Novel X-linked inhibitor of apoptosis inhibiting compound as
sensitizer for TRAIL-mediated apoptosis in chronic lymphocytic
leukaemia with poor prognosis
The disease course of chronic lymphocytic leukaemia (CLL) is
heterogeneous. While patients with a good prognosis can be
managed effectively, although not cured, with conventional
chemotherapy or antibody-based chemo(immuno)therapy,
high-risk patients often suffer early disease progression and
death. Patients with poor prognosis are deﬁned as ZAP-70+
,
IGHV unmutated, CD38+
and/or carry deletions on chromosomes
11q and/or 17p, are grouped in Binet C stage or show an
early relapse after treatment (Binet et al, 2006). For this reason,
novel therapeutic strategies are needed to further reﬁne the
treatment options for patients and improve the efﬁcacy by
exploiting the speciﬁc biology of CLL.
The consensus based on previous experimental and clinical
studies of CLL cells is that the abnormal accumulation of
malignant monoclonal B cells in patients is largely attributed to
defective apoptosis programmes rather than aberrant proliferation.
With the discovery of the death receptors the opportunity
has arisen to directly trigger apoptosis externally of tumour
cells, thereby circumventing the intrinsic apoptotic machinery,
which is mainly triggered by conventional chemotherapeutic
treatments. In particular, tumour necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) has recently received the
most attention because of its unique property to selectively kill
malignant tumour cells but not healthy cells. Recombinant
Lukas P. Frenzel,1–3
Michaela Patz,1
Christian P. Pallasch,1–4
Reinhild
Brinker,1
Julia Claasen,1
Alexandra
Schulz,1
Michael Hallek,1–3
Hamid
Kashkar3,5
* and Clemens-Martin
Wendtner1–3
*
1
Department I of Internal Medicine, 2
Centre of
Integrated Oncology, 3
Cologne Excellence Cluster
on Cellular Stress Responses in Aging-Associated
Diseases, University of Cologne, Cologne,
Germany, 4
David H. Koch Institute for Integrative
Cancer Research, MIT, Cambridge, MA, USA,
and 5
Institute for Medical Microbiology and
Immunology, and Centre for Molecular Medicine
Cologne, University of Cologne, Cologne,
Germany
Received 14 June 2010, accepted for publication
10 August 2010
Correspondence: Lukas P. Frenzel, MD,
Department I of Internal Medicine, Center for
Integrated Oncology Cologne Bonn, University
of Cologne, D-50937 Cologne, Germany.
E-mail: Lukas.frenzel@uk-koeln.de
*These authors contributed equally to this work.
Summary
Given that aggressive DNA damaging chemotherapy shows suboptimal
efﬁcacy in chronic lymphocytic leukaemia (CLL), alternative therapeutic
approaches are needed. Tumour necrosis factor-related apoptosis-inducing
ligand (TRAIL) is able to induce tumour-speciﬁc apoptosis. However,
apoptosis might be inhibited by elevated levels of X-linked inhibitor of
apoptosis (XIAP). Use of XIAP-inhibiting compounds might sensitize
primary CLL cells towards TRAIL-mediated apoptosis. A novel small
molecule, compound A (CA), an inhibitor of XIAP, was used in
combination with TRAIL to induce apoptosis in primary CLL cells
(n = 48). XIAP was signiﬁcantly more highly expressed in primary CLL
cells (n = 28) compared to healthy B cells (n = 16) (P = 0Æ02). Our data
obtained by speciﬁc knock-down of XIAP by siRNA identiﬁed XIAP as the
key factor conferring resistance to TRAIL in CLL. Combined treatment with
CA/TRAIL signiﬁcantly increased apoptosis compared to untreated
(P = 8Æ5 · 10)10
), solely CA (P = 4Æ1 · 10)12
) or TRAIL treated
(P = 4Æ8 · 10)10
) CLL cells. CA rendered 40 of 48 (83Æ3%) primary CLL
samples susceptible to TRAIL-mediated apoptosis. In particular, cells derived
from patients with poor prognosis CLL (ZAP-70+
, IGHV unmutated, 17p-)
were highly responsive to this drug combination. Our highly-effective XIAP
inhibitor CA, in concert with TRAIL, shows potential for the treatment of
CLL cases with poor prognosis and therefore warrants further clinical
investigation.
Keywords: chronic lymphocytic leukaemia, second mitochondria-derived
activator of caspase-mimetic, X-linked inhibitor of apoptosis, tumour
necrosis factor-related apoptosis-inducing ligand, therapy.
research paper
First published online 23 November 2010
ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 152, 191–200 doi:10.1111/j.1365-2141.2010.08426.x
TRAIL or monoclonal antibodies targeting TRAIL receptors are
currently being evaluated in early clinical trials (Ashkenazi,
2008; Johnstone et al, 2008). Human TRAIL can bind to ﬁve
different receptor molecules. TRAIL-R1 and -R2 contain a
cytoplasmic so-called death domain that is capable of recruiting
and promoting caspase-8 activation, which, in turn,
activates downstream executioner caspases, such as caspase-3.
The proteolytic activity of caspase-3 can be inhibited by Xlinked
inhibitor of apoptosis (XIAP) that directly binds
caspase-3 (Eckelman et al, 2006). Therefore, for effective death
receptor-mediated killing in cells with XIAP expression there is
an essential need to disrupt the mitochondrial outer membrane
and release the second mitochondria-derived activator of
caspase (SMAC) which can directly bind and antagonize XIAP,
thereby enabling the enrichment of the critical level of effector
caspase activity (Du et al, 2000; Jost et al, 2009).
Although TRAIL receptors are constitutively expressed, CLL
cells have been previously shown to be resistant to TRAIL
treatment (Olsson et al, 2001; MacFarlane et al, 2002; Secchiero
et al, 2005). However, the underlying molecular mechanisms
giving rise to the resistance to TRAIL in CLL cells are
still undetermined. Given its role in TRAIL-induced apoptosis
and its elevated expression in CLL cells, this study aimed to
inhibit XIAP in order to overcome the TRAIL-resistance in
CLL. One strategy for targeting XIAP involves agents that
mimic the amino terminus of SMAC and thus block critical
XIAP/caspase interactions. Here we used a cell permeable small
molecule XIAP antagonist, compound A (CA), which was
developed based on the crystal structure of the four amino
acids of SMAC which enable SMAC to efﬁciently bind the
BIR3 domain of XIAP. In contrast to the active compound CA,
which consists of an amino terminal methyl alanine, the
inactive compound CB used in our studies as a negative
control has an amino terminal methyl glycine. This speciﬁc
substitution results in a signiﬁcant reduction of IAP binding
capability of CB as CA has binding afﬁnity to XIAP in the
picomolar range and CB is a weak binder with micromolar
binding afﬁnity to XIAP (Chai et al, 2000; Vince et al, 2007).
Materials and methods
Patients and cells
After informed consent was given, blood was obtained from
patients fulﬁlling the diagnostic criteria for CLL. Only patients
without prior therapy or patients who had a period of at least
6 months since their last chemotherapy were included in this
study. Fresh CLL samples were enriched by applying BRosetteSep
(StemCell Technologies, Vancouver, BC, USA) and
Ficoll- Hypaque (Seromed, Berlin, Germany) density gradient
puriﬁcation, resulting in purity of more than 98% of CD19/
CD5 CLL cells (Pallasch et al, 2008a). Control cells were
isolated from healthy blood donors using untouched depletion
(naive B-cell isolation kit II; Miltenyi, Bergisch-Gladbach,
Germany) resulting in at least 90% purity of B cells. Isolated
cells were cultured as previously described (Pallasch et al,
2008a, 2009). This study was approved by the ethics committee
of the University of Cologne and blood samples were given
with informed consent according to the Helsinki protocol.
Transient XIAP knock-down
Transient gene knock-down of XIAP was achieved with 1 lg of
the speciﬁcON-TARGETplusSMARTpool siRNA(Dharmacon,
Lafayette, CO, USA). The non-targeting ON-TARGETplus
siCONTROL pool was used as negative control. Program U-
013 was used with 100 ll Solution V for nucleofection of
1Æ2 · 107
primary CLL cells with siRNA (Lonza, Cologne,
Germany). Two days after transfection protein lysates were
prepared and cytotoxicity assays were performed.
Treatment of cells
Small molecule active compound A (CA) and the negative
control compound B (CB) were dissolved in dimethyl
sulfoxide (DMSO) and were applied at a concentration of
100 nmol/l (Tetralogic Pharmaceuticals, Malvern, PA, USA)
(Vince et al, 2007). Both alanine residues are carbamoylated in
the control compound B in comparison to active compound A
(Li et al, 2004). TRAIL was dissolved in aquaeous buffer and
applied in 100 ng/ml concentration (Enzo Life Sciences
GmbH, Lo¨rrach, Germany).
Apoptosis
Apoptosis was assessed by ﬂow-cytometry using AnnexinV-ﬂuorescein
isothiocyanate (FITC)/7-Aminoactinomycin D
(7-AAD) staining (BD, Heidelberg, Germany). In the ﬁgures,
apoptosis levels are given as Annexin-V positive cells, including
also Annexin-V/7-AAD double-positive cells. Apoptosis
statistics were normalized to untreated controls.
XIAP protein expression analysis
Cellular lystates were processed with radioimmunoprecipitation
assay buffer, sonicated and further blotted onto nitrocellulose
membranes. A XIAP-speciﬁc monocolonal antibody
(clone 48/hILP/XIAP; BD) was used for detection of XIAP
protein. Monoclonal antibody against actin (clone C4;
Millipore, Schwalbach/Ts., Germany) served as housekeeping
gene expression control and was used as an indication of
protein loading. Western blot detection and density measurements
were preformed on an Odyssey infrared imaging system
(Licor, Lincoln, NE, USA).
Assessment of TRAIL receptor expression
Puriﬁed primary CLL cells were incubated for 30 min on ice
with human IgGs to block unspeciﬁc binding and then
incubated with phycoerythrin (PE)-conjugated anti-human
L. P. Frenzel et al
192 ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 152, 191–200
TRAIL receptor antibodies (DR4, clone: DJR1; DR5, DJR2-4)
(NatuTec, Frankfurt, Germany) or an IgG1-PE isotype control
antibody (Beckman Coulter, Krefeld, Germany) for 30 min at
4°C. The samples were washed twice with ice-cold phosphatebuffered
saline (PBS), resuspended in PBS and analysed by
ﬂow cytometry.
Statistics and data processing
Excel (Microsoft, Redmond, WA, USA) and Sigma-Plot
(Stata-Corp, College Station, TX, USA) were applied for
statistical analysis; in particular the Student t-test and Wilcoxon
Ranged Sum Test were used.
Results
Elevated XIAP expression in primary CLL cells
As it is established that primary CLL cells are resistant to
TRAIL treatment and in view of the fact that XIAP can
efﬁciently inhibit TRAIL-induced apoptosis, we ﬁrst examined
the XIAP expression level in primary CLL cells compared to B
cells from healthy donors. Of note, the ﬁrst evidence indicating
the elevated expression XIAP was derived from our microarray
gene expression proﬁles, showing signiﬁcantly higher expression
of XIAP mRNA (2Æ36-fold; P = 0Æ0002) in CLL cells
(n = 8) in comparison to healthy B cells (n = 5) (Pallasch
et al, 2008b). To conﬁrm this result at the protein level, we
performed Western blot analysis on 28 CLL samples and 16
healthy donor B cell samples. Calculation of the ratio between
b-actin and XIAP expression revealed a signiﬁcantly higher
XIAP expression (2Æ22-fold; P = 0Æ02) in CLL cells at the
protein level (Fig 1). This indicated that XIAP protein was
signiﬁcantly over-expressed in puriﬁed primary CLL cells
compared to healthy B cells, suggesting XIAP as a possible key
target for improving death receptor-induced apoptosis in CLL.
XIAP confers resistance to TRAIL-induced cell death in
primary CLL cells
The impact of XIAP on inefﬁcient TRAIL-mediated killing was
further analysed by speciﬁc siRNA-mediated knock-down of
XIAP. Efﬁciency of speciﬁc XIAP knock-down was examined
by Western blotting and showed efﬁcient down-regulation of
XIAP in primary CLL cells transiently transfected with XIAPspeciﬁc
siRNA but not control siRNA after 48 h (Fig 2A).
Additional TRAIL administration for 24 h resulted in significantly
higher cell death of CLL cells (P = 0Æ02) compared to
control siRNA transfected cells (Fig 2B).
Active compound A sensitizes primary CLL cells towards
TRAIL-mediated caspase-dependent apoptosis
Based on these analyses it was tempting to speculate that the
combination of XIAP inhibition with TRAIL treatment might
display an efﬁcient and highly speciﬁc treatment for CLL.
Therefore we next examined the susceptibility of freshly
isolated primary CLL cells from 48 patients towards treatment
with 100 nmol/l of XIAP inhibitor CA or its CB as a negative
control either alone or in combination with TRAIL. Patients’
characteristics are summarized in Table I. Of note, concentrations
of up to 100 nmol/l CA or CB showed no direct
cytotoxicity to healthy B cells and primary CLL cells (Fig 3 and
data not shown). Compared to untreated samples, single
TRAIL-treatment resulted in a mean level of apoptotic cells of
6Æ3% (P = 0Æ00083) (Fig 3A). Meanwhile, simultaneous treatment
with TRAIL and CA dramatically increased the rate of
apoptotic CLL cells with a mean rate of apoptotic cells of
32Æ8% and an overall response (deﬁned by more than 10% of
apoptotic cells) rate of 83Æ3% (40 of 48 samples) (Table I,
Fig 3A). Compared to untreated (P = 8Æ5 · 10)10
), solely CA
(P = 4Æ1 · 10)12
) or TRAIL-treated (P = 4Æ8 · 10)10
) CLL
cells the apoptosis rate was signiﬁcantly increased by combined
treatment with XIAP inhibitor and TRAIL.
To assess whether TRAIL treatment is CLL-cell speciﬁc,
healthy B cells (n = 4) were exposed to TRAIL alone or
CA(CB)/TRAIL and showed signiﬁcantly lower susceptibility
towards CA/TRAIL administration than CLL cells (Fig 3B).
As a potent cellular caspase inhibitor we next examined the
involvement of caspases in CA/TRAIL-mediated apoptosis.
Not surprisingly, co-application of the pan-caspase inhibitor
zVAD.fmk inhibited cell death induced by CA/TRAIL, underscoring
the apoptotic caspase-dependent cytotoxicity of CA/
Fig 1. Elevated XIAP expression in primary CLL cells. To determine
XIAP expression in 28 primary CLL cell samples and 16 B-cell samples
from healthy donors, Western blot was performed with mousemonoclonal
antibody against human XIAP (clone 48/hILP/XIAP; BD).
Monoclonal antibody against actin (clone C4; Millipore) served as
housekeeping gene expression control and was used as an indication of
protein loading. Calculation of the ratio between b-actin and XIAP
expression revealed a signiﬁcantly higher XIAP protein expression
(2Æ22-fold; P = 0Æ02) in CLL cells. Each lane was loaded with 30 lg of
protein. *Statistics calculated by t-test; *P < 0Æ05. Dashed lines indicate
means; solid lines indicate medians.
XIAP Inhibitor in CLL with Poor Prognosis
ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 152, 191–200 193
TRAIL treatment in CLL cells (Fig 3C). Previous studies have
shown that, in addition to functional inhibition, IAP antagonists
may potentiate caspase activation by directing IAP
protein degradation. In order to address this issue we
examined the XIAP expression level in primary CLL cells after
treatment with CA. As shown in Fig 3D, concentrations up to
100 nmol/l CA or CB did not promote any XIAP protein level
reduction, suggesting that the pro-apoptotic effect of CA is a
result of the functional antagonization of XIAP. This is in line
with a previous report that demonstrated that CA does not
promote XIAP protein degradation (Vince et al, 2007).
ZAP-70+
, IGVH unmutated and CD38+
poor-prognosis
patients signiﬁcantly beneﬁt from CA/TRAIL treatment
CLL patients who are ZAP-70+
and CD38+
and harbour
unmutated IGHV genes are known to suffer from early disease
progression and shorter treatment-free intervals.
Strikingly, ZAP-70+
(13%; P = 0Æ002), IGHV unmutated
(12Æ6%; P = 0Æ043) and CD38+
(11Æ2%; P = 0Æ032) CLL
samples were signiﬁcantly more susceptible to TRAIL compared
to ZAP-70)
(2Æ0%), IGHV mutated (3Æ9%) and CD38)
(4Æ5%) CLL cells (Fig 4A,B,C).
Interestingly, ZAP-70+
samples (n = 9) expressed higher
levels of TRAIL-R1 (not signiﬁcant) and signiﬁcantly higher
levels of TRAIL-R2 (P = 0Æ0016) on their surface in comparison
to ZAP-70)
(n = 10) CLL samples (Fig 4D).
Samples derived from CLL patients with poor-prognostic
features were highly susceptible to combined treatment with
TRAIL and CA. Co-treatment with CA and TRAIL dramatically
increased the mean fraction of apoptotic cells in ZAP-70+
(45Æ2 vs. 29Æ2%; P = 0Æ005), IGHV unmutated (48Æ8 vs. 31Æ3%;
P = 0Æ035) and CD38+
(42 vs. 28Æ9%; P = 0Æ09) cohorts
compared to their counterparts (no compound and CB;
Fig 3A) (Fig 4A–C).
Poor-prognosis patients show same response to CA/TRAIL
application compared to low-risk patients in terms of prior
treatment, cytogenetics and Binet stage
In addition, previously treated patients and especially CLL
cases with 17q-deletion often do not show durable responses to
chemotherapeutic approaches. Samples derived from high-risk
patients did not demonstrate signiﬁcant differences concerning
their mean of apoptosis response to the CA/TRAIL regimen in
comparison to CLL samples derived from low-risk patients. In
detail, 14 of 17 (82Æ4%) CLL cells from patients who had
already received at least one chemotherapy, responded equally
compared to leukaemic cells derived from therapy-naive
patients (26/31; 83Æ8%) with respect to combined CA/TRAIL
administration (Fig 5A). Also, no signiﬁcant differences could
be detected regarding the mean of apoptotic cells between
clinical stage Binet A (31Æ9%), B (37Æ6%) and C (24Æ9%)
patients, although the response rate in Binet C group was
lowest (9/12; 75%) compared to the Binet A (18/22; 81Æ8%)
and B (13/14; 92Æ9%) cohorts.
Most importantly, all patients (5/5) with 17q-deletion and
four of ﬁve patients with 11q- deletion responded to CA/
TRAIL treatment with a mean level of apoptosis of 31Æ7% for
17p-del and 41% for 11q-del patients, which was comparable
to those from low-risk patients with normal cytogenetics
(36Æ9%) or 13q deletion (26Æ4%).
Discussion
Here we describe a potent novel therapeutic option for CLL
patients, especially those with poor prognostic features like
ZAP-70+
, IGHV unmutated status, CD38+
and/or 17p-, using
highly efﬁcient compound A to inhibit XIAP function and
thereby to promote TRAIL-mediated apoptosis in CLL cells,
but not in healthy B cells.
(B)(A)
Fig 2. XIAP confers resistance to TRAIL-induced cell death in primary CLL cells. (A) Knock-down efﬁciency of XIAP was determined by Western
blot for XIAP and Actin and shows down-regulation of XIAP in siXIAP samples compared to samples treated with control siRNA. Lysates were
prepared 48 h after nucleofection of XIAP-speciﬁc and control (Ctrl) siRNAs. Each lane was loaded with 30 lg protein. (B) Knock-down of XIAP by
nucleofection of siRNA sensitized primary CLL cells (1Æ2 · 107
cells in 100 ll solution V, Lonza, Cologne, Germany) (n = 4) to TRAIL. 48 h after
nucleofection cells were exposed to TRAIL for additional 24 h. Knock-down of XIAP resulted in signiﬁcantly higher apoptosis (P = 0Æ02). Results are
expressed as the mean ± SD of four independent experiments. Note: *Statistics calculated by t-test; *P < 0Æ05
L. P. Frenzel et al
194 ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 152, 191–200
TRAIL-induced cell death remains a very attractive biologically
targeted therapeutic option, because cancer cells are
killed while normal cells are not affected. In fact, TRAIL
administration in phase I and II trials with monoclonal
antibodies against TRAIL-R1, -R2 or with recombinant TRAIL
were shown to be safe and well tolerated (Plummer et al, 2007;
Table I. Characteristics of patient samples.
ID Age (years) Sex Treatment Binet CD38 ZAP-70 IGHV Cytogenetics Response
1 59 M y C ) ) m 13q del 23
2 70 M n B ) + u Normal 29
3 68 M y C + + n.d. 11q del, trisomy 12 16
4 61 F n B n.d. n.d. n.d. n.d. 18
5 69 M y C + + u 6q del, 13q del 45
6 66 M n C ) ) m 13q del 0
7 69 M n A n.d. n.d. n.d. n.d. 0
8 62 F y C ) ) m Normal 24
9 70 F n A + ) n.d. Normal 61
10 54 F y B n.d. n.d. n.d. 17p del 38
11 51 F n B ) + n.d. Normal 47
12 76 M n B ) + u Trisomy 12 67
13 45 M n A ) ) n.d. n.d. 31
14 54 M y A + ) u 11q del 75
15 63 M y C ) ) n.d. Normal 0
16 73 M y C + + n.d. n.d. 5
17 56 M n A + ) m Normal 33
18 59 F n B ) ) n.d. n.d. 2
19 65 F n A ) ) n.d. n.d. 10
20 69 M n A n.d. ) n.d. n.d. 0
21 53 M y C + + n.d. Normal 32
22 66 M y A ) ) n.d. n.d. 23
23 61 M n A ) + m Normal 86
24 32 M n A ) ) n.d. 17p del 13
25 69 M y B ) ) m 13q del 51
26 73 M n A ) ) m Normal 8
27 79 M n B ) + n.d. 17p del 66
28 46 M n A ) ) m 13q del 17
29 59 M n A n.d. n.d. n.d. 13q del 25
30 65 M y C + + m n.d. 82
31 70 M y B ) ) m 11q del, 13q del 17
32 53 M n A + + u 13q del 25
33 69 M y C ) + n.d. n.d. 33
34 64 M n B + + u 11q del 89
35 63 M y C n.d. ) n.d. 11q del, 13q del 8
36 55 M n A ) + m 13q del 15
37 61 M n B + ) m n.d. 31
38 69 M n A + ) m 17p del 25
39 56 F n B ) + u 13q del 19
40 61 F n B + + u 13q del 39
41 88 M n A ) ) m 13q del 38
42 84 M n A ) ) u Normal 60
43 77 F n A + ) u 13q del 19
44 81 M n A + + u 13q del 56
45 68 M n A + + u n.d. 62
46 75 M y C ) ) m Normal 35
47 90 M y B + ) m 17p del 17
48 72 M n A ) n.d. n.d. n.d. 19
M, male; F, female; y, yes; n, no; m, mutated; u, unmutated; n.d., not determined; del, deletion; response: percentage of apoptotic cells by Annexin-V/
7-AAD staining after combined treatment with compound A and TRAIL; for ZAP-70 and CD38 respectively, a 20% (ZAP-70) and 30% (CD38)
threshold of positive stained cells was applied for deﬁnition of positivity for these prognostic markers.
XIAP Inhibitor in CLL with Poor Prognosis
ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 152, 191–200 195
Greco et al, 2008; Soria et al, 2010; Trarbach et al, 2010).
However, the beneﬁt of these approaches has been critically
discussed, because many primary cancers exhibit distinct
resistance mechanisms against TRAIL-induced cell death in
contrast to cell-lines, which are often more susceptible (Dyer
et al, 2007). Primary CLL cells show low susceptibility towards
TRAIL apoptosis (MacFarlane et al, 2002; Loeder et al, 2009).
However, this paradigm does not seem to be the case for all
CLL samples. Comparing ZAP-70+
versus ZAP-70)
(IGHV
unmutated versus IGHV mutated; CD38+
versus CD38)
)
samples, we report here for the ﬁrst time, that poor-prognosis
samples (ZAP-70+
, IGHV unmutated, CD38+
) were signiﬁcantly
more susceptible to TRAIL-mediated apoptosis than
their counterparts.
The reason for this observation might be the slightly higher
expression of TRAIL-R1 and the signiﬁcantly enhanced
expression of TRAIL-R2 in ZAP-70 positive versus ZAP-70
negative CLL samples (P = 0Æ0016). TRAIL mediates apoptosis
in CLL cells via TRAIL-R1 and -R2 (Natoni et al, 2007).
Initially it was thought that CLL cells exhibit apoptotic
signalling via TRAIL-R1 after CLL cells were sensitized by
HDAC1 inhibitors (MacFarlane et al, 2005). However, this
result might be due to the efﬁcacy of different TRAIL-R1
and -R2 agonistic antibodies and recombinant TRAIL, as
supported by the observation that CLL cells are also efﬁciently
killed by TRAIL-R2 agonistic antibodies after cross-linking
(Natoni et al, 2007). Here we used recombinant TRAIL, which
did not require cross-linking to induce apoptosis in CLL cells.
However, further studies need to determine why ZAP-70+
CLL
samples express higher levels of TRAIL-R1 and -R2 and
whether the higher expression and/or the ZAP-70+
status is
responsible for higher apoptosis by TRAIL.
Given that death-inducing signalling complex (DISC) formation
in CLL cells – although at low level – is mediated by
TRAIL (MacFarlane et al, 2002), further mechanisms might be
involved in low susceptibility of CLL cells towards TRAILinduced
apoptosis. Because CLL is known to show general
anti-apoptotic properties, we hypothesized that effector
caspases-3/-9 might be inhibited by their major inhibitor
XIAP. Indeed, analyses performed with peripheral blood
mononuclear cells from CLL patients suggested an elevated
XIAP expression in CLL cells from patients with progressive
(A) (B)
(C) (D)
Fig 3. Compound A sensitizes primary CLL cells towards TRAIL-mediated caspase-dependent apoptosis. (A) Primary CLL cells from 48 different
patients were treated for 24 h with 100 nmol/l compound A (CA) or negative control compound B (CB) and/or 100 ng/ml TRAIL. Apoptosis was
signiﬁcantly increased in CA/TRAIL treated compared to solely CA, TRAIL or untreated samples Dashed lines indicate means; solid lines indicate
medians. (B) Healthy B cells (n = 4) are signiﬁcantly weaker lysed by CA/TRAIL application than CLL cells. (C) Pan-caspase inhibitor zVAD.fmk
restores survival in primary CLL cells (n = 4) after 24 h CA/TRAIL treatment. Results are expressed as mean ± SD of at least four independent
experiments (for B+C). (D) Primary CLL cells were treated for 24 h with 100 nmol/l of compounds A and B, because this time-point was also chosen
for measurement of apoptosis. Lysates were prepared after 24 h. Each lane was loaded with 30 lg protein. Note: *Statistics calculated by t-test;
*P < 0Æ05; **P < 0Æ001; ***P < 0Æ0001.
L. P. Frenzel et al
196 ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 152, 191–200
disease, whereas those with stable disease had decreased XIAP
protein level compared to lymphocytes from healthy donors
(Grzybowska-Izydorczyk et al, 2010). Our analyses of XIAP
expression however, revealed a signiﬁcantly elevated expression
of XIAP in puriﬁed, primary CLL cells versus non-malignant
B-cells isolated from several healthy donors. Although not
quantiﬁed, Loeder et al (2009) also showed different expression
patterns of XIAP between CLL cells and normal B cells by
Western Blot. Two prior studies that investigated XIAP
expression at the transcript level did not detect signiﬁcant
differences between CLL cells and healthy donor B cells
(Munzert et al, 2002; de Graaf et al, 2005). Moreover, Munzert
et al (2002) revealed that TRAF1 was the only nuclear factorkappa
B-regulated antiapoptotic gene that was signiﬁcantly
over-expressed in CLL, but did not correlate with markers of
disease progression or overall survival. Furthermore, de Graaf
et al (2005) compared apoptosis-regulating genes and IAP
family members by quantitative reverse transcription polymerase
chain reaction between different lymphoid malignancies.
In CLL, XIAP expression was highest compared to the
other malignant entities and to B cells from hyperplastic
tonsils. Peripheral B cells showed similar levels of XIAP
transcript in comparison to CLL cells (de Graaf et al, 2005). In
our study, speciﬁc knock-down of XIAP by siRNA signiﬁcantly
enhanced TRAIL-induced apoptosis identifying XIAP as the
key factor establishing resistance to TRAIL and further
underscored the impact of XIAP as a therapeutic target for
improving death receptor-induced apoptosis in CLL.
These results encouraged us to use CA in order to inhibit
XIAP function. Single treatment with CA did not induce any
toxicity in primary CLL cells, healthy B cells and PBMCs.
However, low-dose CA was highly sufﬁcient to render CLL
cells, in 40 of 48 (83Æ3%) cases, susceptible towards caspasedependent
TRAIL-induced cell death. Our data are in line with
several recent reports demonstrating the synergistic effect of
XIAP antagonizing protocols with TRAIL in different tumour
(A) (B)
(C) (D)
Fig 4. ZAP-70+
, IGHV unmutated and CD38+
poor-prognosis patients signiﬁcantly beneﬁt from compound A/TRAIL treatment. (A) ZAP-70
positive samples showed signiﬁcantly higher rates of apoptosis either by TRAIL alone or by compound A/TRAIL co-treatment compared to ZAP-70
negative CLL patients. (B) Samples with unmutated IGHV showed signiﬁcantly higher apoptosis rates either by TRAIL alone or by compound A/
TRAIL co-treatment compared to CLL patients with mutated IGHV genes. (C) TRAIL treatment alone resulted in CD38 positive individuals in
signiﬁcantly higher apoptosis than in CD38 negative samples. Addition of XIAP inhibitor to TRAIL induced signiﬁcantly higher apoptosis in both
CD38 negative and positive samples. (D) ZAP-70)
(n = 10) and ZAP-70+
(n = 9) CLL samples were stained with PE-conjugated TRAIL-R1 (clone:
DJR1) and -R2 antibodies (clone: DJR2-4) and percentage of positive cells was assessed by ﬂow-cytometry. Cells from ZAP-70+
CLL donors express
higher levels of TRAIL-R1 (not signiﬁcant) and -R2 (P = 0Æ0016) compared to ZAP-70)
CLL donors on their surface. Note: *Statistics calculated by ttest;
*1
Statistics calculated by Wilcoxon Ranged Sum Test; */*1
P < 0Æ05; **P < 0Æ001; ***P < 0Æ0001 Dashed lines indicate means; solid lines indicate
medians.
XIAP Inhibitor in CLL with Poor Prognosis
ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 152, 191–200 197
models including childhood acute leukaemia, pancreatic, colon
and breast cancer (Karikari et al, 2007; Vogler et al, 2008,
2009; Fakler et al, 2009). Interestingly, a recent study using
another XIAP inhibitor in 27 primary CLL samples showed
that this XIAP inhibitor acted in concert with TRAIL to trigger
apoptosis in 18 of 27 (67%) cases (Loeder et al, 2009). In
contrast to the XIAP inhibitor used in the aforementioned
study, the XIAP inhibitor used in our study was applied at 30times
lower concentrations, had almost no direct cytotoxic
effect and did not induce any signiﬁcant cytotoxic activity in
non-malignant B cells at the concentrations used in combination
with TRAIL.
Impressively, the administration of CA in our settings, i.e.
especially using samples derived from CLL patients with
poor prognostic features like ZAP-70+
, CD38+
and/or IGHV
unmutated disease, resulted in signiﬁcantly greater susceptibility
towards TRAIL-mediated cell death. These observations
are supported by Lopez-Guerra et al (2009), who
showed that ZAP-70+
CLL cells were more susceptible
towards selective IkB kinase inhibitor and TRAIL-mediated
apoptosis.
Another major ﬁnding of our study is that samples derived
from patients that had received prior chemotherapy showed
similar response and apoptosis rates comparable to those
samples derived from therapy-naı¨ve patients. This observation
can also be extended to p53-deﬁcient patients, who generally
had the worst clinical outcome (Binet et al, 2006), but showed
maximum response under CA/TRAIL. It can be speculated that
CA/TRAIL treatment might also be an attractive therapeutic
option for all patients, who are not ﬁt enough to receive
standard chemo(immuno)therapy since CA/TRAIL did not
show unspeciﬁc toxicity in vitro. Nevertheless, the deﬁnitive
toxicity proﬁle of CA is currently being deﬁned in ongoing
phase I clinical trials. Besides combination treatment of CA
with TRAIL, another route of investigation will be to test these
targeted drugs in combination with classic alkylating agents or
purine analogues.
Since the discovery of XIAP in the second half of the 1990s,
research on this unique inhibitor of apoptosis has been
exponential giving us a detailed structural and mechanistic
view of its activity in addition to abundant cell biology data.
Through its ability to inhibit caspases, it has been suggested
that XIAP renders cells resistant to multiagent chemotherapy.
As a result, efforts have been undertaken to develop potential
drugs targeting XIAP as a new way to counteract cancer and
overcome drug resistance. In line with previous observations
our results showed that XIAP is highly expressed and efﬁciently
inhibits TRAIL-mediated cell death in CLL cells. The successful
use of a novel potent pharmaceutic inhibitor of XIAP to
restore TRAIL-induced cell death in CLL cells of 48 patients
has a strong therapeutic implication, especially for treatment of
CLL patients with poor prognosis, which needs to be further
investigated in clinical trials to hopefully improve the outcome
for patients with CLL.
(A) (B)
(C)
Fig 5. Poor-prognosis patients show same response to compound A/TRAIL application compared to low-risk patients in terms of prior treatment,
cytogenetics and Binet stage. No signiﬁcant differences are detectable in terms of apoptosis between (A) patients with prior chemotherapy and
untreated patients, (B) patients with different cytogenetic feature and (C) patients with different clinical stages (Binet) of CLL. There is no statistically
difference between the box-plots according to t-test. Dashed lines indicate means; solid lines indicate medians.
L. P. Frenzel et al
198 ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 152, 191–200
Acknowledgement
Compounds A and B were kindly provided by TetraLogic
Pharmaceuticals, Malvern, PA, USA.
Authorship and disclosures
LPF, HK and C-MW conceived and designed the present work;
LPF, MP, CPP, RB, JC and AS performed the research; LPF
and MP conducted statistical analysis; LPF, MH, HK and
C-MW analysed the data; LPF, HK and C-MW wrote the
manuscript. HK and C-MW contributed equally to this work.
The authors declare no competing ﬁnancial interests.
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