Published OnlineFirst November 9, 2011; DOI: 10.1158/1078-0432.CCR-11-1049
Clinical
Cancer
Research
Cancer Therapy: Preclinical
Leflunomide Induces Apoptosis in Fludarabine-Resistant
and Clinically Refractory CLL Cells
€mer5, Esther Hahn1,2, Claudia Scha
€fer5, Thomas Giese3,
Sascha Dietrich1,2, Oliver H. Kra
€llein4, Thorsten Zenz2,4,
Michael Hess1,2, Theresa Tretter2, Michael Rieger2, Jennifer Hu
Anthony D. Ho2, Peter Dreger2, and Thomas Luft1,2
Abstract
Purpose: Environmental conditions in lymph node proliferation centers protect chronic lymphocytic
leukemia (CLL) cells from apoptotic triggers. This situation can be mimicked by in vitro stimulation with
CD40 ligand (CD40L) and interleukin 4 (IL-4). Our study investigates the impact of the drug leflunomide to
overcome apoptosis resistance of CLL cells.
Experimental Design: CLL cells were stimulated with CD40L and IL-4 and treated with fludarabine and
the leflunomide metabolite A771726.
Results: Resistance to fludarabine-mediated apoptosis was induced by CD40 activation alone stimulating high levels of BCL-XL and MCL1 protein expression. Apoptosis resistance was further enhanced by a
complementary Janus-activated kinase (JAK)/STAT signal induced by IL-4. In contrast, CLL proliferation
required both a CD40 and a JAK/STAT signal and could be completely blocked by pan-JAK inhibition.
Leflunomide (A771726) antagonized CD40L/IL-4–induced proliferation at very low concentrations (3 mg/
mL) reported to inhibit dihydroorotate dehydrogenase. At a concentration of 10 mg/mL, A771726
additionally attenuated STAT3/6 phosphorylation, whereas apoptosis of CD40L/IL-4–activated ("resistant") CLL cells was achieved with higher concentrations (IC50: 80 mg/mL). Apoptosis was also effectively
induced by A771726 in clinically refractory CLL cells with and without a defective p53 pathway. Induction
of apoptosis involved inhibition of NF-kB activity and loss of BCL-XL and MCL1 expression. In combination
with fludarabine, A771726 synergistically induced apoptosis (IC50: 56 mg/mL).
Conclusion: We thus show that A771726 overcomes CD40L/IL-4–mediated resistance to fludarabine in
CLL cells of untreated as well as clinically refractory CLL cells. We present a possible novel therapeutic
principle for attacking chemoresistant CLL cells. Clin Cancer Res; 18(2); 417–31. 2011 AACR.
Introduction
Chronic lymphocytic leukemia (CLL) is a heterogeneous
disease, which is still considered incurable with conventional chemotherapy. Although CLL has an indolent behavior in the majority of cases, some patients show an aggressive course and die within few years from diagnosis (1).
Clinically, this "poor-risk" CLL is characterized by resistance
to chemotherapy including modern purine analogue–anti-
Authors' Affiliations: 1German Cancer Research Center, Department of
Molecular Oncology/Hematology; 2Department of Medicine V, University
of Heidelberg; 3National Center for Tumor Diseases and Institute of Immunology, University of Heidelberg; and 4Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research
Center, Heidelberg; and 5Center for Molecular Biomedicine, Institute for
Biochemistry and Biophysics, University of Jena, Jena, Germany
Corresponding Author: Thomas Luft, University of Heidelberg, Department of Medicine V, Im Neuenheimer Feld 410, 69120 Heidelberg,
Germany. Phone: 49-6221-56-8001; Fax: 49-6221-56-4920; E-mail:
[email protected]
doi: 10.1158/1078-0432.CCR-11-1049
2011 American Association for Cancer Research.
body combination regimens (2). Failure to respond to an
induction therapy containing purine analogues (mostly
fludarabine) can hardly be overcome by escalating cytostatic drugs (3–5). Therefore, alternative therapeutic strategies
such as alemtuzumab (anti-CD52; ref. 6), ofatumumab
(anti-CD20; ref. 7), or allogeneic stem cell transplantation
(8) are currently available therapeutic alternatives.
Predicting nonresponse is a major focus of current
research, and poor-risk patients could be identified who
have a defective p53 pathway (9). However, only about 50%
of fludarabine-refractory patients carry a p53 mutation or
17p deletion (9) raising the question of alternative resistance mechanisms in p53 wild-type patients.
It is furthermore well established that chemotherapy
resistance of CLL cells may not only result from CLL cell–
specific intrinsic defects, but may also depend to a large
degree on interactions with its microenvironment (10).
Interactions between CLL cells and CD40 ligand (CD40L)
expressing T cells (11, 12), bone marrow stromal cells (13),
and nurse-like cells producing stromal cell–derived factors
(14) have been shown to increase the apoptotic threshold in
CLL cells.
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417
Published OnlineFirst November 9, 2011; DOI: 10.1158/1078-0432.CCR-11-1049
Dietrich et al.
Translational Relevance
With this study, we provide evidence that the drug
leflunomide may be a promising agent for overcoming
fludarabine resistance mediated by the lymph node
environment, as well as intrinsic resistance due to defective p53 signaling in chronic lymphocytic leukemia
(CLL) cells. Because leflunomide is already approved
for clinical use in rheumatologic diseases and has a welldocumented safety profile, it is available for immediate
clinical studies involving CLL patients.
Although CLL has been considered to be a cumulative
rather than a proliferative B-cell neoplasm, CLL cells have a
proliferation rate that is higher than previously recognized,
particularly in lymphoid tissues (15, 16). Very recently Gine
and colleagues have shown that higher numbers of proliferation centers in lymph nodes as well as higher numbers of
mitoses or percentages of KI-67–positive cells are associated
with a worse prognosis (16). These proliferation centers are
composed of clusters of prolymphocytes and contain
numerous T cells which are predominantly CD4 positive
(17). These T cells supply signals through their CD40L
receptor as well as cytokines such as interleukin 4 (IL-4)
that stimulate CLL cells to proliferate (18).
CD40 signaling has been recognized as a strong antiapoptotic pathway mediating fludarabine resistance in vitro
(19). All CLL cells express CD40, and in vitro resistance can
be induced in 100% of patients. CD40 signaling is induced
upon binding to its ligand (CD40L and CD154), a 39 kDa
type II transmembrane protein member of the TNF gene
superfamily (20). CD40L is expressed on activated CD4þ T
cells and activated platelets, however, a wide variety of other
cells including even malignant B cells can also express the
ligand (21, 22). Binding of CD40L forms a CD40 trimer that
induces the recruitment of adaptor proteins known as
tumor necrosis factor receptor–associated factors (TRAF)
to the cytoplasmatic domain of CD40. The TRAF proteins
activate different signaling pathways including the canonical and the noncanonical NF-kB signaling pathway, the
mitogen-activated protein kinase pathway, and phosphoinositide 3-kinase pathway (23). It was also shown that
CD40 is able to associate with Janus-activated kinase
(JAK)3 leading to STAT3 activation in B cells (24). Activation of these pathways induces expression of antiapoptotic
proteins such as BCL-XL and MCL1, both mediating resistance to purine analogues (11, 19, 25).
We have shown in antigen-presenting cells that the effects
of CD40 signaling can be modulated by complementary
JAK/STAT signals (26) which are triggered through cytokines such as interferon-g and IL-4. IL-4 seems especially
important because B-CLL cells have a constitutively high
expression of the IL-4 receptor (27). IL-4 is a TH2-type
cytokine that signals predominantly via JAK1 and JAK3 and
subsequently induces phosphorylation and activation of
418
Clin Cancer Res; 18(2) January 15, 2012
STAT6 (28). STAT6 itself was shown to enhance BCL-XL
expression which may contribute to the prosurvival effect of
IL-4 (29). Furthermore, the JAK inhibitor PF-956980 has
recently been shown to effectively overcome IL4-mediated
resistance to fludarabine (30). CLL cells, especially those
located in lymphoid tissues, could thus be exposed to
signals that drive proliferation and raise the apoptotic
threshold. These dividing and resistant cells may represent
the origin of relapse. New therapeutic strategies are warranted to overcome the protective effect of these signals.
Candidate targets for interventions addressing fludarabine
resistance are therefore the JAK/STAT and NF-kB pathways
as they represent key players in preventing apoptosis.
An agent known to interfere with JAK/STAT (31, 32) as
well as NF-kB (33) and AKT1 (34) activation is A771726,
the active metabolite of leflunomide. This agent inhibits
dihydroorotate-dehydrogenase (DHODH, EC: 1.3.3.1), an
enzyme involved in pyrimidine synthesis. As a consequence, T- and B-cell proliferation slows down (35). Leflunomide is orally available and approved for treatment of
active rheumatoid and psoriatic arthritis (35). Following
intestinal resorption, this prodrug is converted into its active
metabolite A771726 inside the gastrointestinal tract.
Although a wide interpatient variability was observed,
serum concentrations of A771726 up to 176 mg/mL can be
achieved with a 20 mg tablet per day (36). The mode(s) of
action responsible for the clinical benefits remain controversially discussed (35).
The purpose of this study was to investigate whether the
stimulating effects of CD40 signaling on proliferation and
survival of CLL cells can be modulated by JAK/STAT
signals and whether this breaks fludarabine resistance.
Second, we evaluated the capacity of the JAK/STAT and
NF-kB inhibitor A771726 for treating resistant CLL cells.
For this purpose, we used a CD40L/IL-4 culture system of
primary CLL cells that mimics fludarabine resistance. Our
study shows that A771726 is able to block proliferation
and induce apoptosis in resistant CLL cells that were
activated with CD40L/IL-4. The molecular basis for
this beneficial action dose dependently involves inhibition of DHODH, suppression of the JAK/STAT pathway,
and a block of NF-kB–dependent MCL-1 and BCL-XL
expression.
Materials and Methods
Patients
Peripheral blood (PB) samples were obtained from
patients with CLL after informed consent in accordance
with the Declaration of Helsinki. Patient sample collection
was approved by the local ethics committee of the University Hospital of Heidelberg as part of the tumor bank of the
National Centre for Tumour Diseases (NCT), Heidelberg,
Germany. All CLL cases used in this study matched the
standard diagnostic criteria for CLL. Blood samples of 28
different patients were used. Median age was 65 years
(range: 46–89). 25 patients had not received any therapy
at the time of sample collection. Eighteen of these 25
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Published OnlineFirst November 9, 2011; DOI: 10.1158/1078-0432.CCR-11-1049
Leflunomide in CLL
patients were Binet stage A, 5 of 25 patients Binet stage B,
and 2 of 25 patients Binet stage C. Seven patients had a
normal karyotype, 14 patients had a deletion 13q14, 2
patients had a trisomy 12q13, and 2 patients had a deletion
11q22. Nine patients had received multiple lines of chemotherapy and were clinically highly resistant to either
fludarabine and/or bendamustine. Classical cytogenetic
analysis revealed a 17p deletion in at least 80% of interphase
nuclei in 6 patients. An additional p53 mutation was
identified in 4 of 6 patients.
Cell culture
CLL cells were purified from EDTA PB-samples of CLL
patients by density-gradient separation and subsequent
affinity purification with the MACS CD19 isolation Kit
(Miltenyi Biotech). CLL cells were either frozen in FBS
(Biochrom AG) containing 10% dimethyl sulfoxide and
stored in liquid nitrogen, or cultured at a concentration of
106 per milliliter in Dulbecco’s Modified Eagle’s Medium
supplemented with 10% fetal calf serum, 4 mmol/L Lglutamine, 4.5 g/L glucose, 100 mg/mL penicillin, and
100 mg/mL streptomycin (complete medium) at 37 C in
a 10% CO2 humidified incubator.
CLL cells were activated by an irradiated baby hamster
kidney (BHK) cell line stably expressing CD40L (concentration BHK/CLL 1:20) and/or IL-4 at (50 U/mL; Friesoythe;
Immunotools). Expression of CD40L was confirmed by
flow cytometry with an anti-CD40L monoclonal antibody
(BD Pharmingen), whereas the mock-transfected control
BHK cell line did not express CD40L.
The pan-JAK inhibitor I (pyridone 6, 0.15 mg/mL; Calbiochem; Merck) and fludarabine (2-fluoroadenine-9-b-Darabinofuranoside: 2, 10, 25 mmol/L; Sigma) were added to
the cell cultures 60 minutes prior to stimulation. The active
metabolite of leflunomide, A771726 (Calbiochem; Merck),
was added to the cell cultures 90 minutes prior to stimulation and titrated at a concentration range between 1 and
120 mg/mL.
Western blot analysis
CLL cells were activated as indicated, harvested, washed,
resuspended, and lysed at a density of approximately 3 107 cells per milliliter in Western Blot Sample Buffer (50
mmol/L Tris, pH 7.5, 1% BriJ 96V, 10 mmol/L NaF, 1
mmol/L Na-orthovanadate, 1 mg/mL leupeptin, 1,5 mg/mL
pepstatin, 100 mmol/L phenylmethanesulfonylfluoride)
and snap frozen. Protein concentrations of the lysates were
determined by a modified Bradford method (Bio-Rad Laboratories). Cell lysates were analyzed by SDS-PAGE and
Western blots as described (37) with antibodies specific
for pSTAT1 (tyr701), pSTAT3 (tyr705; Cell Signaling),
pSTAT6 (tyr641), total STAT3, BCL-XL, and actin (Cell
Signaling) and MCL1, BCL-2 (Santa Cruz Biotechnology).
For enhanced chemiluminescence (ECL)-based detection,
Western blots were developed with ECL plus Western blot
system (Santa Cruz Biotechnology) and the secondary
horseradish peroxidase–conjugated antibodies goat antirabbit IgG or goat anti-mouse IgG were used (Santa Cruz
www.aacrjournals.org
Biotechnology). Western blots were quantified by the ImageJ software (version 1.6.0).
Avidin–biotin complex DNA analysis
Avidin-biotin complex DNA (ABCD) assays were carried
out as described (38, 39). Proteins recognized by specific
antibodies were detected with Western blots generated with
X-ray films. NF-kB oligonucleotides for ABCD assays were
Bio 50 -GGAATTTCCGGGAATTTCCGGGAATTTCCG-GGAATTTCCC; 50 -GGAAATTCCCGGAAATTCCCGGAAATTCCCGGAAATTCC (tandem repeat NF-kB consensus site) or
irrelevant biotinylated sequences (mutated NF-kB sequences
from the FasL promoter.
Nucleofection of MEC1 cells with NF-kB-EGFP reporter
plasmid
A total 1 106 to 3 106 cells of the CLL model cell
MEC1 were transfected with the NF-kB-EGFP reporter
plasmid (provided by Dr. S. Drube, University Clinic of
Jena) using an Amaxa Human B cell Nucleofector kit
following the instructions of the manufacturer (Amaxa)
with the program U13. After nucleofection, cells were
incubated in the presence of CD40L-expressing BHK
cells, IL-4 (50 U/mL), and uridine (75mmol/L) with or
without A771726 (80 mg/mL). Expression levels of EGFP
were measured by fluorescence-activated cell sorting
(FACS).
Flow cytometry
Detection of apoptosis. Apoptotic cell death was detected
by flow cytometry with Annexin V–propidium iodide (PI)
or 7-amino-actinomycin (7-AAD) staining. Cells were harvested and resuspended in Annexin V–binding buffer (10
mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl and 2.5
mmol/L CaCl2) containing 10% Annexin V–fluorescein
isothiocyanate and 10% PI staining solution, or 10%
Annexin V–PE and 10% 7-AAD staining solution (BD
Biosciences). After an incubation time of 15 minutes at
4 C, stained cells were analyzed by flow cytometry gating on
lymphocytes. Double-negative cells were counted as viable
cells. The results were confirmed on the basis of changes in
forward light scattering properties of dead cells that have
decreased cell size.
Staining for intracellular proteins. Paraformaldehyde
was added to the cell culture medium at a final concentration of 4% and incubated for 15 minutes at room temperature. Cells were then harvested, washed with PBS, and
permeabilized by dropwise addition of ice-cold ethanol
(80%) to the cell pellet. During addition of ethanol, the
sample was vigorously vortexed. Cells were stored at –20 C
for at least 2 hours. For intracellular staining, cells were
washed twice with PBS containing 1 mmol/L sodium azide
and 1% bovine serum albumin and then stained for BCL-XL
(Santa Cruz), and p-p65 (Ser529; BD Biosciences). Analyses
were conducted on a double laser (488 and 637 nm) 5-color
FACScan flow cytometer (BD Biosciences; upgraded by
Cytek). Living cells were selected on the basis of their side
and forward light scattering properties.
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419
Published OnlineFirst November 9, 2011; DOI: 10.1158/1078-0432.CCR-11-1049
Dietrich et al.
A
Fludarabine 10 µmol/L
Untreated control
1,32%
1
2
10
1
66,62%
10
0
10
0,77%
1
2
3
10
10
0
10
4
10
10
1
59,09%
7,11%
10
1
2
10
3
10
10
0
10
4
10
8,76%
0
0
FL2-H
2
10
10
39,90%
30,95%
0
10
2
10
10
9,92%
23,45%
3
10
1
10
0
8,70%
40,54%
3
10
FL3-H
2
10
10
12,45%
66,95%
3
10
10
4
4
10
22,67%
FL3-H
FL3-H
7-AAD
0,79%
3
10
CD40L + IL-4
CD40L
4
10
FL3-H
i)
IL-4
4
10
10
1
FL2-H
2
10
3
10
0
10
4
10
10
1
10
10
2
3
4
10
10
FL2-H
FL2-H
Annexin V
13,58%
70,72%
2
10
1
10
10
1
2
3
10
10
10
1
FL2-H
2
10
3
10
1
70,56%
9,58%
8,50%
0
10
0
10
4
10
2
10
10
39,12%
22,75%
0
10
0
10
4
10
10
10
1,42%
16,66%
0
10
0
10
3
2
1
10
29,16%
0
13,40%
10
10
2
1
10
7,55%
37,72%
3
3
10
FL3-H
FL3-H
7-AAD
3
10
10
5,11%
49,26%
10
4
4
4
10
4,93%
FL3-H
4
10
FL3-H
ii)
10
1
2
10
3
10
0
10
4
10
10
1
10
10
2
3
4
10
10
FL2-H
FL2-H
FL2-H
Annexin V
6,80%
13,98%
77,21%
10
1
2
3
10
10
4
10
1
47,37%
22,05%
10
0
10
66,69%
6,79%
10
1
2
10
3
10
4
10
10
0
10
10
1
2
10
3
4
10
10
10
0
10
1
10
Annexin V
Control
Fludarabine
Fludarabine + pyridone 6
% 7-AAD/Annexin V–negative cells
B
**
100
2
**
3
10
4
10
80
60
40
20
0
CLL
IL-4
CD40L
Proliferation assay
CLL cells were seeded at a concentration of 105 per well
in 96-well plates in triplicates for 96 hours and stimulated
as indicated. Cells were pulsed with 0.5 mCi (0.0185
MBq) per well [3H] thymidine (TdR; Hartmann Analytik)
for the last 16 hours of culture and harvested on an
semiautomatic cell harvester (Tomtec). [3H]TdR incorporation was quantified in a TopCount Scintillation
Counter (Perkin-Elmer).
Real-time reverse transcriptase PCR quantification
A total of 1 106 cells were collected in 100 mL lysis buffer
from the MagnaPure mRNA Isolation Kit I (Roche Applied
Science) supplemented with 1% (w/v) dithiothreitol, and
420
10
FL2-H
FL2-H
FL2-H
FL2-H
8,62%
0
0
0
2
10
10
10
1,37%
2,98%
0
2
10
1
10
10
FL3-H
2
10
1
1
13,11%
3
10
10
FL3-H
FL3-H
FL3-H
7-AAD
2
11,58%
20,16%
3
10
10
10
0
10
25,69%
69,79%
3
10
10
10
10
5,82%
3
4
4
4
4
10
iii)
Figure 1. CD40L and JAK/STAT
signals synergize to induce
resistance to fludarabine in primary
CLL cells. A, apoptosis of CLL cells
quantified by Annexin V and 7-AAD
staining. Cells were activated with
CD40L and IL-4 with and without
fludarabine (10 mg/mL). Induction
of apoptosis was quantified after
96 hours. Double-negative cells
were considered as viable. FACS
plots of 3 representative patients (i,
ii, and iii) out of 11 are shown. B,
mean percentages of viable CLL
cells after activation with CD40L/
IL-4 with and without pyridone 6
(0.15 mg/mL) and/or fludarabine
(10 mg/mL). Activation by CD40L
but not by IL-4 alone protects CLL
cells from apoptosis induced by
fludarabine. Combined stimulation
of CLL cells with CD40L and IL-4
significantly increased survival.
Means SEM of 11 individual
patients are shown; , P < 0.01.
Clin Cancer Res; 18(2) January 15, 2012
CD40L + IL-4
mRNA was isolated with the MagnaPure-LC device using
the mRNA-I standard protocol. The elution volume was set
to 50 mL. An aliquot of 8.2 mL RNA was reverse transcribed
using avian myeloblastosis virus reverse transcriptase and
oligo-(dT) as primer (First Strand cDNA Synthesis Kit;
Roche Applied Science) according to the manufacturer’s
protocol in a thermocycler. After termination of the cDNA
synthesis, the reaction mix was diluted to a final volume of
500 mL and stored at –20 C until PCR analysis.
Primer sets specific for the sequences of BCL-XL and MCL1 optimized for the LightCycler (RAS) were developed and
provided by SEARCH-LC GmbH, Heidelberg (www.searchlc.com). The PCR was carried out with the LightCycler
FastStart DNA SYBR Green I kit (RAS) according to the
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Published OnlineFirst November 9, 2011; DOI: 10.1158/1078-0432.CCR-11-1049
Leflunomide in CLL
C
ii)
i)
BCL-XL
MCL-1
Actin
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
BCL-XL
iv)
iii)
MCL-1
Actin
1
2
3
4
5
6
7
8
1
2
3
4
5
6
**
Relative BCL-XL/Actin
8
**
1.5
*
1
0.5
0
Figure 1. (Continued ) C, Western blot
analysis of BCL-XL and MCL1 in CLL cells
of 4 different patients (i, ii, iii, and iv)
activated for 48 hours with (1) control, (2)
IL-4, (3) IL-4 þ pyridone 6, (4) CD40L, (5)
CD40L þ pyridone 6, (6) CD40L þ IL-4, (7)
CD40L þ IL-4 þ pyridone 6, (8) CD40L þ IL4 þ fludarabine. Expression levels of
BCL-XL and MCL-1 were quantified and
normalized to Actin and their maximal
expression after activation with CD40L and
6
IL-4; , P < 0.05. D, a total of 1 10 CLL
cells were activated with IL-4, CD40L,
CD40L/IL-4, and CD40L/IL-4/pyridone 6.
mRNA levels of BCL-XL and MCL1 were
quantified by real-time PCR and normalized
to mRNA levels of CD45. Means SEM of 4
individual patients are shown; , P < 0.05.
7
-4
-4
p
p
-4
p
-4
1.5
Relative MCL-1/Actin
**
*
1
0.5
0
-4
-4
p
p
-4
p
-4
D
*
*
*
600
500
400
300
200
100
0
*
250
200
150
100
50
0
Control IL-4 CD40L CD40L CD40L
IL-4
IL-4
pyridone6
www.aacrjournals.org
300
mRNA MCL-1/CD45
mRNA BCL-XL/CD45
700
Control IL-4 CD40L CD40L CD40L
IL-4
IL-4
pyridone6
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Dietrich et al.
A
Untreated control
FL3-H
10
10
10
7-AAD
+ IL4
10
3
10
2
10
1
10
10
3
10
2
1
10
1
10
2
10
3
10
10
1
10
2
10
3
10
10 0
10
4
3
10
2
10
1
10
10
3
10
2
10
1
10
86,16%
0
10
2
10
3
10
10
3
10
4
3
2
1
0
10 0
10
4
2
5,85%
0
10
1
4
81,03%
1
10
FL2-H
4
FL3-H
10
10
1
FL2-H
4
10 0
10
2
0
10 0
10
4
FL3-H
FL3-H
10
3
4,30%
0
10
FL2-H
10
10
4
4,46%
0
10 0
10
10
A771726
4
49,92%
10
CD40L
Fludarabine
4
FL3-H
10
FL3-H
i)
10
1
10
FL2-H
2
10
3
10
10 0
10
4
10
1
10
FL2-H
2
10
3
10
4
FL2-H
Annexin V
FL3-H
10
10
10
4
10
3
10
2
10
1
10
4
3
10
2
10
10
1
10
2
10
3
10
1
10
1
10
2
10
3
10
10 0
10
4
10
1
10
FL2-H
10
3
10
2
10
2
10
3
10
4
FL2-H
4
10
3
10
FL3-H
4
FL3-H
FL3-H
10
2
0
10 0
10
4
FL2-H
10
3
2,96%
0
10 0
10
4
4,46%
0
10
10
1
69,74%
7-AAD
10
FL3-H
10
FL3-H
ii)
2
10
4
3
2
Figure 2. A771726 induces
apoptosis in fludarabine-resistant
CLL cells. A, apoptosis of
untreated and CD40L/IL-4–
activated CLL cells quantified by
Annexin V and 7-AAD staining.
Cells were incubated with or
without fludarabine (10 mmol/L) or
A771726 (80 mg/mL) for 96 hours.
Three representative patients
(i, ii, and iii) of 9 are shown.
CD40L
10
+ IL4
1
10
1
79,66%
10
81,15%
0
11,23%
0
10 0
10
10
1
1
10
2
10
3
10
0
10 0
10
4
10
1
10
FL2-H
2
10
3
10
10 0
10
4
10
1
10
FL2-H
2
10
3
10
4
FL2-H
Annexin V
FL3-H
10
10
7-AAD
10
10
2
1
10
10
4
10
3
10
2
1
10
1
10
2
10
3
10
10 0
10
4
10
10
10
2
10
3
10
10
3
10
2
1
10
10
10
0
3
10
2
1
10
10
2
10
3
FL2-H
10
4
10 0
10
10
2
10
3
10
4
3
2
1
8,89%
0
10
1
4
64,40%
1
10
FL2-H
4
72,73%
10
10 0
10
4
FL3-H
4
10 0
10
1
FL2-H
FL3-H
10
2
5,40%
1
FL2-H
10
3
0
0
10
10
4
0,62%
0
10 0
10
FL3-H
+ IL4
10
3
50,70%
10
CD40L
4
FL3-H
10
FL3-H
iii)
0
10
1
10
2
10
FL2-H
3
10
4
10 0
10
10
1
10
2
10
3
10
4
FL2-H
Annexin V
protocol provided in the parameter specific kits. To control
for specificity of the amplification products, a melting curve
analysis was conducted. No amplification of unspecific
products was observed. The copy number was calculated
422
Clin Cancer Res; 18(2) January 15, 2012
from a standard curve, obtained by plotting known input
concentrations of 4 different plasmids at log dilutions to the
PCR cycle number at which the detected fluorescence intensity reaches a fixed value. This approach dramatically
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Leflunomide in CLL
B
Annexin V/7AAD-negative cells
80
CD40L + IL-4
60
CD40L + IL-4 + fludarabine (10 µmol/L)
40
20
0
0
20
40
C
80
100
i)
120
ii)
iii)
MCL-1
BCL-XL
Actin
5
0
0
0.15
24 h
200
IL
IL
-4
71
72
6
+
+
L
L
C
D
40
40
D
C
+
+
IL
A7
+
-4
D
+
-4
71
72
6
0
IL
-4
D
C
0
0
400
40
L
40
L
0
600
+
C
C
D
D
C
40
L
on
tro
l
+
IL
A7 -4
71
72
6
0
10 80
800
on
tro
l
200
on
tro
l
+
IL
+
-4
IL
+
A7 -4
71
72
6
0
40
L
400
40
L
0
0.15
1,000
C
600
C
0
8h
mRNA MCL1/CD45
800
C
D
0
24 h
1,000
Statistical analysis
Differences of means were calculated by the Student t test
or one-way ANOVA analysis. Statistical analyses were conducted with MedCalc (release 11.0; MedCalc Software
bvba). IC50 values were calculated with GraphPad Prism
software.
10 80
A7
0
+
5
0
-4
8h
0
C
D
0
0.15
IL
0
on
tro
l
80
0
40
L
10
0
C
5
0
D
0
C
A771726 (µg/mL)
Pyridone 6 (µg/mL)
reduced variations due to handling errors over several
logarithmic dilution steps.
To correct for differences in the content of mRNA, the
calculated copy numbers were normalized according to the
average expression of 2 housekeeping genes, CD45 and
b-actin. Values were thus given as input adjusted copy
number per microliters of cDNA.
www.aacrjournals.org
60
A771726 (µg/mL)
mRNA BCL-XL/CD45
Figure 2. (Continued ) B, mean
percentages SEM of viable CLL
cells after activation with CD40L/IL-4
and exposure to different
concentrations of A771726 (0–120
mg/mL). Means SEM of 9 patients
are shown. The IC50 for single-agent
A771726 was 80 mg/mL. The IC50 for
A771726 in combination with
fludarabine was 56 mg/mL. C,
Western blot analysis of BCL-XL and
MCL1 expression in CLL cells
activated with CD40L/IL-4 and
different concentrations of A771726.
A771726 strongly reduces
expression of BCL-XL
and MCL-1. Western lysates were
prepared after 48 hours when
BCL-XL and MCL-1 expression was
maximally induced and significant
numbers of cells treated with
A771726 were still alive. Pyridone 6
partially inhibits BCL-XL and MCL1
expression induced by CD40L/IL-4.
D, a total of 1 106 CLL cells were
activated with CD40L/IL-4 with and
without A771726 (80 mg/mL). mRNA
levels of BCL-XL and MCL1 were
quantified by real-time PCR and
normalized to mRNA levels of CD45.
Means SEM of 8 individual patients
are shown; , P < 0.05.
100
Results
CD40 signals protect CLL cells from fludarabineinduced apoptosis—synergy with JAK/STAT
CD40L stimulation of CLL cells is known to mediate
resistance to purine analogues such as fludarabine. To
evaluate whether resistance to the proapoptotic activities
of fludarabine can be modulated by costimulation of the
CD40L/CD40 and IL-4–JAK/STAT pathways, CD19 purified CLL cells were cultured in the presence of CD40L
and/or IL-4 with or without of fludarabine (10 mg/mL).
Percentages of viable cells were assessed by Annexin V
and 7-AAD FACS staining 96 hours after treatment. As
shown before, we could confirm that CD40L þ IL-4
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423
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Dietrich et al.
E
CD40L + IL-4
CD40L + IL-4 + A771726
127
61
30
37
125
250
375
500
FL2-W
0
0
0
125
250
375
500
0
100
FL2-W
200
300
400
p-p65 (p-REL-A)
F
ii)
G
protected primary CLL cells from fludarabine-induced
apoptosis (Fig. 1A). To assess the individual contribution
of IL4- and CD40 ligation to this resistance effect, we
have stimulated in vitro fludarabine treated patient
samples with IL-4 and CD40L alone as well as in combination (Fig. 1B). CD40L stimulation rescued approximately half of the cells from apoptosis induced by
fludarabine. In contrast, IL-4 alone was not sufficient to
protect a significant number of CLL cells from apoptosis
induced by this drug. However, adding IL-4 to CD40L
stimulation significantly augmented the percentage of
viable cells almost up to levels observed without fludarabine. The additive effect of IL-4 could be reversed by
inhibiting JAK/STAT activation with the established panJAK inhibitor pyridone 6 as a positive control (Fig. 1B).
424
Clin Cancer Res; 18(2) January 15, 2012
0
100
200
FL2-W
FL2-W
i)
63
32
18
0
0
95
55
64
32
0
iv)
Count
Count
Count
iii)
95
91
126
73
ii)
Count
121
i)
iii)
300
400
Figure 2. (Continued ) E, CLL cells
were activated with CD40L/IL-4
with and without A771726. After 48
hours, cells were harvested, fixed
with formaldehyde, permeabilized,
and stained for phospho-p65.
Experiments of 4 different patients
(i, ii, iii, and iv) are shown. Based on
forward and side light scattering
properties of CLL lymphocytes
only living cells were evaluated.
Higher concentrations of A771726
(80 mg/mL) blocked
phosphorylation of RELA (p65). F,
A771726 inhibits p65 and RELB
DNA binding. ABCD assay was
used to visualize the binding of NFkB p65 and RELB to a kB
consensus site. CD40L-induced
p65 and RELB DNA binding were
reduced after A771726 treatment.
One representative out of 2
experiments is shown. Three of 5
individual patients (i, ii, and iii) are
shown. G, A771726 inhibits NFkB–dependent EGFP expression in
MEC1 cells. MEC1 cells were
transfected with an NF-kB–
dependent EGFP plasmid and
incubated with IL-4, CD40L, and
uridine (75mmol/L)—with and
without A771726 (80 mg/mL). EGFP
expression was quantified by
FACS. One representative out of 2
experiments is shown.
Pyridone 6 was used at a concentration of 0.15 mg/mL
and did not augment apoptosis of CLL control cells
(n ¼ 3).
The antiapoptotic proteins BCL-XL and MCL1 are known
to mediate fludarabine resistance (11, 19, 25). Expression of
BCL-XL and MCL1 was therefore measured in CLL cells by
quantitative Western blotting. CD40L induced expression
of BCL-XL and MCL1 (but not of BCL-2, n ¼ 3, data not
shown). IL-4 alone induced MCL1 but did not cause accumulation of significant levels of BCL-XL (Fig. 1C). Of note,
combined IL-4 and CD40 ligation synergistically enhanced
BCL-XL and MCL1 expression (Fig. 1C). The enhancing
effect of IL-4 on CD40L-induced BCL-XL and MCL1 expression was reversed by pyridone 6 (Fig. 1C). Of note, fludarabine had no effect on BCL-XL and MCL1 expression in the
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Leflunomide in CLL
presence of CD40L/IL-4 costimulation. Real-time PCR analyses revealed that upregulation of BCL-XL and MCL1 protein levels appears due to a strong transcriptional upregulation of BCL-XL and MCL1 mRNA levels in CLL cells
(Fig. 1D).
Our results show that JAK/STAT enhances CD40-induced
BCL-XL and MCL1 protein expression levels and this correlates with apoptosis rates measured by FACS as shown
in Fig. 1B.
The active metabolite A771726 of leflunomide induces
apoptosis in CD40L/IL-4–activated fludarabineresistant CLL cells at clinically achievable
concentrations
Because A771726 (the active metabolite of leflunomide) is known to interfere with the JAK/STAT pathway
(induced by IL-4) and the NF-kB pathway (induced by
CD40L; refs. 31, 33), we have tested whether A771726 is
able to overcome CD40L/IL-4–mediated fludarabine
resistance in CLL cells. Figure 2A shows 3 representative
out of 9 experiments carried out with CLL samples from 9
individual patients, illustrating that fludarabine as well as
A771726 induce apoptosis in unstimulated CLL cells.
Simultaneous exposure to CD40L and IL-4 reproducibly
caused resistance to fludarabine, but not to A771726 (Fig.
2A). To determine the effective concentration range for
the proapoptotic effect of A771726, CD40L/IL-4–activated CLL cells were incubated with various concentrations
of A771726 yielding an IC50 of 80 mg/mL for A771726
alone, however, nearly all cells were apoptotic already at
the next concentration step (100 mg/mL; Fig. 2B). In
combination with fludarabine, A771726 was significantly
more effective than on its own in lower concentrations
(IC50 for A771726 in combination with fludarabine: 56
mg/mL; Fig. 2B). Induction of apoptosis by A771726 at 80
mg/mL correlated with strongly reduced BCL-XL and
MCL1 mRNA expression levels, which translated into
lower protein levels of these antiapoptotic factors (Fig.
2C and D). BCL-2 expression was lost in response to
CD40L activation as shown by others (40) and no additional effects of A771726 were observed on BCL-2 expression (data not shown).
A771726 was previously shown to inhibit NF-kB (33).
Accordingly, 80 mg/mL leflunomide completely blocked
phosphorylation of the NF-kB family member p65
(p-p65; Fig. 2E), diminished expression levels of its
target genes BCL-XL and MCL1, and caused cell death
(Fig. 2A–C). In parallel, A771726 inhibited DNAbinding activity of the NF-kB transcription factors p65
(RELA) and RELB in ABCD assays (Fig. 2F). Finally, we
transfected the model CLL cell line MEC1 with an NF-kBEGFP response plasmid and incubated these cells in the
presence of CD40L-expressing BHK cells, IL-4 with and
without A771726. A771726 (80 mg/mL) was able to
almost completely block NF-kB–dependent EGFP expression after 12 hours (Fig. 2G). Therefore, we conclude that
A771726 causes NF-kB inhibition and apoptosis of CLL
cells by concentrations of 80 mg/mL.
www.aacrjournals.org
The active metabolite A771726 of leflunomide induces
apoptosis in clinically refractory CLL cells and CLL cells
with a defective p53 pathway
Next, we evaluated whether A771726 also induced apoptosis in CLL cells of clinically refractory patients. We
incubated unstimulated and CD40L/IL-4–stimulated CLL
cells of 9 patients who were clinically refractory to either
fludarabine and/or bendamustine with A771726 in vitro.
Representative Annexin V/7-AAD FACS plots of 3 different
patients are shown in Fig. 3A. Refractoriness to fludarabine
could be confirmed in vitro, whereas in contrast A771726
induced apoptosis in a dose-dependent manner.
P53 alteration analyses revealed that 6 patients had 17p
deletions in more than 80% of evaluated interphase nuclei.
Four of the 6 refractory patients harbored additional p53
mutations in the remaining alleles. A771726 also effectively
induced apoptosis of CLL cells with p53-defective pathways
(Fig. 3B). In 3 out of the 9 patients who have been clinically
refractory according to iwCLL criteria (41), no p53 alteration could be detected. A771726 was equally an effective
inducer of apoptosis in CLL cells of such patients (Fig. 3C).
Refractory CLL cells were also simulated with CD40L and
IL-4 and incubated with fludarabine or A771726. CD40L/
IL-4 stimulation protected CLL cells of clinically refractory
patients from spontaneous apoptosis in vitro (Fig. 3B and
C). More importantly, A771726 effectively induced apoptosis in CD40/IL-4 activated and clinically refractory CLL
cells. In line with previous results, BCL-XL expression was
also inhibited in CLL cells of clinically refractory patients
(Fig. 3D).
Proliferation of CLL cells requires complementary
CD40 and JAK/STAT signals
The proliferating compartment of CLL is located in lymphoid tissue (16), the anatomic site where CD40/IL-4
stimulation of CLL cells in vivo appears most likely. CD40L
and IL-4 stimulation of CLL cells is an established model to
mimic the microenvironment within lymph nodes.
To assess the individual contribution of CD40- and/or IL4 ligation to induce proliferation of primary CLL cells, we
stimulated CD19 purified CLL cells for 96 hours with
CD40L and/or IL-4. Proliferation was quantified by thymidine incorporation in cultures of 6 different patients (Fig.
4A). CD40L but not IL-4 induced proliferation of CLL cells.
In contrast to the CD40L-mediated antiapoptotic effects,
CD40L-induced proliferation could be completely blocked
by pyridone 6, a specific pan-JAK inhibitor (Fig. 4A). Thymidine incorporation could be further enhanced by combining CD40L and IL-4. Proliferation of CLL cells induced
by these stimuli was also inhibited effectively by pyridone
6 (Fig. 4A). Absolute counts of incorporated thymidine
varied widely between individual cultures. Therefore, we
normalized counts to the protocol achieving maximal proliferation (CD40L þ IL-4, range: 11,590–61,373 cpm).
Taken together, a complementary CD40 and JAK/STAT
signaling is required for the induction of proliferation.
Interfering with only one of the pathways could completely
inhibit proliferation.
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425
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Dietrich et al.
Untreated control
i)
Fludarabine
(10 µmol/L)
3%
10
11%
10
19%
2
10
2
10
82%
71%
3%
3%
7%
1
10
2
10
3
10
0
10
4
10
10
17%
90
0
0
0
2
10
10
10
10
0
10
72%
1
1
1
10
8%
3
10
10
7-AAD
7-AAD
4%
3
3
10
C
4
4
4
10
7-AAD
A771726
(120 µg/mL)
7-AAD
A
1
10
2
10
3
10
0
10
4
10
10
1
10
2
10
3
4
10
10
Annexin V
Annexin V
Annexin V
80
4
4
10
2%
29%
3
1%
10
30%
3
1
2
10
1
10
56%
10
12%
53%
0
16%
9%
0
1
10
2
10
3
10
21%
0
10
0
10
4
10
2
10
1
10
10
0
10
63%
10
7-AAD
2
10
6%
3
10
7-AAD
7-AAD
7-AAD
4
10
10
Percentage of Annexin
V/7-AAD–negative cells
Annexin V
ii)
1
2
10
3
10
Annexin V
10
0
10
4
10
10
1
2
10
3
10
Annexin V
4
10
10
Annexin V
Annexin V
4
10
10
27%
3
7-AAD
7-AAD
7-AAD
2
1
2
10
9%
2
10
3
10
4
10
10
0
10
30
20
Control
Flu
40
80
120
A771726 (µg/mL)
2
10
1
10
46%
11%
0
1
10
40
0
82%
3
10
42%
0
4%
10
1
10
10
0
10
10
33%
10
10
CD40L + IL-4
50
4
11%
3
10
No activation
60
10
4
22%
7-AAD
iii)
70
2%
12%
0
1
10
Annexin V
2
10
3
10
4
10
10
0
10
1
10
Annexin V
2
10
3
10
4
10
Annexin V
Annexin V
B
D
80
CD40L + IL-4
CD40L + IL-4 + A771726
70
Percentage of Annexin
V/7-AAD–negative cells
Untreated control
No activation
60
CD40L + IL-4
50
40
30
20
10
0
Control
Flu
40
80
120
BCL-XL
A771726 (µg/mL)
Figure 3. The active metabolite A771726 of leflunomide induces apoptosis in clinically refractory CLL cells with defective p53 pathway. A, apoptosis of clinically
refractory CLL cells was quantified by Annexin V and 7-AAD staining. Cells were incubated with or without fludarabine (10 mmol/L) or A771726 (40, 80, and 120
mg/mL). Three representative patients of 9 (i, ii, and iii) are shown. B, viability of p53-defective CLL cells that were unstimulated as well as CD40L/IL-4 activated
and exposed to different concentrations of A771726 (0–120 mg/mL). Means SEM of 6 patients are shown. C, viability of refractory CLL patients without p53
alterations that were unstimulated as well as CD40L/IL-4 activated and exposed to different concentrations of A771726 (0–120 mg/mL). Means SEM of 3
patients are shown. D, purified CLL cells of 2 clinically refractory CLL patients were activated with CD40L/IL-4 in the presence of A771726 (80 mg/mL).
After 48 hours, cells were harvested, fixed with formaldehyde, permeabilized, and stained for BCL-XL. Expression of BCL-XL was quantified by FACS.
KI-67 staining measured by FACS analysis was used to
verify proliferation induction by CD40L and IL-4 with or
without JAK inhibition. KI-67 FACS plots of 3 representative
patient (n ¼ 6) are shown in Fig. 4B. Percentage of KI-67–
positive cells induced by CD40L and IL-4 also varied
between individual patients (median 8.5%, range: 3.5%–
17.8%). Incubation with pyridone 6 significantly reduced
percentages of KI-67–positive cells (median: 0.6%, range:
0.1%–1.5%, P ¼ 0.03). These observations reveal that both,
426
Clin Cancer Res; 18(2) January 15, 2012
CD40 signaling and the JAK/STAT pathways, are critically
involved in the CD40L inducible growth of primary CLL
cells.
The active metabolite A771726 of leflunomide
effectively blocks CD40L/IL-4–induced proliferation of
CLL cells by two different mechanisms
Leflunomide (A771726) is known to inhibit lymphocyte proliferation and is therefore used to treat
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Leflunomide in CLL
100
80
No pyridone6
60
Pyridone6 0.15 µg/mL
40
20
0
CLL
B
IL-4
CD40L + IL-4
CD40L CD40L + IL-4
CD40L + IL-4
+ pyridone6
Percentage of KI-67–positive cells
i)
7-AAD
Figure 4. Complementary CD40L
and JAK/STAT signaling induces
proliferation of B-CLL cells.
A, proliferation of B-CLL cells
3
measured by H-thymidine
incorporation. Cells were activated
with CD40L and IL-4 with and without
the pan-JAK inhibitor pyridone 6 for
96 hours. 3H-thymidine was added
for the last 16 hours. Each data set
were normalized to its maximum
count rate at stimulation with CD40L
plus IL-4. Means SEM of 7
individual patients are shown;
, P < 0.01. B, after activation with
the indicated protocols for 96 hours,
CLL cells were stained for KI-67
expression and DNA content and
analyzed by flow cytometry. FACS
plots of 3 representative patients
(i, ii, and iii) out of 6 are shown. Please
note that due to the fixation
procedure all cells are 7-AAD
positive.
Percentage of normalized [3H] TdR (cpm)
A
ii)
iii)
CD40L
IL-4
CD40L
IL-4
pyridone6
KI-67
rheumatologic diseases. The drug is well tolerated by
patients for periods of several years. A long-term exposure
of CLL patients to A771726 could therefore be used to
inhibit CLL cell proliferation and progression of the
disease. Thus, we have tested whether A771726 is able
to inhibit CD40L/IL-4–induced CLL cell proliferation
already at lower concentrations not inducing apoptosis.
We therefore costimulated CLL cells of 6 different patients
with CD40L and IL-4 and added different concentrations
of A771726 for 96 hours (Fig. 5A). Proliferation of CLL
cells was effectively blocked by 3 mg/mL of this drug.
A771726 blocks dihydroorotate dehydrogenase generating orotate, which is metabolized to uridine triphosphate
(UTP) in following steps. CTP-Synthase (EC 6.3.4.2) generates cytidine triphosphate from UTP. Thus, supplementing cells with uridine can antagonize biological effects of
A771726 on DHODH. Indeed, addition of uridine (75
www.aacrjournals.org
mmol/L) restored proliferation of CLL cells incubated with
3 and 5 mg/mL of A771726. Thus, these cells are growth
arrested but not fully apoptotic.
At A771726 concentrations of 10 mg/mL or more, cell
growth could no longer be completely rescued by uridine,
suggesting that alternative inhibitory mechanisms are operational. Accordingly, STAT3 and STAT6 phosphorylation
was already inhibited by 10 mg/mL A771726, and higher
concentrations of this agent (80 mg/mL) completely blocked
phosphorylation of these transcription factors (Fig. 5B).
Similarly, the pan-JAK inhibitor pyridone 6 induced growth
arrest of CLL cells that could not be reversed by the nucleoside uridine (Fig. 5A). In view of the results that CLL cell
proliferation requires both, CD40 and JAK/STAT signaling,
we conclude that inhibition of STAT3/6 phosphorylation
represents a second inhibitory mechanism of A771726 on
proliferation of CLL cells. Taken together, CLL cell
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427
Published OnlineFirst November 9, 2011; DOI: 10.1158/1078-0432.CCR-11-1049
Dietrich et al.
Percentage of normalized [3H]
TdR (cpm)
A
0
1
3
5
10
20
80 0.03 0.15
A771726 (µg/mL)
B
i)
Pyridone 6
(µg/mL)
ii)
iii)
P-STAT3
STAT3
P-STAT6
STAT6
Actin
A771726 (µg/mL)
0
5
10
Pyridone-6 (µg/mL) 0
0
0
80
0
0
5
10
80
0
5
10
80
0
0.15
0
0
0
0
0
0
0
0
3
Figure 5. A771726 blocks proliferation induced by CD40L and IL-4 by 2 different mechanisms. A, proliferation of CLL cells quantified by H-thymidine
incorporation. Cells were activated by CD40L and IL-4 and incubated for 96 hours with different A771726 concentrations. 3H-thymidine was added for
the last 16 hours, counts were normalized to the CD40L plus IL-4 protocol. Proliferation was effectively blocked by A771726 starting at a concentration of
3 mg/mL (black bars). Addition of uridine (75 mmol/L) restored proliferation of CLL cells incubated with 3 and 5 mg/mL of A771726. At A771726-concentrations
of 10 mg/mL or more, proliferation could not be completely restored by uridine. The inhibitory effect of pyridone 6 could also not be overcome by uridine.
Means of 7 individual patients are shown; , P > 0.05. B, Western blot analyses of CLL cells activated with CD40L and IL-4 for 48 hours with and
without A771726. A771726 inhibits STAT phosphorylation at a minimal concentration of 10 mg/mL. Pyridone 6 (0.15 mg/mL) inhibits STAT phosphorylation.
Three representative Western blots (i, ii, and iii) out of 4 are shown.
proliferation is inhibited by A771726 at much lower
concentrations than CD40L/IL-4–mediated apoptosis
resistance.
Discussion
CD40 signaling stays at the interface of various vital
functions of CLL cells, such as proliferation, survival, and
immunogenicity. Accordingly, numerous attempts are
undertaken to use CD40 as therapeutic target for CLL. These
strategies aim for apoptosis induction, opsonization, or
immune modulation applying various biological strategies
including antibody-based and gene-therapeutic approaches
(42–44). However, none of these modalities has left the
experimental stage to date.
Our group has recently reported that CD40 and cytokine-mediated JAK/STAT pathways provide strictly complementary signals for the induction of IL-12p70 in
human dendritic cells (26). Despite the complexity of
intracellular signaling networks, cells are likely to use and
reuse functional motives in a modular way (45). We have
therefore investigated whether CD40 and JAK/STAT signals exert similar complementary effects in human CLL
cells using proliferation and apoptosis resistance as func-
428
Clin Cancer Res; 18(2) January 15, 2012
tional endpoints. Indeed, we could show that IL-4 and
CD40 had an additive antiapoptotic effect rescuing more
than 90% of CLL from undergoing fludarabine-induced
apoptosis after 96 hours. In contrast to CD40 ligation
alone, which rescued approximately half of the CLL cells,
IL-4 had no independent effect on fludarabine resistance
on its own.
The antiapoptotic effect of CD40L relies on the induction
of apoptosis inhibitors such as BCL-XL and MCL1 (11, 40).
We show here that IL-4 in combination with CD40L augments BCL-XL and MCL1 levels in comparison with CD40L
alone. In contrast, IL-4 alone, although inducing phosphoSTAT6 and low MCL1 levels, did not alter BCL-XL expression nor enhance CLL cell survival in the context of
fludarabine.
Fludarabine mediates apoptosis of CLL cells via induction of the proapoptotic transcription factor p53 (46).
However, CD40L inhibits the proapoptotic effects of this
agent without reducing p53 levels (own unpublished
results). As CLL cells resistant to fludarabine express high
levels of BCL-XL, direct interaction between BCL-XL and
p53 might be a potential mechanism of CD40L-induced
resistance to p53-mediated apoptosis (47). BCL-XL is an
NF-kB target gene (48), and increased p65 phosphorylation
Clinical Cancer Research
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Published OnlineFirst November 9, 2011; DOI: 10.1158/1078-0432.CCR-11-1049
Leflunomide in CLL
coinciding with increased p65 DNA binding could indeed
be shown by us in CD40L-stimulated cells.
In contrast to antiapoptotic effects, stimulating induction
of measurable in vitro growth of CLL cells strictly required
the presence of both CD40 and JAK/STAT signaling. IL-4
alone or CD40L in the presence of a pan-JAK inhibitor could
not induce proliferation. This suggests that it should be
possible to block CLL proliferation if only one of the 2
complementary signaling pathways is impeded. It is not
clear which JAK/STAT family members are involved in the
growth-promoting effects of IL-4 in CLL. As siRNA studies in
human CLL cells are lacking, the problem cannot be finally
resolved at the time being.
Given the synergistic interactions of NF-kB and JAK/STAT
in CLL apoptosis and proliferation resistance, a drug that
interferes with both signaling pathways represents a promising candidate to overcome survival signals provided by the
microenvironment. We have therefore investigated the antirheumatic drug A771726 known to have inhibitory effects
on T lymphocytes, B lymphocytes (35), CLL cells (49) and
myeloma cell lines (50). We could show that this drug
induces apoptosis in CD40L/IL-4–activated, resistant CLL
cells. This apoptosis induction coincided with reduced BCLXL and MCL1 expression. This seems especially important
because MCL1 and BCL-XL expression levels have been
associated with resistance to standard treatments such as
fludarabine (51) in vivo or novel approaches such as BH3
mimetics (ABT-737 and ABT-263; ref. 52). Most interestingly, we could also show that clinically refractory, p53defective CLL cells remain sensitive to A771726, supporting
a p53-independent mechanism of apoptosis induction by
A771726.
Transcriptional regulation of BCL-XL is complex but has
been shown to depend on NF-kB as well as STAT activity
(48). Accordingly, we could show that A771726 effectively
reduces both, NF-kB activation as well as phosphorylation
of STAT3 and STAT6. Such a finding is especially important
because STAT3 and NF-kB collaborate in oncogenesis and
apoptosis resistance (53). Moreover, CLL cells need NF-kB
signaling for their survival and the NF-kB subunit p65/RelA
was shown to be associated with CLL cell progression (54).
Accordingly, we could show that p65/RelA phosphorylation can be inhibited by A771726 and consequently does
not bind to cognate DNA.
The concentration of A771726 required to induce apoptosis of CD40L/IL-4–activated CLL cells was significantly
lower in the presence of fludarabine (IC50 56 mg/mL),
arguing for a synergistic effect of both drugs on CLL apoptosis. These concentrations are achievable by an oral intake
of 20 mg A771726 per day in patients treated for active
rheumatoid or psoriatic arthritis (36). A combination of
methotrexate and leflunomide (A771726) is already a clinical routine in rheumatology which suggests that clinical
experience of combination treatment should rapidly
become attainable.
The antiproliferative effect of A771726 in vivo is primarily
thought to be attributable to its ability to block the enzyme
DHODH. This results in depletion of intracellular pyrimi-
www.aacrjournals.org
dines which can be reversed by addition of uridine. However, it has also been reported that A771726 can inhibit the
JAK/STAT pathway induced by cytokines (31, 32, 55).
Accordingly in our hands, this compound induced uridine-reversible inhibition of CD40L/IL-4–stimulated CLL
proliferation at low levels (3–5mg/mL) without interfering
with STAT phosphorylation. At intermediate levels (>10 mg/
mL), however, A771726 inhibited STAT3 and 6 phosphorylation. This coincided with a reduced sensitivity to uridine
rescue. Therefore, this agent affects proliferation of CLL
cells by blocking DHODH at very low concentrations
and by additionally inhibiting JAK/STAT at intermediate
concentrations.
Our results advocate the exploration of leflunomide
(A771726) in patients with CLL. In comparison with other
novel drugs currently being studied in this condition, this
compound has remarkable advantages: on one hand,
the drug seems to be highly effective in the setting of
fludarabine resistance, although this still has to be confirmed in vivo. On the other hand, translation into clinical
application in the field of CLL seems to be readily achievable
because the drug has already been approved for clinical use
for more than 10 years. Leflunomide (A771726) is an oral
drug that can be taken over prolonged periods of time. It is
known that serum levels can differ strongly after intake of a
20 mg tablet a day. Therefore, monitoring serum levels will
be important to achieve apoptosis-inducing concentrations
in all patients. However, in a significant proportion of
patients with rheumatologic diseases, these dosages are well
tolerated (36). In addition, lower concentrations should
still be capable of inhibiting proliferation of CLL. Therefore,
clinical evidence should quickly be attainable with this
promising drug that inhibits both, proliferation and apoptosis resistance by blocking JAK/STAT and NF-kB
activation.
In conclusion, complementary JAK/STAT signaling is
required for CD40-mediated proliferation of CLL cells and
strongly augments CD40-mediated apoptosis resistance of
CLL cells. Accordingly, pan-JAK inhibition can block proliferation and reduces the apoptosis resistance of CLL cells
conditioned with CD40L and IL-4. However, a much stronger abrogation of chemoresistance can be achieved with
therapeutic doses of the clinically approved DHODH blocker leflunomide (A771726). This drug affects both, JAK/STAT
and NF-kB signaling, resulting in reduced BCL-XL and
MCL1 expression. In particular, the potent inhibition of
CD40L-induced fludarabine resistance raises hopes that
leflunomide (A771726) might set the stage for a novel
therapeutic principle complementing our growing armature against chemotherapy resistant malignancies.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank Dr. S. Drube, Institute of Immunology, Jena for
providing NF-kB-EGFP.
Clin Cancer Res; 18(2) January 15, 2012
Downloaded from clincancerres.aacrjournals.org on June 14, 2017. © 2012 American Association for Cancer Research.
429
Published OnlineFirst November 9, 2011; DOI: 10.1158/1078-0432.CCR-11-1049
Dietrich et al.
Grant Support
This study was supported by a grant of F€
orderkreis Stammzell-Forschung Blut e.V., Weingarten, Germany and a grant from the Deutsche Krebshilfe
(German Cancer Aid; FKZ102362) to O.H. Kr€amer.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received May 3, 2011; revised October 3, 2011; accepted October 30,
2011; published OnlineFirst November 9, 2011.
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Leflunomide Induces Apoptosis in Fludarabine-Resistant and
Clinically Refractory CLL Cells
Sascha Dietrich, Oliver H. Krämer, Esther Hahn, et al.
Clin Cancer Res 2012;18:417-431. Published OnlineFirst November 9, 2011.
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