Table Of ContentApo2L/TRAIL
Avi Ashkenazi
*
Department of Molecular Oncology, Genentech Inc., 1 DNA Way, South San Francisco,
CA 94080-4918, USA
*corresponding author tel: 650-225-1853, fax: 650-225-6443, e-mail: [email protected]
DOI: 10.1006/rwcy.2000.05012.
SUMMARY and lymphotoxin (cid:11) indicate that this region contains
aseriesof10(cid:12) strandsthatformthebasicfoldofthe
ECD. TNF-related ligands bind to specific receptors
Apo2L/TRAILisamemberoftheTNFligandfamily
that belong to the TNF receptor (TNFR) gene
that is closely related to FasL. Many tissues express
superfamily (Smith et al., 1994). Most TNFR family
the Apo2L/TRAIL mRNA, and T cells upregulate
members are type 1 transmembrane proteins; their
Apo2L/TRAIL expression upon T cell receptor stim-
common structural feature is the presence of 2–6
ulation. The distinct biologic function of Apo2L/
cysteine-rich domains in the extracellular portion.
TRAIL has yet to be defined. There is evidence that
The crystal structure of the complex between
Apo2L/TRAILmight play arolein T cell activation-
lymphotoxin-(cid:11) and the ECD of TNFRI reveals that
induced cell death (AICD), in target-killing by cyto-
three receptor molecules bind to the homotrimeric
toxic lymphocytes and macrophages, and perhaps in
ligand, each docking in the groove between two
the immune-privilege of certain tumors. Apo2L/
ligand subunits (Banner et al., 1993). TNFR family
TRAIL bindsto a complex system of receptors: DR4
proteins fall into two subgroups on the basis of
and DR5 have death domains that signal apoptosis,
intracellular sequence: receptors that contain death
whereas DcR1, DcR2, and OPG lack functional
domains (a 70 amino acid homophilic protein
deathdomainsandcaninhibitapoptosisinductionby
interaction motif), and receptors that lack death
the ligand. Apo2L/TRAIL potently activates apop-
domains. The receptors that have death domains,
tosisin awide variety oftumor cell lines, butitis not
dubbed‘deathreceptors’,triggercellularresponsesby
cytotoxic towards most normal cell types studied so
engaging specific adapter molecules such as TRADD
far. Experiments in mouse models of cancer suggest
(TNFR-associated death domain) or FADD (Fas-
that Apo2L/TRAIL might be useful as an anticancer
associated death domain) through homophilic death
agent that kills tumor cells without damaging normal
domain interactions (Ashkenazi and Dixit, 1998).
tissues.
TNFR family members that lack death domains
signal by engaging specific adaptor molecules called
TRAFs (TNFR-associated factors).
BACKGROUND
The best characterized death receptor and ligand
systems (Simonet et al., 1997)are thoseofFas/Apo1/
The members of the tumor necrosis factor (TNF) CD95 with Fas ligand (FasL), and the p55 TNF
gene superfamily modulate diverse biological func- receptor (TNFRI) with TNF. There is a growing
tions, including cell differentiation, proliferation, and interest in a third, more recently discovered apop-
apoptosis (Gruss and Dower, 1995). Most TNF tosis-inducing ligand, called Apo2 ligand (Apo2L)
familyligandsareexpressedastype2transmembrane or TNF-related apoptosis-inducing ligand, which is
proteins, some of which are processed by specific structurally related to FasL and TNF. Much of the
proteases into soluble homotrimeric cytokines. The interest stems from the existence of an unusually
sequence homology between TNF and its known complex system of death and decoy receptors that
relatives occurs primarily in the C-terminal extra- interact with this ligand (Ashkenazi and Dixit, 1998,
cellulardomain(ECD).ThecrystalstructuresofTNF 1999).
504 Avi Ashkenazi
Discovery Third, Chinese hamster ovary cells transfected with
an expression plasmid encoding full-length Apo2L/
TRAIL release a soluble form of the ligand that
Investigators (Wiley et al., 1995; Pitti et al., 1996)
migrates on SDS-PAGE gels under reducing condi-
identifiedexpressedsequencetags(ESTs)thatshowed
tions as a 24kDa protein band (Figure 2).
homology to the TNF ligand family. On the basis of
the ESTs, a full-length cDNA was isolated that
encoded a previously unknown member of the TNF
Main activities and
family. The novel protein, which showed highest
homologytoFas/Apo1ligand,wasdesignatedApo2L pathophysiological roles
(Pitti et al., 1996) or TRAIL (Wiley et al., 1995).
ThebiologicfunctionsofApo2L/TRAILarenotfully
understood; however, the ligand’s ability to trigger
Alternative names
apoptosis in a variety of transformed cell lines sug-
gests that it may be a physiologic modulator of
Alternative names are TL2 (Tan et al., 1997) and apoptosis. Apo2L/TRAIL also is capable of activat-
TNFSF10. ing the proinflammatory transcription factor NF(cid:20)B;
however, in comparison to TNF, it is a very weak
NF(cid:20)B activator (Sheridan et al., 1997; Ashkenazi
Structure
et al., 1999). Several lines of evidence suggest that
Apo2L/TRAIL may play a role in the AICD of peri-
The human Apo2L/TRAIL cDNA predicts a poly- pheralTcells,aprocessinwhichFasLplaysacentral
peptide of 281 amino acids. Hydropathy analysis role (Nagata, 1997).
suggests that Apo2L/TRAIL has the topology of a First, a subset of peripheral T cells acquires sen-
type 2 transmembrane protein, with a 15–17 amino sitivity to Apo2L/TRAIL-induced apoptosis after a
acid-longN-terminalcytoplasmicregion,followedby 3-day stimulation by IL-2 (Marsters et al., 1996) or a
a transmembrane region of 21–23 residues, and an 5-day stimulation by IL-2 and PHA (Snell et al.,
extracellular C-terminal region. The protein has one 1997).
potential N-linked glycosylationsite (Asn109).Align- Second, Apo2L/TRAIL mRNA expression is in-
ment of the extracellular region with that of other duced upon the stimulation of CD4+ and CD8+
family members suggests that the (cid:12) strand region peripheralbloodTcellswithPHAorwiththeprotein
starts around amino acid 120 (Figure 1). FACS kinase C activator phorbol 12-myristate 13-acetate
staining of cells, transfected with the full-length (PMA) and the calcium ionophore ionomycin
Apo2L/TRAIL cDNA tagged by a C-terminal Myc (Jeramiasetal.,1988;Screatonetal.,1997;Martinez-
epitope, with anti-Myc antibody indicates that the Lorenzo et al., 1998).
molecule is expressed at the cell surface as a type 2 Third, neutralizing anti-Apo2L/TRAIL antibody
protein(Pittietal.,1996).FACSstainingofcellsthat reduces the cytotoxicity of the supernatants of PHA-
express endogenous Apo2L/TRAIL with polyclonal stimulated peripheral blood mononuclear cells or
antiligand antibodies also indicates cell surface ex- JurkatTcellstowardsunstimulatedTcells(Martinez-
pression (Mariani and Krammer, 1998a, 1998b), as Lorenzo et al., 1998). In addition, T cells from HIV-
does immunohistochemical staining of tissue sections infectedpatientsshowincreasedsensitivitytoApo2L/
with monoclonal anti-Apo2L/TRAIL (Ashkenazi TRAIL,whichsuggestsapotentialrolefortheligand
et al., 1999). The full-length Apo2L/TRAIL protein in the killing of virus-infected cells (Jeramias et al.,
migrates on SDS-PAGE gels as a 32–33kDa protein 1998).
(Mariani and Krammer, 1998a, 1998b). There is evidence also for the involvement of
There is evidence that cells can release Apo2L/ Apo2L/TRAILintarget-killingbyCD4+cytotoxicT
TRAIL in soluble form, apparently through proteo- lymphocytes (CTLs) and by natural killer (NK) cells,
lytic processing of the ECD. First, supernatants from bothofwhichexpressApo2L/TRAILmRNA(Thomas
phytohemagglutinin (PHA)-stimulated human Jurkat and Hersey, 1998; Zamani et al, 1998). Further
T cells contain cytotoxic activity that can be blocked evidence suggests that Apo2L/TRAIL is involved in
partially by a neutralizing monoclonal anti-Apo2L/ tumor cell killing by macrophages (Griffith et al.,
TRAIL antibody (Martinez-Lorenzo et al., 1998). 1999).Humanastrocyticbraintumors,butnotnormal
Second, the surface expression of Apo2L/TRAIL on glia, express Apo2L/TRAIL mRNA (Rieger et al.,
Jurkat cells increases upon treatment with inhibitors 1998),asdoseveralcelllinesderivedfromothertypes
ofcysteineproteases(Mariani andKrammer,1998a). of cancer (Ashkenazi et al., 1999); this expression
Apo2L/TRAIL 505
Figure 1 Alignment of the putative extracellular (cid:12) strand region of Apo2L/TRAIL with that of other TNF family
members. The (cid:12) strand regions are marked above with lines and letters.
suggestsapotentialinvolvementofApo2L/TRAILin transcription of the Apo2L/TRAIL gene (Screaton
the immune-privilege of tumors. et al., 1997; Martinez-Lorenzo et al., 1998).
The apparent involvement of Apo2L/TRAIL in
T cell AICD, in target-killing by cytotoxic lympho- Accession numbers
cytes,andintheimmune-privilegeoftumorssuggests
that this ligand resembles FasL not only in sequence,
Human mRNA: HSU37518, HSU57059
but also in certain functions. Recent work suggests
Mouse mRNA: MMU37522
that Apo2L/TRAIL, but not FasL or TNF, plays a
key role in the killing of dendritic cells by auto-
Chromosome location
logousCD4+CTLs(Wanget al.,1999),suggestinga
potentially unique role for Apo2L/TRAIL in mod-
ulating the immune system. Human chromosome 3q26 (Wiley et al., 1995).
Relevant linkages
GENE AND GENE REGULATION
Apo2L/TRAIL receptors
PublishedinformationonthestructureoftheApo2L/ Osteoprotegerin
TRAIL gene is not yet available. The PHA- FasL
stimulation of peripheral blood T cells induces TNF
506 Avi Ashkenazi
Figure2 Westernblotanalysisofsol- PROTEIN
uble Apo2L/TRAIL shed by Chinese
hamster ovary cells after transfection
Accession numbers
with cDNA encoding the full-length
protein. Cell supernatants were col-
lected 24 hours after transfection, Human protein: GEN13977
resolvedbySDS-PAGE,andanalyzed Human protein: P_W19777
byimmunoblotwithmonoclonalanti- Mouse protein: GEN13978
Apo2L/TRAIL antibody 5C2.
Sequence
See Figure 3.
60
Description of protein
Apo2L/TRAIL is a 281 amino acid, type 2 trans-
42 membraneprotein,withacalculatedmolecularweight
of 32.5kDa and an isoelectric point of 7.63. The full-
lengthproteinmigratesasa32–33kDabandonSDS-
PAGE gels (Mariani and Krammer, 1998a, 1998b);
30
a shed form of the protein migrates as a band of
approximately 24kDa (see Figure 2). Several recom-
binant soluble versions of the protein have been
22 generated. A polyhistidine-tagged soluble form of
Apo2L/TRAIL (amino acids 114–281) forms homo-
trimers and is biologically active (Pitti et al., 1996;
17 Ashkenazi et al., 1999). In contrast, a Flag-epitope-
tagged soluble form of the protein (amino acids 95–
281) is poorly active, and requires oligomerization by
anti-Flag antibody for potent biologic activity (Wiley
et al., 1995; Walczak et al., 1999). A fusion protein,
containingthesameApo2L/TRAILaminoacids(95–
281) fused N-terminally to a modified leucine-zipper
Cells and tissues that express
that promotes trimerization, forms trimers and is
the gene biologically active (Walczak et al., 1999).
Northern blot analyses with probes that are based Important homologies
upon the Apo2L/TRAIL cDNA reveal a single
mRNA transcript of (cid:24)2kb (Wiley et al., 1995; Pitti
The C-terminal portion of the Apo2L/TRAIL ECD
et al., 1996). The Apo2L/TRAIL message is present
shows 28% identity to the FasL ECD, 23% identity
constitutively in many tissues, including fetal lung,
to the ECDs of TNF and lymphotoxin (cid:11), and 22%
liver, and kidney, and adult spleen, prostate, thymus,
identity to the ECD of lymphotoxin (cid:12) (see Figure 1).
ovary, small intestine, colon, PBLs, heart, placenta,
The loop that connects the first and second putative
lung, skeletal muscle, and kidney. In situ hybridiza-
(cid:12) strands is substantially longer in the ECD of
tionanalysisindicatesApo2L/TRAILmRNAexpres-
Apo2L/TRAIL ECD than in other family members.
sion in human astrocytic brain tumors and in glial
The murine Apo2L/TRAIL protein is 291 amino
cancer cell lines (Rieger et al., 1998). In addition, the
acids long, and it shows 65% identity to the human
stimulation of CD4+ and CD8+ peripheral blood
protein (Wiley et al., 1995).
T cells with PHA or with PMA and ionomycin
induces expression of the Apo2L/TRAIL mRNA
Posttranslational modifications
(Screatonetal.,1997;Jeramiasetal.,1998;Martinez-
Lorenzo et al., 1998). Stimulation of macrophages
with IFN(cid:13) induces cell surface expression of the Human and murine Apo2L/TRAIL each have one
Apo2L/TRAIL protein (Griffith et al., 1999). potential N-linked glycosylation site (Asn109 and
Apo2L/TRAIL 507
Figure 3 Amino acid sequence of Apo2L/TRAIL.
Sequence
MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIA
CFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQ
NISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFL
SNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPIL
LMKSARNSCWSKDAEYGLYSIYQCGIFELKENDRIFVSVTNEHLIDMDHEASFFG
AFLVG
Asn52 respectively). Apo2L/TRAIL is made as a RECEPTOR UTILIZATION
type 2 transmembrane protein; a soluble form that
contains the ECD can be released proteolytically
Apo2L/TRAIL interacts with four cell surface
by cells that express the full-length protein (see
receptors that form a distinct subgroup within the
Description of protein).
TNFR gene superfamily (for a review, see Ashkenazi
and Dixit, 1998, 1999). Death receptor 4 (DR4) (Pan
et al., 1997) and DR5 (Pan and Dixit, 1997; Sheridan
et al., 1997) (also called TRICK2, TRAIL-R2 or
CELLULAR SOURCES AND
KILLER)(Screatonetal.,1997;Walczaketal.,1997;
TISSUE EXPRESSION Wuetal.,1997)havecytoplasmicdeathdomainsand
signal apoptosis. Decoy receptor 1 (DcR1) (Sheridan
Cellular sources that produce et al., 1997) (also called TRID, TRAIL-R3, or LIT)
(Degli-Esposti et al., 1997a; Mongkolsapaya et al.,
1998; Pan et al., 1998a), is a phospholipid-anchored
Apo2L/TRAILappearstobeexpressedconstitutively
cell surface protein that lacks a cytoplasmic tail.
on the surface of mouse and human tumor cells of
DcR2 (Marsters et al., 1997) (also called TRAIL-R4
TandBorigin,includingmouseEL4TcellsandA20
orTRUNDD)(Degli-Espostietal.,1997b;Panetal.,
B cells, and human Jurkat T cells and Bjab B cells
1998b) has a substantially truncated death domain
(Mariani and Krammer, 1998a). Freshly isolated
that does not signal apoptosis induction. Upon over-
murine splenocytes, concanavalin A/IL-2-activated
expression, DcR1 or DcR2 inhibits apoptosis induc-
T cells, and lipopolysaccharide-activated B cells were
tionbyApo2L/TRAIL.Apo2L/TRAILbindsalsoto
analyzedbysurfacestainingwithanti-Apo2L/TRAIL
a secreted, soluble member of the TNF receptor
antibody (Mariani and Krammer, 1998b). Activated,
family, osteoprotegerin (OPG) (Simonet et al., 1997;
but not resting, CD3+ cells expressed Apo2L/
Emery et al., 1998). The binding affinity of Apo2L/
TRAIL. Freshly isolated B220+ cells displayed sur-
TRAIL for OPG is 3–5-fold lower than the affinity
face Apo2L/TRAIL and CD95L that were retained
for DR5 or DcR1 (Emery et al., 1998). OPG is not
following activation. In CD3+ TCR(cid:11)(cid:12) cells with the
closely related to the other four Apo2L/TRAIL
CD4+orCD8+phenotype,restimulationwithPMA
receptors, and it binds also to another TNF family
and ionomycin or an agonistic anti-CD3 monoclonal
member, OPGL (also called RANKL or TRANCE).
antibody induced a significant upregulation of sur-
face Apo2L/TRAIL and CD95L. Apo2L/TRAIL
upregulation was inhibited by cyclohexamide and by
cyclosporin A. IN VITRO ACTIVITIES
In vitro findings
Eliciting and inhibitory stimuli,
including exogenous and The main in vitro biologic activity of Apo2L/TRAIL
so far reported is the induction of caspase-dependent
endogenous modulators
apoptosis in tumor cell lines from various tissue
origins (Wiley et al., 1995; Pitti et al., 1996; Mariani
See Gene and gene regulation. et al., 1997; Snell et al., 1997; Jeramias et al., 1998;
508 Avi Ashkenazi
Thomas and Hersey, 1998). There is also evidence and Dixit, 1998; Emery et al., 1998). Intracellularly,
that Apo2L/TRAIL can induce apoptosis in a subset the c-FLIP molecule, which blocks apoptosis signal-
of activated T cells (Marsters et al., 1996; Snell et al., ing by FasL and TNF, is also capable of inhibiting
1997; Martinez-Lorenzo et al., 1998), and in a subset apoptosisinductionbyApo2L/TRAIL(Thomeetal.,
ofTcellsfromHIV-infectedchildren(Jeramiasetal., 1997). Certain chemotherapeutic drugs, including
1998). In addition, Apo2L/TRAIL is capable of inhibitors of transcription or translation and DNA-
activating NF(cid:20)B; the magnitude of this activation is damaging agents, sensitize cancer cell lines to the
much less than that of activation by TNF, and it cytotoxic activity of Apo2L/TRAIL (Keane et al.,
requires a substantially higher concentration of 1999; Mori et al., 1999). In addition, IL-1 protects
Apo2L/TRAIL relative to TNF (Sheridan et al., transformed keratinocyte cell lines against apoptosis-
1997; Ashkenazi et al., 1999). Apo2L/TRAIL is also induction by Apo2L/TRAIL (Kothny-Wilkes et al.,
capable of inducing the activation of c-Jun 1998).
N-terminal kinase (JNK): in HeLa cells, but not in
Kym-1 cells, the activation can be blocked by the
caspase inhibitor zVAD-fmk, which suggests the Bioassays used
involvement of caspase-dependent and caspase-
independent mechanisms in this effect (Muhlenbeck
Apo2L/TRAIL bioactivity can be conveniently
et al., 1998).
assayed in standard cytotoxicity or cell viability
Morethanhalfofthe60tumorcelllinesinapanel
assays. Alternatively, Apo2L/TRAIL bioactivity can
developed by the US National Cancer Institute,
be assayed by measuring apoptosis induction, on the
which contains cell lines derived from leukemia,
basis of parameters such as DNA fragmentation, or
melanoma, non-small cell lung cancer, colon cancer,
phosphatidylserine-flip, as measured by FACS stain-
central nervous system cancer, ovarian cancer, renal
ing with annexin V (Pitti et al., 1996). Apoptosis can
cancer, prostate cancer, and breast cancer, showed
also be assayed on the basis of caspase activation,
sensitivitytoApo2L/TRAIL(Ashkenazietal.,1999).
measured by the cleavage of synthetic caspase sub-
In contrast, several normal cell types were resistant
strates, or by the cleavage of cellular caspase sub-
in culture to Apo2L/TRAIL (Ashkenazi et al., 1999;
strates such as poly-ADP ribose polymerase (PARP)
Walczak et al., 1999), with the exception of fetal
or caspase 3.
astrocytes (Walczak et al., 1999).
Neutralizing anti-Apo2L/TRAIL antibodies atten-
uate the cytotoxic activity of supernatants from
activated T cells towards unstimulated T cells IN VIVO BIOLOGICAL
(Martinez-Lorenzo et al., 1998), which implicates
ACTIVITIES OF LIGANDS IN
Apo2L/TRAIL in the AICD of T lymphocytes. This
ANIMAL MODELS
notion gains further support from the observation
that blocking anti-Apo2L/TRAIL antibody inhibits
the AICD of T cells from HIV-infected patients Species differences
(Katsikis et al., 1997). Soluble Apo2L/TRAIL recep-
tors in the form of an Fc-fusion protein (immuno- Studies on a limited number of cell lines indicate that
adhesin) inhibit the killing of target tumor cells by human Apo2L/TRAIL is cytotoxic towards murine
macrophages, implicating the ligand in mediating the celllinessimilarlytomurineApo2L/TRAIL,andthat
cytotoxic activity of macrophages (Griffith et al., murine Apo2L/TRAIL is cytotoxic towards human
1999). cell lines similarly to human Apo2L/TRAIL (Wiley
etal.,1995;Marianietal.,1997;Walczaketal.,1999).
Regulatory molecules: Inhibitors
Endogenous inhibitors and
and enhancers
enhancers
Extracellular,aswellasintracellular,mechanismsmay
modulate apoptosis induction by Apo2L/TRAIL. The mRNA for Apo2L/TRAIL is expressed consti-
Extracellularly, three decoy receptors that belong to tutively in many tissues, as are the mRNAs for DR4
the TNFR gene superfamily can bind to Apo2L/ and DR5; hence, there may be endogenous mecha-
TRAIL and inhibit the ligand’s apoptosis-inducing nisms that control cellular sensitivity to the cytotoxic
activity:theseareDcR1,DcR2,andOPG(Ashkenazi action of Apo2L/TRAIL. One such mechanism may
Apo2L/TRAIL 509
involve the expression of the decoy receptors DcR1 injections of the protein in nonhuman primates
and DcR2 in normal tissues. Because DcR1 and did not cause detectable toxicity to the tissues and
DcR2areexpressedinfrequentlyandgenerallyatlow organs examined. Apo2L/TRAIL exerted cytostatic
levels in cancer cell lines, it is unlikely that they or cytotoxic effects in vitro on 32 of 39 cell lines
regulate the sensitivity to the ligand in transformed from colon cancer, lung cancer, breast cancer, kidney
cells. Whether the decoys are important inhibitors of cancer, brain cancer, and skin cancer. The treatment
Apo2L/TRAIL cytotoxicity in normal tissues that of athymic mice with Apo2L/TRAIL shortly after
express DcR1 or DcR2 more abundantly remains to tumor xenograft injection markedly reduced tumor
be investigated. incidence. The Apo2L/TRAIL treatment of mice
Consistent with the latter hypothesis is the bearing solid tumors induced tumor cell apoptosis
observation that T cell activation downregulates and tumor regression, suppressed tumor progression,
DcR1 expression, perhaps as a mechanism for and improved survival. Apo2L/TRAIL cooperated
regulating the sensitivity to Apo2L/TRAIL during synergistically with the chemotherapeutic drugs
AICD (Mongkolsapaya et al., 1998). In addition, the 5-fluorouracil and CPT-11 at causing substantial
treatment of human umbilical cord endothelial cells tumor regression or complete tumor ablation. These
with phosphatidylinositol phospholipase C sensitizes studies suggest that Apo2L/TRAIL may turn out to
the cells to apoptosis induction by Apo2L/TRAIL, be useful as an anticancer agent that induces apop-
consistent with the removal of GPI-anchored DcR1 tosis in tumors while sparing normal tissues.
from the cell surface (Sheridan et al., 1997). Another
mechanism to regulate the sensitivity of cells to
Pharmacokinetics
Apo2L/TRAIL may involve the expression of anti-
apoptoticfactorssuchasc-FLIP(Thomeetal.,1997)
or BclXL (Mori et al., 1999). It is also possible that The leucine-zipper Apo2L/TRAIL fusion protein
thereisregulationoftheApo2L/TRAILproteinitself exhibited a distribution half-life of 1.3 hours and an
at the level of translation, cellular transport, and/or elimination half-life of 4.8 hours after intravenous
proteolytic processing. Further, in certain tumor cell injection in mice (Walczak et al., 1999). The native
lines, activation of the p53 tumor suppressor up- sequence form of recombinant soluble Apo2L/
regulates the expression of DR5 mRNA, perhaps TRAIL exhibited a pharmacologic half-life of 0.5
increasing sensitivity to Apo2L/TRAIL (Wu et al., hours in cynomolgus monkeys (Ashkenazi et al.,
1997). 1999).
Toxicity
IN THERAPY
Preclinical – How does it affect The systemic injection of up to 0.5mg of the human
leucine-zipper Apo2L/TRAIL fusion protein and
disease models in animals?
1mg of the corresponding murine fusion protein in
mice had no detectable adverse effects on viability,
Apo2L/TRAIL has been studied in mouse-based tissue integrity, or blood cell count (Walczak et al.,
xenograft models of human cancer (Ashkenazi et al., 1999). Intravenous injection of the native sequence
1999; Walczak et al., 1999). In nude mice, systemic form ofrecombinant soluble Apo2L/TRAILat up to
treatment with a leucine-zipper-fused Apo2L/TRAIL 10mg/kg/day for 7 days in cynomolgus monkeys
proteinbegunshortlyafterthesubcutaneousinjection showednoevidenceoftoxicityusingacomprehensive
of tumor cells substantially reduced tumor incidence set of clinical tests. Apo2L/TRAIL did not affect
(Walczak et al., 1999). Treatment with the fusion body weight, body temperature, blood pressure or
proteinaftersolidtumorshadbeguntoformreduced heart rate. There were no Apo2L/TRAIL-associated
tumor volume and in some cases eliminated the changes in leukocyte count, liver enzyme activity,
tumors if started within a few days, delaying tumor coagulation profiles, serum chemistry or urine anal-
progression if started later. Histologic examination ysis. Histologic evaluation ofliver, lung, heart, brain,
indicated that the systemic injection of the fusion kidney,ovary,testis,spleen,bonemarrow,andlymph
protein in tumor-bearing mice induced apoptosis in nodes did not reveal Apo2L/TRAIL-related adverse
tumor epithelial cells (Walczak et al., 1999). changes. In particular, there was no morphologic
In another study, a native sequence version of evidence of hepatotoxicity or lymphotoxicity, and
recombinant solubleApo2L/TRAIL wasinvestigated bone marrow sections displayed normal cellularity
(Ashkenazi et al., 1999). Repeated intravenous andprogressivematurationofalllineages(Ashkenazi
510 Avi Ashkenazi
et al., 1999). These data suggest that, unlike its Mariani, S., and Krammer, P. (1998a). Differential regulation of
relatives TNF and FasL, Apo2L/TRAIL is not sig- TRAIL and CD95 ligand in transformed cells of the T and B
lymphocytelineage.Eur.J.Immunol.28,973–982.
nificantly toxic in experimental animal models; care-
Mariani, S., and Krammer, P. (1998b). Surface expression of
ful testing will, however, be needed to evaluate the
TRAIL/Apo2 ligand in activated mouse Tand Bcells. Eur. J.
safety of this molecule in human beings. Immunol.28,1492–1498.
Mariani, S. M., Matiba, B., Armandola, E. A., and
Krammer, P. H. (1997). Interleukin 1-b-converting enzyme
References related proteases/caspases are involved in TRAIL-induced
apoptosis of Myeloma and leukemia cells. J. Cell. Biol. 137,
221–229.
Ashkenazi,A.,andDixit,V.M.(1998).Deathreceptors:signaling Marsters,S.,Pitti,R.,Donahue,C.,Ruppert,S.,Bauer,K.,and
andmodulation.Science281,1305–1308. Ashkenazi,A.(1996).ActivationofapoptosisbyApo-2ligand
Ashkenazi, A., and Dixit, V. M. (1999). Apoptosis control isindependent ofFADDbutblocked byCrmA.Curr.Biol.6,
by death and decoy receptors. Curr. Opin. Cell Biol. 11, 255– 750–752.
260. Marsters, S. A., Sheridan, J. P., Pitti, R. M., Huang, A.,
Ashkenazi, A., Pai, R., Fong, S., Leung, S., Lawrence, D., Skubatch, M., Baldwin, D., Yuan, J., Gurney, A.,
Marsters,S.,Blackie,C.,Chang,L.,McMurtrey,A.,Hebert,A., Goddard, A. D., Godowski, P., and Ashkenazi, A. (1997).
DeForge,L.,Khoumenis,I.,Lewis,D.,Harris,L.,Bussiere,J., A novel receptor for Apo2L/TRAIL contains a truncated
Koeppen,H.,Shahrokh,Z.,andSchwall,R.(1999).Safetyand deathdomain.Curr.Biol.7,1003–1006.
anti-tumoractivityofrecombinantsolubleApo2ligand.J.Clin. Martinez-Lorenzo, M. J., Alava, M. A., Gamen, S., Kim, J. K.,
Invest.104,155–162. Chuntharapai, A., Pineiro, A., Naval, J., and Anel, A. (1998).
Banner,D.W.,D’Arcy,A.,Janes,W.,Gentz,R.,Schoenfeld,H.J., Involvement of Apo2 ligand/TRAIL in activation-induced
Broger, C., Loetscher, H., and Lesslauer, W. (1993). Crystal death of Jurkat and human peripheral blood T cells. Eur. J.
structure of the soluble human 55kd TNF receptor-human Immunol.28,2714–2725.
TNF beta complex: implications for TNF receptor activation. Mongkolsapaya, J., Cowper, A., Xu, X. N., Morris, G.,
Cell73,431–435. McMichael, A., Bell, J. I., and Screaton, G. R. (1998).
Degli-Esposti, M., Smolak, P. J., Walczak, H., Waugh, J., Lymphocyte inhibitor of TRAIL: a new receptor protecting
Huang,C.P.,Dubose,R.F.,Goodwin,R.G.,andSmith,C.A. lymphocytes from the death ligand TRAIL. J. Immunol. 160,
(1997a). Cloning and characterization of TRAIL-R3, a novel 3–6.
memberoftheemergingTRAILreceptorfamily.J.Exp.Med. Mori,S.,Murakami-Mori,K.,Nakamura,S.,Ashkenazi,A.,and
186,1165–1170. Bonavida, B. (1999). Sensitization of AIDS-Kaposi’s sarcoma
Degli-Esposti,M.A.,Dougall,W.C.,Smolak,P.J.,Waugh,J.Y., cells to Apo2 ligand-induced apoptosis by actinomycin D.
Smith,C.A.,andGoodwin,R.G.(1997b).Thenovelreceptor J.Immunol.162,5616–5623.
TRAIL-R4 induces NF-(cid:20)B and protects aginst TRAIL- Muhlenbeck,F.,Haas,E.,Schwenzer,R.,Schubert,G.,Grell,M.,
mediated apoptosis, yet retains an incomplete death domain. Smith, C., Scheurich, P., and Wajant, H. (1998). TRAIL/
Immunity7,813–820. Apo2L activates c-Jun NH2-terminal kinase (JNK) via cas-
Emery,J.G.,McDonell,P.,Burke,M.C.,Deen,K.C.,Lyn,S., pase-dependent and caspase-independent pathways. J. Biol.
Silverman, C., Dul, E., Appelbaum, E. R., Eichman, C., Chem.273,33091–33098.
DiPrinzio, R., Dodds, R. A., James, I. E., Rosenberg, M., Nagata,S.(1997).Apoptosisbydeathfactor.Cell88,355–365.
Lee,J.C.,andYoung,P.R.(1998).Osteoprotegerinisarecep- Pan, G., and Dixit, V. M. (1997). An antagonist decoy receptor
torforthecytotoxicligandTRAIL.J.Biol.Chem.273,14363– and a new death domain-containing receptor for TRAIL.
14367. Science277,815–818.
Griffith,T.,Wiley,S.,Kubin,M.,Sedger,L.,Maliszewski,C.,and Pan,G.,O’Rourke,K.,Chinnaiyan,A.M.,Gentz,R.,Ebner,R.,
Fanger,N.(1999).Monocyte-mediatedtumoricidalactivityvia Ni,J., andDixit, V. M.(1997). The receptorfor the cytotoxic
the tumor necrosis factor-related cytokine, TRAIL. J. Exp. ligandTRAIL.Science276,111–113.
Med.189,1343–1353. Pan, G., Bauer, J. H., Haridas, V., Wang, S., Liu, D., Yu, G.,
Gruss, H. J., and Dower, S. K. (1995). Tumor necrosis factor Vincenz, C., Aggarwal, B., Ni, J., and Dixit, V. M. (1998a).
ligandsuperfamily:involvementinthepathologyofmalignant Identification and functional characterization of DR6, a novel
lymphomas.Blood85,3378–3404. deathdomain-containingTNFreceptor.FEBSLett.431,351–
Jeramias, I., Herr, I., Boehler, T., and Debatin, K. M. (1998). 356.
TRAIL/Apo-2-ligand-induced apoptosis in human T cells. Pan,G.,Ni,J.,Yu,G.L.,Wei,Y.F.,andDixit,V.M.(1998b).
Eur.J.Immunol.28,143–152. TRUNDD,anewmemberoftheTRAILreceptorfamilythat
Keane, M. M., Ettenberg, S. A., Nau, M. M., Russel, E., and antagonizesTRAILsignaling.FEBSLett.424,41–45.
Lipkowitz,S.(1999).ChemotherapyaugmentsTRAIL-induced Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J.,
apoptosisinbreastcelllines.CancerRes.59,734–741. Moore, A., and Ashkenazi, A. (1996). Induction of apoptosis
Katsikis, P. D., Garcia-Ojeda, M. E., Torres-Roca, J. F., by Apo-2 Ligand, a new member of the tumor necrosis factor
Tijoe, I. M., Smith, C. A., and Herzenberg, L. A. (1997). receptorfamily.J.Biol.Chem.271,12697–12690.
TNF-relatedapoptosis-inducingligandcanmediateactivation- Rieger, J., Naumann, U., Glaser, T., Ashkenazi, A., and
inducedTcelldeathinHIVinfection.J.Exp.Med.186,1365– Weller,M.(1998).Apo2ligand:anovellethalweaponagainst
1372. malignantglioma?FEBSLett.427,124–128.
Kothny-Wilkes, G., Kulms, D., Poppelmann, B., Luger, T. A., Screaton, G. R., Mongkolsapaya, J., Xu, X. N., Cowper, A. E.,
Kubin, M., and Schwartz, T. (1998). Interleukin-1 protects McMichael,A.J.,andBell,J.I.(1997).TRICK2,anewalter-
transformed keratinocytes from tumor necrosis factor-related natively spliced receptor that transduces the cytotoxic signal
apoptosis-inducingligand.J.Biol.Chem.273,29247–29253. fromTRAIL.Curr.Biol.7,693–696.
Apo2L/TRAIL 511
Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., prevent apoptosis induced by death receptors. Nature 386,
Skubatch, M., Baldwin, D., Ramakrishnan, L., Gray, C., 517–521.
Baker, K., Wood, W. I., Goddard, A. D., Godowski, P., and Walczak,H.,Degli-Esposti,M.A.,Johnson,R.S.,Smolak,P.J.,
Ashkenazi,A.(1997).ControlofTRAIL-inducedapoptosisby Waugh, J. Y., Boiani, N., Timour, M. S., Gerhart, M. J.,
afamilyofsignalinganddecoyreceptors.Science277,818–821. Schooley, K. A., Smith, C. A., Goodwin, R. G., and
Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Rauch, C. T. (1997). TRAIL-R2: a novel apoptosis-mediating
Chang, M. S., Luthy, R., Nguyen, H. Q., Wooden, S., receptorforTRAIL.EMBOJ.16,5386–5397.
Bennet,L.,Boone,T.,Shimamoto,G.,DeRose,M.,Elliot,R., Walczak,H.,Miller,R.E.,Ariail,K.,Gliniak,B.,Griffith,T.S.,
Colombero, A., Tan, H. L., Trail, G., Sullivan, J., Davy, E., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T.,
Bucay, N., and Boyle, W. J. (1997). Osteoprotegerin: a novel Smith, C., Smolak, P., Goodwin, R. G., Rauch, C. T.,
secretedproteininvolvedintheregulationofbonedensity.Cell Schuh,J.C.L.,andLynch,D.H.(1999).Tumoricidalactivity
89,309–319. of tumor necrosis factor-related apoptosis-inducing ligand in
Smith,C. A.,Farrah, T.,and Goodwin,R. G.(1994).TheTNF vivo.NatureMed.5,157–163.
receptor superfamily of cellular and viral proteins: activation, Wang, J., Zheng, L., Lobito, A., Chan, F., Dale, J., Sneller, M.,
costimulation,anddeath.Cell76,959–962. Yao,X.,Puck,J.,Straus,S.,andLenardo,M.(1999).Inherited
Snell, V., Clodi, K., Zhao, S., Goodwin, R., Thomas, E. K., humancaspase10mutationsunderliedefectivelymphocyteand
Morris, S. W., Kadin, M. E., Cabanillas, F., Andreeff, M., dendritic cell apoptosis in autoimmune lymphoproliferative
and Younes, A. (1997). Activity of TNF-related apoptosis- syndrometypeII.Cell98,47–58.
inducing ligand (TRAIL) in haematological malignancies. Br. Wiley,S.R.,Schooley,K.,Smolak,P.J.,Din,W.S.,Huang,C.P.,
J.Haematol.99,618–624. Nicholl, J.K.,Sutherland,G.R.,Davis-Smith, T.,Rauch,C.,
Tan,K.,Harrop,J.,Reddy,M.,Young,P.,Terrett,J.,Emery,J., Smith, C. A., and Goodwin, R. G. (1995). Identification and
Moore,G.,andTruneh,A.(1997).Characterizationofanovel characterization of a new member of the TNF family that
TNF-like ligand and recently described TNF ligand and TNF inducesapoptosis.Immunity3,673–682.
receptorsuperfamilygenesandtheirconstitutiveandinducible Wu,G.S.,Burns,T.F.,McDonald,E.R.,Jiang,W.,Meng,R.,
expressioninhematopoieticandnon-hematopoieticcells.Gene Krantz,I.D.,Kao,G.,Gan,D.D.,Zhou,J.Y.,Muschel,R.,
204,35–46. Hamilton, S. R., Spinner, N. B., Markowitz, S., Wu, G., and
Thomas, W. D., and Hersey, P. (1998). TNF-related apoptosis- El-Deiry, W.(1997).KILLER/DR5 isaDNAdamage-induci-
inducingligand(TRAIL)inducesapoptosisinFasligand-resis- ble p53-regulated death receptor gene. Nature Genet. 17,
tant melanoma cells and mediates CD4 T cell killing of target 141–143.
cells.J.Immunol.161,2195–2200. Zamani,L.,Ahmad,M.,Bennet,I.M.,Azzoni,L.,Alnemri,E.S.,
Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., andPerussia,B.(1998).Naturalkiller(NK)cell-mediatedcyto-
Meinl, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J., toxicity:differentialuseofTRAILandFasligandbyimmature
Schroter, M., Scaffidi, C., Krammer, P., Peter, M., and andmatureprimaryhumanNKcells.J.Exp.Med.188,2375–
Tschopp, J. (1997). Viral FLICE-inhibitory proteins (FLIPs) 2380.
