dose was not reached in a phase I study, but reversible dose-limiting transaminitis was observed in one patient. Although no objective responses as assessed by RECIST have been noted for apomab, one patient with colorectal cancer and one with ovarian cancer had minor responses. 65 Early-phase studies of apomab in combination with a range of different chemotherapies have commenced, including for colorectal cancer, follicular non-hodgkin lymphoma, and previously untreated advanced non-small-cell lung cancer (table 3). The half-life of AMG-655 is similar to apomab. AMG- 655 was tolerated well at doses up to 20 mg/kg every 14 days. 66 An objective response was noted in a patient with adenocarcinoma of the lung, and a PET response in a patient with colorectal cancer. 66 chemotherapy combination studies of AMG-655 in patients with advanced non-small-cell lung cancer are planned. Detailed phase I results of a fourth TRAIL-R2 agonist antibody, LBY-135, are awaited (table 3). 68 Proapoptotic ligands Recombinant human TRAIL (rhtrail) has been developed in a form suitable for clinical investigation as a soluble zinc-coordinated homotrimer. It seems to trigger apoptosis in a P53-independent way in up to 50% of cancer cell lines tested and has little effect on most nonmalignant cells. Preclinical data show minimum toxic effects across species, including monkeys. 69,70 A phase I study 71 of rhtrail given as a 1-h intravenous infusion daily for five consecutive days every 3 weeks showed that it was tolerated well at the doses tested. One confirmed partial response was recorded in a patient with metastatic synovial chrondrosarcoma. Assessment of its role in sarcoma and colorectal cancer at the recommended phase II dose is ongoing. 71 Chemotherapy combination studies with rhtrail in non-hodgkin lymphoma and non-small-cell lung cancer are also under way (table 3). APO010 is a recombinant form of human Fas ligand, designed as a fusion protein of three Fas-ligand extracellular domains linked to a protein backbone comprising a dimer-forming collagen domain. A phase I trial in solid cancers has commenced (table 3). Fasaret is a recombinant adenovirus that encodes Fas ligand. Given intratumorally, a phase I single-agent trial of two injections of fasaret is under consideration. TNFerade is a replication-deficient adenovector that expresses human TNF-α under the control of a radiationinducible promoter. TNFerade is given intratumorally and activated with radiotherapy. TNFerade was tolerated well in two phase I studies, 72,73 and phase II combination studies of TNFerade with chemoradio therapy are under way (table 3). Proapoptotic discussion Stimulation of TRAIL receptors by single agents such as TRAIL or agonist monoclonal antibodies leads to substantial and rapid proapoptotic activity against up to 50% of cancer lines in vitro. However, the clinical activity of these single agents seems rare in heavily pretreated patients and perhaps slow. Several of the reported objective responses to date eg, in colorectal cancer and sarcomas take many months and many cycles of treatment to evoke a response. 51,65,66,69 The determinants that might predict response to TRAIL-based therapy remain under investigation. Although standard immunohistochemical testing to quantify TRAIL receptor expression in clinical tumour samples is being developed, surface expression in cell lines does not seem to be associated with susceptibility (apart from lack of susceptibility in those with no discernible receptor expression). 51,58 Resistance to TRAIL might occur by various mechanisms. Surface expression Vol 9 October 2008

8 of either of two decoy receptors (TRAIL-R3 and TRAIL-R4) is a potential resistance factor in vitro. Unlike TRAIL-R1 and TRAIL-R2, decoy receptors do not have an active intracytoplasmic domain: they act as nonfunctional binding partners for TRAIL, protecting cell lines against TRAIL-induced apoptosis. 74 Although their importance is unknown clinically, in theory the decoy receptors could act as ligand sinks decreasing the effectiveness of rhtrail. Furthermore, because of the potential for the decoy receptors to heteromulti merise with TRAIL-R1 and TRAIL-R2, they could also change the function of the death-inducing signalling complex, affecting both ligand and agonist-antibody activity. Invitro O-glycosylation of death receptors and expression of O-glycosyl transferases by cancer cells has also been reported to affect response to rhtrail. 75 A similar effect on sensitivity to agonist antibodies is expected, but has not yet been reported. The clinical relevance of O-glycosylation is not yet known. Other mechanisms of cancer-cell resistance can occur, including mutations in death receptors and upregulation of the intrinsic apoptotic pathway (eg, IAP or BCL2 overexpression). 59,74 Understanding of resistance mechanisms will be important for prioritisation of combination studies of TRAIL-receptor agonists with chemotherapy, radiotherapy, and other targeted apoptosis-manipulating agents (eg, BCL2 inhibitors, smac mimetics, or proteasome inhibitors). 40,58 Conclusion In many cancers, the normal apoptotic process for elimination of damaged cells is dysregulated, which can lead to uninhibited growth and the development of resistance to conventional chemotherapy and radiotherapy. Drugs that restore apoptosis might selectively kill cancer cells that depend on dysregulated apoptotic pathways, or might make them more sensitive to traditional cytotoxic treatment. Assuming that a treatment aim is to generate enough caspase drive in cancer cells, these cells probably need to be attacked with a combination of proapoptotic stimuli (eg, chemotherapy, radiotherapy, or extrinsic-pathway stimulation) and antisurvival stimuli (eg, BCL2 or IAP manipulation). Maximisation of apoptotic responses might extend tumour control, and might increase the effectiveness of radical radiotherapy, chemoradiotherapy, and adjuvant chemotherapy after surgery. New agents that selectively target various components of apoptosis are in clinical development, many of which have shown early signs of single-agent activity and fairly few effects on healthy tissue. Furthermore, these agents seem to be tolerated well when combined with traditional chemotherapy. However, as with all new anticancer approaches, it will be important to determine: which patients are most likely to respond to a particular agent; the optimum treatment schedule; and the most effective treatment combination. Search strategy and selection criteria We used Medline and PubMed to search for articles using the search terms: cancer ; apoptosis ; TRAIL ; inhibitor of apoptosis (IAP) protein ; BCL-2 ; XIAP ; survivin ; proteasome inhibitors ; heat-shock protein-90 inhibitors ; tumor-necrosis factor ; and fas ligand. No date limits were used in these searches. Further references were identified from these articles. Material was also obtained by handsearching of work published in abstract form presented at the annual meetings of the American Society for Clinical Oncology, American Association for Cancer Research, and European Organisation for Research and Treatment of Cancer meetings from 2005 to Only articles and abstracts published in English were included. 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