Oncogene (2013) 32, 1341–1350 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

REVIEW On the TRAIL to successful therapy? Predicting and counteracting resistance against TRAIL-based therapeutics

LY Dimberg1, CK Anderson1, R Camidge2, K Behbakht1, A Thorburn3 and HL Ford1,3

Tumor necrosis factor-related -inducing ligand (TRAIL) and agonistic antibodies against TRAIL death receptors (DR) kill tumor cells while causing virtually no damage to normal cells. Several novel drugs targeting TRAIL receptors are currently in clinical trials. However, TRAIL resistance is a common obstacle in TRAIL-based therapy and limits the efficiency of these drugs. In this review article we discuss different mechanisms of TRAIL resistance, and how they can be predicted and therapeutically circumvented. In addition, we provide a brief overview of all TRAIL-based clinical trials conducted so far. It is apparent that although the effects of TRAIL therapy are disappointingly modest overall, a small subset of patients responds very well to TRAIL. We argue that the true potential of targeting TRAIL DRs in cancer can only be reached when we find efficient ways to select for those patients that are most likely to benefit from the treatment. To achieve this, it is crucial to identify biomarkers that can help us predict TRAIL sensitivity.

Oncogene (2013) 32, 1341–1350; doi:10.1038/onc.2012.164; published online 14 May 2012 Keywords: TRAIL; pro-apoptotic receptor agonists; apoptosis; TRAIL resistance; clinical trials

INTRODUCTION TRAIL SIGNALING The holy grail of cancer therapy is to find drugs that will TRAIL is a member of the death receptor (DR) ligand family, a specifically and efficiently kill cancer cells while having little to no subclass of the family. The TRAIL protein is effect on normal cells. The variability between and within different expressed on the membrane of a limited number of immune cells kinds of cancer and the cancer cells’ inherent ability to adapt are and is also present in a soluble form. It binds to at least five obstacles in obtaining this goal. Thus, there is a significant need to receptors. Two of these, DR4 (also known as TRAIL receptor 1) and define those individuals that will benefit from a specific therapy DR5 (TRAIL receptor 2), are transmembrane receptors with a while experiencing few side effects. cytoplasmic death domain that transmits apoptotic signals into Since the tumor necrosis factor-related apoptosis-inducing the cells. Two decoy receptors (DcR), DcR1 (TRAIL receptor 3) and ligand (TRAIL) (also known as APO2 ligand, APO2L) signaling DcR2 (TRAIL receptor 4), do not have functional death domain and pathway was initially discovered,1,2 the plausibility of exploiting it do not enable apoptosis activation.3 TRAIL also binds weakly to a in cancer therapy has been under debate. Initial promising studies fifth receptor, osteoprotegerin. Several pro-apoptotic receptor demonstrated a remarkable specificity for inducing apoptosis in agonists that can trigger TRAIL signaling have been developed, tumor cell lines but not in normal cells. Whereas clinical trials including recombinant human TRAIL ligand and agonistic using TRAIL therapies have shown low toxicity in patients, antibodies against DR4 and DR5, as discussed further below. disappointingly small therapeutic effects have been observed TRAIL signaling induces apoptosis mainly through the extrinsic when TRAIL agonists are used as a monotherapy. It is becoming or DR-mediated pathway. When TRAIL binds to DR4 or DR5, the increasingly apparent that TRAIL therapy may indeed be very receptors homotrimerize, enabling the receptors’ death domain to beneficial, but perhaps only for a small subset of patients. recruit the adaptor protein Fas-associated death domain and the Therefore, it is crucial to identify biomarkers that can predict inactive, uncleaved form of caspase 8, pro-caspase 8. The patient response and to maximize the therapeutic efficacy receptors, Fas-associated protein with death domain, and pro- through drug combinations that not only synergize with TRAIL caspase 8 or pro-caspase 10 together form the death-inducing but that can also overcome resistance as it arises. This review signaling complex, (DISC). At the DISC pro-caspase 8 is activated, a covers some of the mechanisms of TRAIL resistance that have process found to be dependent on both dimerization and been reported and presents an overview of all the TRAIL-based cleavage.4 Activated caspase 8 then cleaves downstream clinical trials performed to date. We argue that lessons learned substrates resulting in, ultimately, the cleavage and activation of from preclinical research should be much more integrated effector caspase 3. In some cell types, called type I cells, this into clinical trial design as a way to select the patients most activation of the extrinsic pathway is sufficient to induce likely to respond to therapy. Only then can we truly evaluate the apoptosis. However, in other cell types, type II cells, activation of efficacy of this drug and see the extensive research already done the intrinsic (mitochondrial) apoptosis pathway is required as well. in this field come to fruition in the form of increased cancer The intrinsic pathway is typically triggered by DNA damage or patient survival. other cell stressors, but it can also be activated through caspase

1Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USA; 2Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA and 3Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA. Correspondence: Dr HL Ford, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, RC2, Room 3100C, MS8613, Aurora, CO 80045, USA. E-mail: [email protected] Received 26 January 2012; revised 19 March 2012; accepted 21 March 2012; published online 14 May 2012 On the TRAIL to successful cancer therapy LY Dimberg et al 1342 8- or caspase 10-mediated cleavage of the pro-apoptotic BCL-2 which p53 is deleted.6 Therefore, TRAIL therapeutics may be an family protein BH3 interacting-domain death agonist (BID). When alternative way of eradicating tumors that are resistant to a wide cleaved, the activated, truncated form of BID can translocate to the range of conventional therapies. mitochondrial membrane, where it interacts with the pro-apoptotic In addition to the classical apoptosis-inducing TRAIL pathway, it Bcl-2 family members BAX and BAK, enabling these proteins is becoming increasingly evident that TRAIL signaling can also to induce permeabilization of the mitochondrial membrane. The activate other pathways including the nuclear factor kB, protein pro-apoptotic proteins cytochrome c and SMAC/DIABLO are then kinase B/AKT and mitogen-activated protein kinase pathways released from the mitochondria. Analogous to the DISC, cytochrome through the formation of signaling complexes secondary to the c forms a protein complex, the apoptosome, with SMAC/DIABLO, DISC consisting of different proteins including receptor-interacting APAF-1 and pro-caspase 9, enabling the cleavage of pro-casapase 9 protein, Fas-associated protein with death domain, caspase 8 and into active caspase 9. Caspase 9 cleaves downstream effector tumor necrosis factor receptor-associated protein (reviewed in caspases such as caspase 3, thus converging with and amplifying Falschlehner et al.7). Some of these pathways promote survival DR-mediated caspase activation (Figure 1). and proliferation and importantly, in cells that are resistant to One important distinction between TRAIL-induced apoptosis apoptosis induced by TRAIL, these pathways may still be intact. and apoptosis induced by conventional and For example, in TRAIL-resistant primary leukemia cells, TRAIL radiotherapy is that the latter is largely dependent on cellular induces proliferation in previously resting cells, accelerates the damage recognition by, for example, the p53 tumor suppressor doubling time of proliferating cells and reduces spontaneous cell protein. The dependence on p53 to elicit an apoptotic response death in a nuclear factor kB-dependent manner.8 Furthermore, poses a problem in cancer therapy, as loss of p53 occurs in more TRAIL promotes metastasis from TRAIL-resistant human pancreatic than half of all cells because of inactivating mutations.5 ductal carcinoma xenografts.9 Thus, if patients that have TRAIL- Although the TRAIL pathway is enhanced by p53 activation resistant tumors are treated with TRAIL, it may actually lead to an through upregulation of DR4, it can induce apoptosis in cells in increased tumor burden and metastasis. This highlights the critical

TRAIL α−DR4

α−DR5

DR4/5 DcR2 DcR1

D D D D D D DISC

FADD FLIP CASP 8 PRO

PRO CASP 3/6/7 CASP 8 BID

BCL2 BCLXL tBID MCL1 CASP 3/6/7 CASP 9 BAX

PRO CASP 9 cyt C CLEAVAGE OF CASPASE SUBSTRATES BAX DNA FRAGMENTATION APAF1

APOPTOSOME cIAP Survivin APOPTOSIS SMAC XIAP

Figure 1. The TRAIL signaling pathway. The TRAIL ligand binds to functional receptors DR4 and DR5 and non-signaling receptors osteoprotegerin (not shown), DcR1 and DcR2. Binding of TRAIL ligand or receptor-specific agonistic antibodies to DR4 and DR5 induces trimerization of the receptors. The cytoplasmic part of the DR4 and DR5 receptors contain death domain that enable recruitment of Fas-associated protein with death domain(FADD) and pro-caspase 8 (proCASP8), enabling cleavage and activation of proCASP8 to its active form caspase 8 (CASP8). CASP8 activates downstream effector caspases both directly and, in some cells, through the activation of the mitochondrial apoptosis pathway through BID cleavage. Once activated, effector caspases cleave downstream substrates and induce DNA fragmentation, ultimately leading to apoptosis. For additional details, please refer to the text.

Oncogene (2013) 1341 – 1350 & 2013 Macmillan Publishers Limited On the TRAIL to successful cancer therapy LY Dimberg et al 1343 importance of identifying patients whose tumors will be responsive used in this study were tagged with polyhistidine and also had a to TRAIL-induced death, and to develop means to counteract TRAIL suboptimal level of zinc, changing the tertiary structure of the resistance through combinations with other drugs. protein and causing it to over aggregate. The tagged preparation was not only toxic to normal hepatocytes but was also less efficient at inducing apoptosis in cancer cells.21 Untagged trimeric THE PHYSIOLOGICAL ROLE OF TRAIL proteins with optimal zinc content showed an absence of The physiological function of TRAIL is reported to be in immune hepatocyte toxicity both in primary human hepatocyte culture22 surveillance and immune-mediated tumor suppression. TRAIL and in systemic administration to non-human primates.6,21 is exclusively expressed on immune cells and can be triggered The physiological role of TRAIL signaling in tumor suppression by antigens such as lipopolysacharides and cytokines such and its specificity for cancer cells versus normal cells has as interferons, suggesting that TRAIL has a regulatory role in important implications. As TRAIL signaling is a physiological the immune system.10–12 TRAIL-deficient (TRAIL À / À ) mice are 13,14 means of preventing cancer, it is plausible that induction of this viable, developmentally normal and fertile. However, pathway would be an efficient way to combat existing tumors in a TRAIL À / À mice have a decreased level of lymphoid and clinical setting. Furthermore, TRAIL-induced killing should be myeloid cell death and are less sensitive to infection by Listeria 15 specific to cancer cells, sparing normal cells. However, as TRAIL- monocytogenes. In addition, mice that lack the functional mouse induced killing is likely part of the body’s first-line defense against TRAIL receptor mDR5 exhibit an enhanced innate immune 16 cancer, successful tumors may need to acquire resistance against response and are more resistant to murine cytomegalovirus, TRAIL-induced apoptosis. Indeed, a large proportion of tumor cells demonstrating a role of TRAIL in the negative regulation of both that metastasize are resistant to TRAIL, and TRAIL resistance may innate and adaptive immunity. The first evidence for a role of also arise during the course of cancer treatment. Thus, we have a TRAIL in tumor suppression came from a study by Walczak and potential ‘Catch-22’ situation, where the attributes that make colleagues that demonstrated a reduction in the size of human TRAIL receptor-targeted drugs attractive (their tumor selectivity mammary adenocarcinoma xenografts in mice administered 6,17 and the fact that they are boosting a physiological tumor soluble recombinant TRAIL. Accordingly, in both TRAIL À / À suppression mechanism) are likely to be selected against during mice and mice treated with a neutralizing anti-TRAIL antibody, tumor evolution. Therefore, one would expect that the ability to inoculation with renal carcinoma and mammary carcinoma cells predict and circumvent TRAIL resistance will be essential for the results in a faster growing and more metastatic tumors than in successful clinical use of these drugs. wild-type mice,13 and aged TRAIL À / À mice have an increased incidence of lymphoma.18 TRAIL RESISTANCE TRAIL AS A SELECTIVE KILLER OF CANCER CELLS Multiple mechanisms of TRAIL resistance have been identified. Interest in TRAIL as an anticancer agent was initiated by early Theoretically, resistance mechanisms could target any of the studies in which TRAIL-induced killing could be demonstrated in a events starting from TRAIL ligand-binding through to the end wide variety of tumor cells in vitro1,2 and in vivo,6,17,19 whereas point of apoptosis by affecting the expression and/or function normal cells were unaffected by TRAIL treatment. Early on, an of TRAIL pathway components (Figure 2). Some of the major alarming study indicated that TRAIL was toxic to normal human mechanisms are discussed below. hepatocytes, seemingly disqualifying TRAIL for clinical applications because of the risk of hepatotoxicity or fulminant hepatic failure.20 The group attributed the discrepancy to a difference in model TRAIL RECEPTORS AS REGULATORS OF TRAIL RESISTANCE systems, but the toxicity was later shown to be an artifact caused Early on, it was postulated that TRAIL resistance was mediated by by the particular preparation of TRAIL. The recombinant proteins the non-functional DcR, DcR1 and DcR2, competing with the

Dulanermin AMG 655 HDAC INHIBITORS CS-1008 PROTEASOME INHIBITORS DR4 / DR5 PRO95780 LBY-135

AS-PTO CASPASE 8 FLIP QUERCETIN

HA14-1 BID BCL2, MCL1 CASPASE 3 ABT-737

CISPLATIN APOPTOSIS CASPASE 9 SURVIVIN SM-164 Figure 2. TRAIL signaling pathway and some points of therapeutic intervention. The functional TRAIL receptors DR4 and DR5 can be activated by proapoptotic receptor agonists such as mapatumumab (DR4), lexatumumab (DR5) and dulanermin (both DR4 and DR5). Some drugs, such as histone deacetylases inhibitors and proteasome inhibitors, augment apoptosis by upregulation of receptor expression. Caspase 8 is activated downstream of receptor activation. This activation is prevented by FLIP, which in turn is inhibited by antisense phosphorothioate oligonucleotide and quercetin. The mitochondrial activation through BID cleavage is counteracted by pro-survival BCL2 protein family members such as BCL2 and MCL1, which can be downregulated by HA14-1 and ABT-737. Further downstream, caspase 9 activation is prevented by survivin, which can be pharmacologically inhibited by cisplatin and SM-164. For additional details, please refer to the text.

& 2013 Macmillan Publishers Limited Oncogene (2013) 1341 – 1350 On the TRAIL to successful cancer therapy LY Dimberg et al 1344 functional receptors DR4 and DR5 for TRAIL binding. The of receptor stimulation and the abundance of the shorter FLIP 41 expression of DR4 and DR5 is detectable in most tissues but is isoforms. In addition, FLIPL has a role in survival by forming a lower in normal cells than in cancer cells.23 However, studies have complex with pro-caspase 8 that inhibits receptor-interacting 42 failed to find a consistent correlation between the expression of protein K3-dependent necrosis. Notably, the FLIPL/ caspase 8 functional receptors, DcR and TRAIL sensitivity in cancer cell lines heterodimer results in a lower degree of activation that alters the and tumors.24–27 In contrast, downregulation of DR4 and/or DR5 substrate specificity, favoring proliferation and differentiation by epigenetic changes such as silencing by hypermethylation28 or pathways rather than apoptosis pathways.43 FLIP short and FLIP by deletions or point mutations29–31 is frequently associated with Raji are truncated forms that lack the caspase-like domain and act TRAIL resistance in cancer, suggesting that a functional receptor is, by competing with the caspases at the DISC to prevent binding 44,45 at least, necessary for the activity. Despite the poor correlation and activation. A novel cleavage form of FLIPL, p22, can between exact levels and sensitivity to TRAIL, upregulation of DR4 augment anti-apoptotic signaling pathways by inducing nuclear and/or DR5 appears to mediate enhanced TRAIL sensitivity by factor kB activation.46 Overexpression of FLIP has been linked to several drugs including the quercetin derivative LY303511,32 TRAIL resistance in many cancers and conversely, downregulation histone deacetylase inhibitors such as trichostatin33 and the of FLIP enhances TRAIL-induced apoptosis (reviewed in Yang47). proteasome inhibitor .23 Importantly, the response to Several TRAIL-sensitizing drugs, including withaferin A,48 TRAIL-stimulating drugs can not generally be predicted from the quercetin,49 agonists of peroxisome proliferator-activated expression of DR4 and DR5. Even in cells where both DR4 and DR5 receptor g50 and a novel epidermal growth factor receptor- expression are intact, there is often a preference for use of one targeted diphteria toxin51 all act at least in part by downregulating receptor over another. For example, in chronic lymphocytic FLIP. Expression of MYC is directly correlated to TRAIL sensitivity leukemia and mantle cell lymphoma, both DR4 and DR5 are and involves transcriptional repression of FLIP.52 In a recent study, expressed and normal, but TRAIL signaling occurs almost an antisense phosphorothioate oligonucleotide-targeting FLIP exclusively through DR4,34 as signaling through DR5 requires sensitized several cancer cell lines but not a normal lung cell crosslinking of the agonistic antibody,35 which can be impaired in line to TRAIL-induced apoptosis. This FLIP-targeted antisense immuncompromised patients owing to the lack of endogenous Fc phosphorothioate oligonucleotide also efficiently enhanced receptors. Intriguingly, a point mutation in DR5 was found to have apoptosis in xenograft models.53 These studies suggest that a dominant negative effect in the sense that the mutated direct targeting of FLIP may be beneficial as therapy in dysfunctional receptor competes with the functional DR4 combination with TRAIL to circumvent and prevent TRAIL receptor for binding of the TRAIL ligand.31 In light of these resistance. However, because of the complexity of the functions findings, it is clear that soluble TRAIL and agonists to the respec- of FLIP, where subtle changes in concentration of the FLIP proteins tive receptors may have different effects in different tumors themselves as well as other DISC components may lead to depending on which of the DR4 and DR5 receptors is active. If, in a opposing effects on apoptosis sensitivity, the usefulness of c-FLIP given tumor, the importance of one receptor over another can be overexpression as a biomarker for TRAIL resistance may be limited. assessed, it may be possible to enhance TRAIL-induced apoptosis by using receptor agonists that are crosslinked35 or that have a high selective affinity for one receptor over the other.36 It is, BCL2 FAMILY PROTEINS however, unlikely that DR4 and DR5 expression per se is a good The BCL2 family consists of proteins that either promote or inhibit enough predictor of TRAIL sensitivity to be used clinically for mitochondria-dependent apoptosis by influencing the perme- patient pre-evaluation. ability of the mitochondrial membrane and/or by regulating the In addition to the receptor levels having a role in TRAIL activity of each other. In TRAIL signaling, pro-apoptotic BID sensitivity, their distribution on the membrane can also affect the activates BAX and BAK, inducing permeabilization of the activity. Cholesterol- and sphingolipid-enriched microdomains in mitochondrial membrane and release of cytochrome c. Loss of the plasma membrane, called lipid rafts, facilitate pre-clustering of pro-apoptotic BAX54 and/or BAK55 as well as overexpression of receptors on the cell membrane, which enhances ligand binding anti-apoptotic BCL2,56,57 BCLXL58 and MCL159–61 all confer and recruitment of receptor-associated signaling molecules. resistance to TRAIL. Pro-survival BCL2 family proteins can be Sensitization to TRAIL by the formation of lipid rafts can be targeted clinically using BH3 mimetics (reviewed in Ghiotto accomplished by treatment with quercetin37 and cox-2 et al.62) or small molecule inhibitors such as the BCL2 inhibitor inhibitors.38 TRAIL resistance may also be acquired through HA14-1.57 The BH3 mimetic ABT-737 enhances TRAIL killing in inefficient transportation of the receptors to the cell membrane. multiple cancer cell types, regardless of MCL1 status, but this This process involves glycosylation, as glycosylation inhibitors effect requires expression of BAX.63 ABT-737 induces upregulation restore receptor transport and TRAIL sensitivity.39 Glycosylation of DR5, suggesting that induction of apoptosis by TRAIL is also influences the clustering of receptors so that highly augmented through both the intrinsic and extrinsic pathways by glycosylated receptors are more prone to dimerize and, this drug.63 Another BH3 mimetic, the BCLXL inhibitor BH3I-20, consequently, form the signaling complex required for caspase synergizes with TRAIL in prostate carcinoma cells by increasing the activation. Interestingly, the protein N-acetylgalctosaminyl- amount of BAX/BAK available to be activated by BID.64 Despite the transferase-14 (GALNT14), which promotes receptor glycosyla- encouraging preclinical data, the complexity and redundancy of tion and clustering, is associated with TRAIL sensitivity, as the BCL2 family proteins makes pre-evaluation of patients based discussed in more detail below.40 on the expression of these proteins cumbersome, and thus may not be practically feasible. FLICE-LIKE INHIBITORY PROTEIN (FLIP) The degree by which caspase 8/10 is regulated by FLIP is another THE INHIBITOR OF APOPTOSIS PROTEINS determinant of TRAIL sensitivity. FLIP is structurally similar to The extrinsic and the intrinsic pathway converge at the level of caspases and exists in two shorter forms, FLIP short and FLIP Raji activation of effector caspases such as caspase 3, 7 and 9. The and a long form, FLIP long (FLIPL). These forms of FLIP interfere inhibitor of apoptosis proteins are a family of proteins that can with TRAIL signaling in distinct ways. FLIPL has a caspase-like inhibit apoptosis by directly inhibiting effector caspases, thus domain that allows it to dimerize with pro-caspase 8 and 10 at the influencing apoptosis signaling from both pathways of apoptosis DISC. It can have either a pro-apoptotic function or an anti- induction. The inhibitor of apoptosis proteins are antagonized by apoptotic function depending on the amount of FLIPL, the degree the protein Smac/Diablo. The most studied IAP, X-linked inhibitor

Oncogene (2013) 1341 – 1350 & 2013 Macmillan Publishers Limited On the TRAIL to successful cancer therapy LY Dimberg et al 1345 of apoptosis (XIAP), mediates TRAIL resistance when over- TRAIL resistance, and are attempting to find ways to define and expressed,65 providing a rationale for the inhibition of XIAP as a circumvent this resistance. strategy to enhance TRAIL-induced apoptosis. Indeed, TRAIL resistance in prostate cancer cells can be overcome by addition of a zinc chelator, which downregulates XIAP66 and a small- HUMAN CLINICAL TRIALS TO DATE molecule XIAP antagonist synergizes with TRAIL in vitro and Several proapoptotic receptor agonists, including the recombinant in vivo.67 In a study by Xu et al.,68 sensitization to TRAIL by cisplatin human TRAIL ligand (dulanermin), which targets both DR4 and involved downregulation of the IAP survivin. Similarly, DR5, and agonistic antibodies against the functional receptors DR4 overexpression of SMAC/DIABLO sensitizes prostate cancer cells (mapatumumab) and DR5 (lexatumumab, drozitumab, conatumu- to TRAIL coinciding with a reduction in XIAP, cIAP1 and cIAP2.69 In mab, tigatuzumab and LBY-135) have been assessed within a recent, more clinically relevant approach, the non-peptide SMAC clinical trials for cancer treatment. At the time of this review, mimetic SM-164 was highly synergistic with TRAIL in breast, there were a total of 27 human clinical trials published on pro- prostate and colon cancer cells in vitro, and in breast cancer apoptotic receptor agonist therapies (see Table 1 and references xenografts in vivo.70 Interestingly, SM-164 did not only act by therein). Several of these clinical investigational studies have been inhibiting inhibitor of apoptosis proteins, but also by enhancing published and summarized in prior reviews.78,79 Table 1 provides DISC formation and recruitment of caspase 8 in a receptor- an updated summary of all published human clinical trials to date interacting protein 1-dependent manner. of both monotherapy and combination therapy utilized in clinical trials. Some trials have been tumor-type focused, whereas others, particularly the first-in-man studies, have explored the pro- O-GLYCOSYLATION AND TRAIL SENSITIVITY apoptotic receptor agonists in general advanced cancer popula- In an attempt to identify determinants of TRAIL sensitivity, Wagner tions. No trials to date have involved any degree of molecular et al.40 investigated TRAIL sensitivity in 119 human cancer cell preselection. lines and performed whole-genome microarray profiling to detect differences in gene expression between TRAIL-sensitive and TRAIL-resistant cell lines. The most prominent molecule found SINGLE AGENT TRIALS to correlate with TRAIL response in this study was the There have been several trials80–90 evaluating efficacy of O-glycosylation-initiating enzyme GALNT14. Indeed, presence of conatumumab AMG 655 ( DR5 agonist), the enzyme predicted sensitivity to TRAIL 61% of the time, CS-1008 (humanized monoclonal antibody DR5 agonist), dulanermin whereas absence of the enzyme predicted resistance in 88% of all (rhApo2L/TRAIL pro-apoptotic receptor agonist), lexatumumab cases. The authors found that GALNT14 enhanced recruitment and (monoclonal antibody DR5 agonist), PRO95780 (fully human activation of caspase 8 by promoting ligand-induced clustering of monoclonal antibody DR5 agonist) and mapatumumab (mono- the DR4 and DR5 receptors, but not of Fas or tumor necrosis clonal antibody DR4 agonist). These agents clearly have factor receptor-1, demonstrating a specific role in sensitization to monotherapy activity with isolated responses reported in TRAIL. The same group subsequently developed an immuno- follicular lymphoma,81 adenocarcinoma of the lung85,91 and histochemical assay that measured the levels of GALNT14 and of synovial sarcoma.80 Although these responses appear rare, in fucosyltransferase 3/6, other enzymes involved in O-glycosylation, some cases they could be very long lasting, with one patient still in formalin-fixed, paraffin-embedded human tumor tissues and in on study at the time of publication receiving 106 doses over 4 human cell lines. These assays are currently being evaluated in years.91 The most commonly reported toxicity that could be phase II clinical trials as diagnostic tools to predict sensitivity to attributed as a class effect, as it occurred across several different the TRAIL receptor agonists dulanermin (recombinant TRAIL) and agents, was rare transaminitis that was reversible on cessation of drozitumab (agonistic anti-DR5 antibody),71 as discussed below. dosing.82

SIX1 COMBINED TRIALS Recently, the homeoprotein Six1 was identified as a novel Several trials have been carried out utilizing combined therapy to mediator of TRAIL resistance.72 Six1 is aberrantly expressed in evaluate the efficacy of conatumumab, dulanermin, lexatumumab, many forms of cancer and contributes to the malignant PRO95780 and mapatumumab in combination with other antic- phenotype by promoting tumor initiation, progression and ancer therapy.80,82,92–103 Four separate trials assessed the effect of metastasis.73–76 Overexpression of Six1 specifically induces TRAIL adding a PARA to standard first-line and resistance in TRAIL-sensitive cells, and knockdown (alone or with ) for advanced non-small cell lung of Six1 sensitizes previously resistant ovarian cancer cells to cancer.101,104–106 Toxicity was minimally affected by the addition TRAIL.72 In concordance with this finding, expression of microRNA- of their pro-apoptotic receptor agonists. However, none of these 185 overcomes TRAIL resistance in ovarian cancer cells specifically trials achieved their primary end points of improving response through downregulation of Six1.77 Importantly, high Six1 rate or progression-free survival. expression occurs in 460% of women with metastatic ovarian These results are not surprising given the rarity of monotherapy cancer, and is strongly associated with worse survival in these activity. Perhaps, this indicates the rarity of a sensitive subpopula- patients.72 These data suggest that many ovarian cancers may be tion. Moreover, we should not view the agents themselves as TRAIL resistant, and thus refractory to multiple different inactive, but only inactive in unselected patients. Within several of therapeutic regimens. Indeed, as Six1 correlates with adverse the randomized phase II studies in non-small cell , a outcomes, including metastasis, in many different tumor types, it prolonged tail in the progression-free survival curves amounting is tempting to speculate that this correlation is, at least in part, due to B15% of the treated population is apparent. Although this is to an acquired resistance to TRAIL, and that a high Six1 expression not enough to sway the median results,104,105 it is enough in tumors may be a negative predictor of TRAIL sensitivity. that with patient selection, these drugs could replicate the rare Because Six1 overexpression occurs in a large percentage of but sustained responses seen in isolated patients in the ovarian and breast cancers, there is a significant potential for monotherapeutic studies. screening of this biomarker to aid in patient selection and, ideally, A large proportion of the drugs used in the combination trials in applying more educated treatment regimes. Thus, we are prevent cell division and DNA replication, for instance by currently evaluating the feasibility of using Six1 as a biomarker for preventing DNA from unwinding (topoisomerase inhibitors such

& 2013 Macmillan Publishers Limited Oncogene (2013) 1341 – 1350 On the TRAIL to successful cancer therapy LY Dimberg et al 1346 Table 1. Clinical trials of pro-apoptotic receptor agonists (PARAs)

Agonist Site Drug Response Reference no.

Conatumumab AMG 655 (monoclonal NSCLC Paclitaxel and carboplatin 1 CR, 3 PR 101 antibody DR5 agonist) Sarcoma Doxorubicin 2 PR 92 CRC mFOLFOX and bevacizumab 6 PR 93 Pancreatic Gemcitabine 2 PR 98 CRC None 93 CRC Panitumumab None 102 Advanced solid Phase 1: single agent 1 PR 85 tumors Advanced solid Phase 1: single agent 1 PR 91 tumors CS-1008 (humanized monoclonal Advanced solid Phase 1: single agent None 86 antibody DR5 agonist) tumors NSCLC Randomized phase 2: paclitaxel/carboplatin — — CRC Randomized phase 2: irinotecan — — Ovarian Single arm phase 2: paclitaxel/carboplatin — — Pancreatic Single arm phase 2: gemcitabine — — cancer Dulanermin rhTRAIL (pro-apoptotic NHL 2 CR, 1 PR 95 receptor agonist) CRC Phase 1: irinotecan/ or FOLFIRI± 3PR 94 bevacizumab ADV TUMORS None 2 PR 80 NSCLC Paclitaxel and carboplatin±bevacizumab 1 CR, 13 PR 99 NSCLC Randomized phase 2: paclitaxel and —99 carboplatin±bevacizumab Lexatumumab (monoclonal antibody Advanced solid Gemcitabine, pemetrexed, doxorubicin, or PRs reported 100 DR5 agonist) tumors FOLFIRI Advanced solid Phase 1: single agent None 81 tumors Advanced solid Phase 1: single agent None 87 tumors PRO95780 (fully human monoclonal Advanced solid Phase 1: single agent None 82 antibody DR5 agonist) tumors CRC Phase 1: cetuximab/irinotecan or FOLFIRI/ —82 bevacizumab CRC Phase 1b: FOLFOX/bevacizumab — 82 NSCLC Randomized phase 2: paclitaxel/ —82 carboplatin/bevacizumab NHL Single arm phase 2: rituximab — 82 NSCLC Randomized phase 2: paclitaxel/ 106 carboplatin/bevacizumab Mapatumumab (monoclonal antibody Advanced solid Gemcitabine and cisplatin 12 PR 103 DR4 agonist) tumors Advanced solid Paclitaxel and carboplatin 5 PR 97 tumors Advanced solid None 19 had stable 83 tumors disease Advanced solid Phase 1: single agent None 84 tumors Hepatocellular carcinoma Phase 1: Single arm phase 2: sorafenib NHL Phase 2: single agent 1 CR, 2 PR 90 Multiple Randomized phase 2: bortezomib — — myeloma CRC Phase 2: single agent None 89 NSCLC Phase 2: single agent None 88 NSCLC Randomized phase 2: paclitaxel/carboplatin — 105 Abbreviations: CRc, ; NHL, non Hodgkin lymphoma; NSCLC, non-small cell lung cancer.

as irinotecan), by disruption of microtubule function (taxanes such antibodies targeting growth factor-signaling pathways relevant to as paclitaxel) or by inducing apoptosis or cell cycle arrest through different tumor types (for instance, bevacizumab, which targets several pathways (histone deacetylases inhibitors such as vorino- vascular endothelial growth factor A and cetuximab, which targets stat). Other drugs used in combination therapy are specific epidermal growth factor receptor). Importantly, although many

Oncogene (2013) 1341 – 1350 & 2013 Macmillan Publishers Limited On the TRAIL to successful cancer therapy LY Dimberg et al 1347 preclinical drugs synergize with TRAIL in cells that are already It must be emphasized that most resistance mechanisms have sensitive to TRAIL, a much smaller proportion of drugs can be been discovered in cell lines and that screening patients for the expected to actually reverse resistance to TRAIL that is already same resistance mechanisms and, even more so, inhibiting these present or that evolves during treatment. If we hypothesize that mechanisms is not always a plausible route to take. Indeed, in a most cancers are actually TRAIL resistant, the results from these recent study by Menke et al.,109 selective inhibition of TRAIL unselected clinical trials become almost predictable. In a recent receptors reduced chemosensitivity in vivo but not in vitro.In study performed by our group, synergism with TRAIL was addition, because of the complexity and the redundancy of the obtained with all 10 drugs tested, whereas only one, the apoptosis machinery, too focused an approach may be as proteasome inhibitor MG132, was able to overcome resistance ineffective as no approach. One additional approach is to in cell lines that had been made resistant to TRAIL by long-term evaluate apoptosis susceptibility further downstream in the in vitro selection in increasing concentrations of lexatumumab, by machinery, taking into account not only known players of the the overexpression of Six1 or decoy receptor overexpression.107 intrinsic as well as the extrinsic pathway, but also as yet The conclusion that we should draw from these data is not that unidentified resistance mechanisms. The principle behind this is TRAIL holds little promise as a cancer drug, but rather that as that if a tumor cell is more primed for death, regardless of the resistance to TRAIL is such a common occurrence, suboptimal mechanisms involved, it is more likely to undergo apoptosis in patient selection is likely to have had a negative effect on the response to TRAIL. In a study by Zhang et al.,110 the basal accurate evaluation of these drugs. apoptotic rate of tumors was measured by the cleavage of the apoptosis substrate keratin 18 before and after treatment with TRAIL. The results were encouraging, showing that an initial high DISCUSSION basal apoptotic rate was more likely to lead to a total basal It is apparent that despite the extensive research on TRAIL- apoptotic rate increase after treatment. resistant mechanisms, we have somehow failed to incorporate this Perhaps, some combination of approaches will be required. For preclinical data into the selection of patients and tailored example, an initial estimation of the likelihood of a positive treatment regimens in clinical trials. Because of this failure, we response to TRAIL therapy could involve a screening for positive are still lacking an accurate picture of how efficient TRAIL markers of sensitivity such as high GALNT14 expression, the treatment can be in the right setting. It is crucial that drug presence of at least one of the functional TRAIL receptors, and low regimens that show no response, when used in studies with or absent Six1 expression. This could be combined with a more suboptimal selection of patients, are not disqualified, in the event general assessment of the tumor cells’ propensity to undergo that the treatment would be highly beneficial in another patient apoptosis by using the basal apoptotic rate assay. When deciding group. It is highly noteworthy that there is to date not a single on a combination treatment, we would ideally like to see a clinical trial of TRAIL published that has attempted to preselect connection between known modes of action of drugs and patient patients using specific biomarkers that may predict response to a expression profiles, that is, if a drug enhances expression of a specific treatment regimen.108 Because of this, the results of TRAIL specific pro-apoptotic protein it may be more fruitful to use it in clinical trials performed to date may greatly underestimate the patients where the expression of that particular pro-apoptotic potential of drugs that activate this pathway. Complicating the protein is initially low. design of any such preselection study in the future is the fact that In conclusion, we have greatly expanded our knowledge about multiple different resistance mechanisms may occur, potentially TRAIL signaling and the factors that regulate it. At the same time, influencing the optimal biomarkers to use depending on the novel drugs that have the potential to specifically target these specific receptor agonist and partner drug involved. For example, regulators have been developed. With these advancements in the in a recent preclinical study evaluating the mechanisms field and with an understanding of the importance of individua- of cisplatin and TRAIL in combination, cisplatin was shown lized patient selection, we have an obligation to take the to enhance the TRAIL-induced apoptosis by survivin down- evaluation of TRAIL-based treatments to the next level, where regulation.68 Thus, the ideal future scenario might be that in a this cancer drug can finally receive a fair trial. clinical trial involving cisplatin treatment, we should carefully select patients in which the prevailing TRAIL-resistant mechanism exhibited in tumors is an overexpression of survivin. As we learn more about what governs the susceptibility to DR4 stimulation CONFLICT OF INTEREST versus DR5 stimulation, it may even be possible to preselect an HL Ford, K Behbakht and A Thorburn hold a patent on the use of Six1 to identify optimal TRAIL-targeting agent for some TRAIL-sensitive patients. TRAIL-sensitive tumor cells. Although the theoretical side of a biological approach to patient selection is very attractive, the practical issue of selecting and assaying appropriate biomarkers is a challenge. Optimally, we ACKNOWLEDGEMENTS would like to be able to select patients that are already sensitive to We apologize to the many investigators whose important works were not cited here TRAIL and/or to define TRAIL-resistant markers that are already owing to space limitations. This work was supported by NIH Grant CA124545 present or that may arise as a result of treatment. One exciting (A Thorburn, K Behbakht and H Ford), Department of Defense (DOD) postdoctoral approach to this is a GALNT14/fucosyltransferase 3/6 assay, which fellowship BC093627 and Swedish Research Council postdoctoral fellowship 2009- is currently being evaluated in phase II clinical trials as a diagnostic 618 (L Dimberg), DOD Ovarian Cancer Idea Award OC06143 (K Behbakht), and tool to predict sensitivity to the TRAIL receptor agonists, Department of Obstetrics and Gynecology Academic Enrichment Fund (AEF), dulanermin and drozitumab.71 University of Colorado-Denver Hospitals (C Anderson). However, if there is one thing we have learned from clinical trials it is that, in patients, TRAIL sensitivity is the exception to the rule. In addition, if sensitivity can be predicted 61% of the time REFERENCES from expression of GALNT14,71 it is likely that an additional biomarker would be necessary to increase the success rate. 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