From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
LYMPHOID NEOPLASIA
Trisomy 12 and elevated GLI1 and PTCH1 transcript levels are biomarkers for
Hedgehog-inhibitor responsiveness in CLL
*Sarah Decker,1,2 *Katja Zirlik,1 Lauritte Djebatchie,1 David Hartmann,1 Gabriele Ihorst,3 Annette Schmitt-Graeff,4
Dieter Herchenbach,1 Hassan Jumaa,2 Markus Warmuth,5 Hendrik Veelken,1,6 and Christine Dierks1
1Department
of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany; 2Faculty of Biology, University of Freiburg, Freiburg, Germany;
Trials Unit, Department of Medical Biometry and Statistics and 4Department of Pathology, University Medical Center Freiburg, Freiburg, Germany;
5H3 Biomedicine, Cambridge, MA; and 6Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands
3Clinical
Hedgehog (HH) signaling is activated in
various lymphoid malignancies, but conflicting results exist about its role in
chronic lymphocytic leukemia (CLL). Here,
we demonstrate that the expression of
essential HH pathway components like
GLI1, PTCH1, and the HH ligands is highly
diverse in CLL. A subset of 36.7% of
60 tested CLL samples responded to all
3 SMOOTHENED (SMO) inhibitors,
whereas 40% were completely resistant.
Responsiveness correlated with elevated
GLI1 and PTCH1 transcript levels and the
presence of trisomy 12, whereas no other
karyotype correlated with responsiveness. All trisomy 12 CLLs displayed constitutive HH pathway activation driven by
autocrine DESERT HH (DHH) ligand secretion, which could be blocked by the
HH-blocking Ab 5E1. Cocultures with
DHH-expressing BM stromal cells reduced sensitivity of CLLs to SMOinhibitor treatment by activation of
noncanonical ERK phosphorylation directly downstream of the PTCH1 receptor
without involvement of SMO and could be
overcome by the HH-blocking Ab 5E1 or a
combination of SMO and ERK inhibitors.
Our results demonstrate that the HHsignaling pathway is an interesting therapeutic target for a subset of patients with
CLL, characterized by high GLI1 and PTCH1
transcript levels, and all patients with
trisomy 12 and indicate HH-blocking Abs
to be favorable over SMO inhibitors in
overcoming stroma-mediated protective
effects. (Blood. 2012;119(4):997-1007)
Introduction
B-cell chronic lymphocytic leukemia (CLL) is the most prevalent
adult leukemia in Western countries and is characterized by a
progressive accumulation of functionally incompetent Blymphocytes in the peripheral blood, BM, and lymphoid organs.1,2
Several prognostic markers such as Rai and Binet staging systems,3,4 immunoglobulin VH gene mutational status,5 ␨-associated
protein 70 (ZAP70) expression6,7 and cytogenetic abnormalities8
such as trisomy 12 and del 13q14 can be used to predict the survival
outcome and the need for treatment of patients with CLL.9 Despite
encouraging new therapeutic strategies10 CLL cannot be cured by
conventional therapy at present, and only hematopoietic stem cell
transplantation has led to complete and ongoing remissions in some
patients.11 The exact pathogenesis of this clinically heterogenous
leukemia is still unknown,12 but it is well established that B-CLL
cells are habitually interacting with stromal cells, indicating the
importance of the microenvironment for survival of CLL cells.13-15
In our previous work, we demonstrated that the hedgehog (HH)
ligands sonic (Shh), indian (Ihh), and desert (Dhh) are produced by
stromal cells in lymphoid organs and that the BM promote
expansion of murine lymphomas in vitro and in vivo.16
HH ligands activate the transmembrane receptor PATCHED
(PTCH), which abrogates the suppression of the 7-transmembranehelix protein SMOOTHENED (SMO), the key player for signal
transduction of the canonical HH pathway.17 SMO activation leads
to production of activating forms of the glioma-associated oncoproteins 1-3 (GLI1-3), the HH transcription factors. With exception of
GLI1, which functions exclusively as a transcriptional activator,
the HH transcription factors exist as full-length transcriptional
activators or as truncated transcriptional repressors.18 The activation process of GLI leads to expression of HH target genes such as
PTCH1, GLI1, BCL2, BMI1, and Cyclin D1 and hence to increased
proliferation, survival, and self-renewal.19-21 Sustained canonical
HH pathway activation has been associated with cancers of the
brain,22,23 the skin,24-26 the gastrointestinal tract,27 the prostate,28,29
the pancreas,30,31 and the lung.32 Noncanonical HH signaling was
described in mammary gland cells, inducing direct ERK activation
downstream of the PTCH1 receptor without involvement of the
SMO receptor.33
Diverse roles of HH signaling are described in hematologic
malignancies. Although HH signaling mediated by SMO is required for maintenance of the leukemic stem cell population in
chronic myeloid leukemia,34,35 it is dispensable for the development of acute leukemias induced by the MLL-AF936 fusion gene or
T-cell acute lymphoblastic leukemia induced by an activated form
of NOTCH.37 In our previous work, we could show that HH
pathway inhibition by SMO inhibitors could block the expansion of
murine lymphomas in vitro and in vivo.16 In addition, human
lymphoid malignancies, such as diffuse large B-cell lymphoma,38
NPM-ALK–driven anaplastic large cell lymphoma,39 mantle cell
lymphoma,40 and multiple myeloma16,41 show activation of HH
signaling and responsiveness to SMO inhibitors in vitro. Conflicting results exist about its role in CLL. Although Hedge et al argue
Submitted June 9, 2011; accepted November 2, 2011. Prepublished online as
Blood First Edition paper, November 30, 2011; DOI 10.1182/blood-2011-06-359075.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
*S.D. and K.Z. contributed equally to this study.
The online version of this article contains a data supplement.
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
© 2012 by The American Society of Hematology
997
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
998
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
DECKER et al
for activation of HH signaling on the level of SMO in all CLL
cases,42 Desch et al reported dependence of CLL cells on the GLI1
transcription factor, but not on the SMO receptor.43
Therefore, the aim of the present study was to identify
biomarkers and clinical parameters, which can discriminate in
between SMO-inhibitor responsive and resistant CLLs and to
identify mechanisms of HH pathway activation in CLL.
before adding CLL cells, to block stroma-produced ligands, before they can
bind to the PTCH1 receptor of the CLL cells. After 4 hours of incubation,
cells were stained with CD19 Ab, fixed with 1.85% formalin, and
permeabilized with 90% methanol. Endogenous levels of p44 and p42 MAP
kinase were detected with the phospho-p44/42 MAPK mouse mAb (no.
9106; Cell Signaling) and anti–mouse IgG Alexa Fluor-conjugated Ab (no.
4408; Cell Signaling). Cells and data were analyzed as described in
“Apoptosis and viability assays.”
Immunohistochemistry staining
Methods
CLL patient samples
This study was approved by the Institutional Review Board of the
University Medical Center Freiburg. Peripheral blood samples were
obtained with informed consent in accordance with the Declaration of
Helsinki from patients with B-CLL who were either untreated or off therapy
for ⱖ 6 months (supplemental Table 2, available on the Blood Web site; see
the Supplemental Materials link at the top of the online article). CLL cases
were characterized for IgVH mutational status, ZAP70 expression, disease
stage according to Binet and Rai criteria, and history of treatment.
Furthermore, genetic aberrations were analyzed by chromosomal analysis
and FISH analysis and copy number changes were verified by single
nucleotide polymorphism arrays44 (patient characteristics are shown in
supplemental Table 2). PBMCs were separated by Ficoll gradient centrifugation and either used fresh or cryopreserved in FCS with 10% DMSO until
use. Cells were maintained in DMEM/10% FCS and 1% penicillinstreptomycin. PBMCs for quantitative PCR (qPCR) contained ⬎ 88%
CD20⫹/CD5⫹ CLL cells.
Apoptosis and viability assays
PBMCs were plated into 96-well plates at a concentration of 1 ⫻ 105 cells/
well with or without support of the murine stromal cell line M2-10B4
(ATCC) or the human stromal cell line HS-5 (ATCC). In case of stromal
coculture, on day ⫺1, 5 ⫻ 103 stromal cells/well were plated. After
24 hours, CLL cells were added and treated with 3 different SMO inhibitors
cyclopamine45,46 (Calbiochem), NVP-LDE22547 (Novartis), or IPI-92648,49
(Infinity Pharmaceuticals) at indicated concentrations. For apoptosis detection, CLL cells with or without stromal coculture were plated as indicated
earlier and treated with SMO inhibitors, 10-40 ␮g/mL HH-blocking Ab
5E150 (Developmental Studies Hybridoma Bank), 1␮M ERK inhibitor
UO126 (Sigma-Aldrich) or combinations. After 24, 48, and 72 hours of
incubation at 37°C in 5% CO2, cells were stained with a CD19allophycocyanin Ab (BD Biosciences), followed by annexin V/7-aminoactinomycin D (7-AAD) staining with anti–annexin V–PE and 7-AAD (BD
Biosciences) according to the manufacturer’s instructions. Cells were
analyzed with the CyanADP flow cytometer (Beckman Coulter). Flow
cytometric data were analyzed with the FlowJo 7.6 software (TreeStar). To
verify the annexin V/7-AAD assay results, we performed a second assay
with the first 60 patients after incubation with compounds for 48 and
72 hours and assessed vital cell numbers with the use of Guava ViaCount
technology. The Guava ViaCount assay is a fluorescence-based assay,
which is performed in a 96-well format and therefore allows the use of the
compounds in a broad range of concentrations. Besides distinguishing
among necrotic, apoptotic, and viable cells (1 membrane-permanent dye
stains all nucleated cells, and a membrane-impermanent dye stains only
damaged cells with breaches in the plasma membrane), the results also
include total cells per volume, which allows one to calculate total numbers
of viable or apoptotic or necrotic cells per well (Millipore; Guava
Technologies; Guava ViaCount Assay). Measurements were performed
with the Guava Technologies PCA-96 system.
Intracellular phospho-ERK staining
CLL cells or stromally cocultured CLL cells were plated as in “Apoptosis
and viability assays” and treated with 5␮M cyclopamine, 1␮M UO126, or a
combination of both. HH-blocking Ab 5E1 was added to stromal cells
Paraffin-embedded BM slides were acquired from the pathology department of the University of Freiburg. Diaminobenzidine immunoperoxidase
staining (EnVision⫹System-HRP/DAB; Dako) on paraffin sections was
performed for 10 minutes. Primary Abs to SHH/IHH (IgG rabbit polyclonal; N-19; Santa Cruz Biotechnology) 1:50, DHH (IgG goat polyclonal;
H-85; Santa Cruz Biotechnology) 1:250, GLI1 (N-16; Santa Cruz Biotechnology) 1:100, and SMO 1:50 (H-300; Santa Cruz Biotechnology) were
incubated overnight, followed by a 30-minute incubation time with
secondary Abs (EnVision⫹System-HRP [DAB]; Dako). Immunodetection
was performed with 3,3⬘ diaminobencidin (EnVision⫹System-HRP [DAB];
Dako). Photographs were taken with Axioplan 2 (Zeiss). Staining intensity
was scored in categories (grade 0 ⫽ ⬍ 10%; 1 ⫽ 10%-20%; 2 ⫽ 20%-50%, and
3 ⫽ ⬎ 50% positive cells; supplemental Table 3).
qPCR
Total cellular RNA was isolated from PBMCs from patients with CLL
containing ⬎ 88% of CD20⫹CD5⫹ CLL cells (single patient information;
supplemental Table 4), reverse transcribed, and amplified for 50 cycles at an
annealing temperature of 60°C. PCR primers and probes for human GLI1,
PTCH1, SMO, and GAPDH and mouse Gli1, Ptch1, Dhh, and Gapdh were
obtained from Applied Biosystems. qPCR from these genes was assessed
by TaqMan PCR (summary of primers and probes in supplemental Table 1).
Results were quantified according to the ␦-␦-CT method that was based on
the relative expression of a target gene versus a reference gene (GAPDH)
and normalized to the median of all samples.
Statistical analysis
Data are represented as the mean ⫾ SEM. Comparisons between parameters were performed with a 2-tailed, paired Student t test. For all analyses,
P ⬍ .05 was considered statistically significant. Correlations were assessed
with the Spearman rank correlation coefficient. The Cochran-Armitage test
for trend is a modified ␹2 test, which was used for calculation of the
association of different genetic subgroups or risk factors with response to
SMO-inhibitor treatment.
Results
Level of HH pathway activation is highly diverse in CLL
To evaluate the presence of the HH-signaling pathway in CLL, we
performed IHC stainings for the downstream target GLI1, the
receptor SMO and the HH ligands SHH, IHH, and DHH, in BM
samples of 24 patients with CLL. Expression levels were scored
from grade 0 to grade 3 (grade 0 ⫽ 0%-10%; 1 ⫽ 10%-20%;
2 ⫽ 20%-50%, and 3 ⫽ ⬎ 50% positive cells). IHC stainings for
the HH-downstream target gene GLI1 showed expression (grades
1-3) of this protein in 33% of the analyzed patient samples (8 of
24), whereas in the rest of the samples the GLI1 transcription factor
was completely absent (Figure 1A-B; supplemental Table 3). The
receptor SMO, which plays a central role in signal transduction of
the ligand-mediated canonical HH signal, was present in 46%
(11 of 24) of the tested CLL samples (Figure 1A-B; supplemental
Table 3). At least 1 of the 3 HH ligands SHH, IHH, and DHH was
present in 33% of the patient samples (SHH/IHH 4 of 24; DHH 6 of
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
BIOMARKERS FOR HH-INHIBITOR RESPONSIVENESS IN CLL
999
Figure 1. The activation status of HH signaling is
highly diverse in CLL. (A) IHC stainings from CLL BM
samples for GLI1, SMO, and the HH ligands SHH/IHH.
Negative (left) and positive (right) examples for expression of GLI1, SMO, and the HH ligands in CLL. Original
magnification ⫻40 for GLI1 (scale bar, 50 ␮m) and
⫻100 for others (scale bar, 25 ␮m). Microscope: Axioplan 2 (Zeiss); GLI1 40⫻/0.95 NA; SMO ⫹ SHH/IHH
100⫻/1.25 NA oil objective, room temperature; microscope software: AxioVision LE. (B) Percentage of
samples positive or negative for IHC staining for GLI1,
SMO, and the HH ligands SHH/IHH and DHH in 24 CLL
cases (single patient information, including scoring;
supplemental Table 3). (C) qPCR for GLI1 and PTCH1 in
peripheral blood samples from patients with CLL. Transcript levels are shown relative to GAPDH and compared with the median of all samples (patient single
data; supplemental Table 4). (D) Correlation in between
GLI1 and PTCH1 transcript levels in same patients with
CLL as in panel C shows a positive correlation between
both genes (n ⫽ 18; P ⫽ .0022, Spearman correlation
coefficient, r ⫽ 0.67321).
24), which might indicate an autoregulatory activation loop in this
CLL subset (Figure 1A-B; supplemental Table 3). There is a direct
positive correlation between GLI1 expression and SMO expression
(n ⫽ 24; P ⫽ .0204, r ⫽ 0.4801, Spearman correlation coefficient) and
between GLI1 expression and ligand expression (GLI1 vs SHH/IHH,
P ⫽ .0111, r ⫽ 0.5194; GLI1 vs DHH, P ⫽ .0020, r ⫽ .6103). To
validate the activation status of the HH-signaling pathway, we
performed qPCR for the direct HH target genes GLI1 and PTCH1
on isolated white blood cells (WBCs) from patients with CLL
(n ⫽ 18; supplemental Table 4). In confirming the IHC results, we
found a great heterogeneity for the expression levels of the
HH-target genes GLI1 and PTCH1 within different CLL patient
samples but a direct positive correlation between the transcript
levels of the 2 target genes (n ⫽ 18; P ⫽ .0022, r ⫽ 0.67321;
Figure 1C-D). In individual patient samples the GLI1 and PTCH1
transcript levels were elevated ⬎ 100-fold compared with the
median of all samples. This indicates a highly diverse activation
level of HH signaling in CLL, with a subset of ⬃ 30% of CLL
samples showing high transcript levels (⬎ 3-fold than the median)
for GLI1 and PTCH1.
SMO-inhibitor treatment of primary human CLL samples results
in 3 different response groups
To further investigate the role of the HH-signaling pathway in CLL,
we examined the effect of 3 different SMO inhibitors (cyclopamine, NVP-LDE225, IPI-926) on apoptosis induction (annexin
V/7-AAD staining) and viable cell counts in 60 different CLL
patient samples. Responses to SMO-inhibitor treatment were
strongly diverse, ranging from dose-dependent apoptosis induction
by SMO inhibitors at already low inhibitor concentrations to
complete apoptosis resistance to SMO inhibition (Figure 2A,
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1000
DECKER et al
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
Figure 2. SMO inhibition in primary human CLL
samples (n ⴝ 60) resulted in 3 different response
groups. (A) Apoptosis assay showing annexin V–positive
cells after treatment of primary CLL cells with the SMO
inhibitor NVP-LDE225 (0␮M, 1␮M, and 3␮M) for 24 hours.
Top panel shows one representative example for a SMO
inhibitor–sensitive CLL (patient 2), bottom panel an example for a SMO inhibitor–resistant CLL (patient 9).
(B) Viable cell counts from the same 2 patients with the
use of Guava ViaCount after 24 hours of treatment with
the SMO inhibitors cyclopamine and NVP-LDE225 in
increasing concentrations. Left side shows CLL cells
treated with cyclopamine; right side CLL cells treated with
NVP-LDE225. Top panel shows a SMO inhibitor–sensitive example (patient 2), bottom panel shows a SMO
inhibitor–resistant CLL (patient 9). (C) Diagram displays
the distribution of 60 CLL patient samples (supplemental
Table 2; patients 1-60) among the 3 different response
groups to SMO inhibitor treatment. Response was determined as good if the CLL sample responded to all 3 SMO
inhibitors and with IC50s for cyclopamine ⬍ 5␮M and for
NVP-LDE225 and IPI-926 ⬍ 2.5␮M. The intermediate
response group was represented by patients who responded to only 1 or 2 of the 3 used SMO inhibitors, and
nonresponders showed no response to any SMO inhibitor or only responded at very high concentrations.
(D) Treatment of CLL cells with 5␮M cyclopamine and
extraction of RNA after defined treatment periods. qPCR
for GLI1 and PTCH1 in comparison to GAPDH and
relative to the time point before treatment were performed
in triplicates and show a significant reduction of GLI1
transcript levels after 6 hours (n ⫽ 3; P ⫽ .0045, Student
t test) and of PTCH1 transcript levels after 2 hours (n ⫽ 3;
P ⫽ .0041, Student t test). Target gene reduction stays
significant until 72 hours.
1 example for responsive [P1] and 1 for nonresponsive [P9]
patient). Those results could be reconfirmed by analysis of viable
cell counts after 24 and 48 hours of treatment with the use of the
Guava ViaCount Assay (Figure 2B, 1 responsive [P1] and
1 nonresponsive [P9] patient). IC50 values for NVP-LDE225 and
IPI-926 were generally lower than for cyclopamine. Responsiveness to SMO-inhibitor treatment was defined as “good” if the CLL
sample responded to all 3 SMO inhibitors and with IC50s for
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
BIOMARKERS FOR HH-INHIBITOR RESPONSIVENESS IN CLL
1001
Figure 3. Elevated GLI1 and PTCH1 transcript levels
and trisomy 12 and DHH expression are biomarkers
for SMO inhibitor responsiveness in CLL. (A) qPCR
for GLI1 and PTCH1 in CLL samples of the different
response groups. High GLI1 (n ⫽ 24; P ⫽ .0260,
Cochran-Armitage trend test) or PTCH1 (n ⫽ 24;
P ⫽ .0006, Cochran-Armitage trend test) transcript levels (⬎ 3-fold than the median) compared with GAPDH
and relative to the median of all samples are correlated
with good response to SMO inhibitor treatment in CLL
(single data information in supplemental Table 4, patients 2-11, 13-17, 23-25, 36, 37, 42, 46, 47, and 60).
(B) Distribution of trisomy 12 between the different
response groups. Presence of trisomy 12 (n ⫽ 11;
P ⫽ .0006, Cochran-Armitage trend test) correlates with
good response to SMO inhibitor treatment. (C) Comparison of DHH expression (⬎ 20% positive CLL cells by
IHC) or elevated relative PTCH1 and GLI1 transcript
levels compared with GAPDH (⬎ 3-fold than the median) between patients positive for trisomy 12 and other
patients. Single data for IHC are included in supplemental Table 3, and single data for TaqMan results are
included in supplemental Table 4. (D) Percentage of
viable cells (annexin V/7-AAD staining) after treatment
of primary CLL cells with the SMO inhibitor cyclopamine
or the HH-blocking Ab 5E1 for 48 hours compared with
control. Patients positive for trisomy 12 (n ⫽ 4) show a
dose-dependent decrease in viable cells after treatment
with SMO inhibitors or the 5E1 Ab, whereas samples
positive for del 13q14 (n ⫽ 3) show only a decrease in
viable cells after SMO inhibitor treatment, but an increase in viable cells after treatment with the 5E1 Ab
(analyzed patients 1, 7, 18, 60, 66, 67, and 68).
cyclopamine ⬍ 5␮M and for NVP-LDE225 and IPI-926 ⬍ 2.5␮M.
The intermediate response group was represented by patients who
responded to only 1 or 2 of the 3 used SMO inhibitors, and
nonresponders showed no response to any SMO inhibitor or only
responded at very high inhibitor concentrations. Of the CLL patient
samples 36.7% were classified in the good response group, 23.3%
in the intermediate response group, and 40% of the samples
showed no response to SMO-inhibitor treatment (Figure 2C). SMO
inhibition by cyclopamine and NVP-LDE225 in responsive samples
resulted in a time-dependent and significant down-regulation of the
HH target genes GLI1 and PTCH1, indicating that the SMO
inhibitors target the HH-signaling pathway in the CLL cells (Figure
2D; supplemental Figure 1). Although treatment with cyclopamine
significantly suppressed GLI1 and PTCH1 transcript levels for
⬎ 72 hours, NVP-LDE225 suppressed GLI1 transcript levels for
only 24 hours and PTCH1 transcript levels for only 12 hours,
indicating that this compound might be less stable under in vitro
culture conditions than cyclopamine.
Elevated GLI1 and PTCH1 transcript levels, trisomy 12, and
DHH expression are biomarkers for SMO-inhibitor
responsiveness in CLL
To identify biomarkers or clinical parameters, which can discriminate between SMO inhibitor-responsive and -resistant CLLs, we
correlated transcript levels from HH pathway members and clinical
characteristics with the 3 different response groups. Elevated
transcript levels for the 2 HH target genes GLI1 and PTCH1
strongly correlated with the good response group (Figure 3A;
supplemental Table 4). Increases ⬎ 3-fold over the median of all
samples in either PTCH1 or GLI1 transcription was present in
87% of the CLL samples in the good response group, but only in
13% in the intermediate response group and in 0% in the
nonresponder group, which indicates this cutoff as a good parameter to discriminate between good and intermediate/no response.
Transcript levels for other pathway members and target genes such
as SMO, STK36, BMI1, PTCH2, SHH, and IHH showed no
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1002
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
DECKER et al
Table 1. Responsiveness to SMO inhibitor treatment in different CLL subgroups
Responsiveness to SMO inhibitor treatment
Del 13q14
Trisomy 12
Del 17p
Good, % (n/N)
Intermediate, % (n/N)
Resistant, % (n/N)
37.9 (11/29)
37.9 (11/29)
24.1 (7/29)
100 (11/11)
0 (0/11)
33 (2/6)
16.6 (1/6)
0 (0/11)
50 (3/6)
Del 11q
28.5 (2/7)
28.5 (2/7)
42.8 (3/7)
Normal karyotype
31.2 (5/16)
12.5 (2/16)
56.2 (9/16)
Response rates to SMO inhibitor treatment in the different genetic subgroups in CLL are shown. Some patients have several chromosomal aberrations and are therefore
represented in several subgroups (single patient information, including risk factors and response group, are shown in supplemental Table 2). Cochran-Armitage trend test for
trisomy 12 was highly statistically significant (P ⫽ .0006; n ⫽ 11). No other genetic subgroup showed a statistically significant correlation with any response group.
association with any response group (data not shown). In addition,
clinical parameters and risk factors, such as ZAP70 expression,
mutational status, WBC counts, stage of disease, or previous
therapy did not correlate with any response group (supplemental
Figure 2; supplemental Table 5). The comparison of chromosomal
aberrations in the first 60 patients with response groups identified
no correlation between response and presence of del 13q14
(n ⫽ 29), no chromosomal aberrations (n ⫽ 16), del 11q (n ⫽ 7),
or del 17p (n ⫽ 6; Table 1). But all patients with CLL with trisomy
12 (n ⫽ 4 within the first 60 patients), which is present in 12%-16%
of all CLL cases, were represented in the good response group
(Table 1). We therefore increased the number of patients with
trisomy 12 to 11 and found the presence of trisomy 12 to be a
highly predictive marker for good response to SMO inhibitors in
CLL (Cochran Armitage trend test P ⫽ .0006; Figure 3B, Table 1).
Interestingly, chromosome 12 contains the gene locus for GLI1 and
DHH, which are both positive regulators for the HH-signaling
pathway. According to the DHH IHC staining from 24 randomly
taken CLL cases, only 2 contained ⬎ 20% DHH-positive CLL cells
(grading 2 and 3), with the only trisomy 12–positive case to have
⬎ 50% DHH-positive cells. Therefore, we added 5 more cases for
trisomy 12. All 5 additional cases with trisomy 12 showed DHH
staining of grades 2 and 3, including 4 of 5 with ⬎ 50% positive
cells (grade 3). Taken together, high DHH expression is nearly
exclusively present in cases positive for trisomy 12 (Figure 3C;
supplemental Figure 3). Furthermore, in most trisomy 12 patient
samples the GLI1 transcript levels were elevated ⬎ 3-fold compared with the median of all samples (8 of 9; Figure 3C;
supplemental Table 4), indicating activation of the HH-signaling
pathway in those samples. PTCH1 transcript levels were elevated
⬎ 3-fold in 66.6% of the measured samples (6 of 9; Figure 3C;
supplemental Table 3). A previous study from Desch et al had
already shown an up-regulation of SMO transcripts in trisomy
12 cases compared with other genotypes.43 To further verify that
the autocrine DHH expression and secretion might be the reason
for HH-pathway activation in patients with trisomy 12 and might
be important for the survival of those cells, we used the HH
ligand–blocking Ab 5E1 to block the autocrine loop, that means the
interaction between the DHH ligand and the PTCH1 receptor. All
4 patients with trisomy 12 treated with increasing amounts of the
HH ligand–blocking Ab showed a dose-dependent apoptosis induction, which was even more effective than treatment with the SMO
inhibitor (Figure 3D). In contrast, treatment of patients with del
13q14 (n ⫽ 3), which were highly responsive to SMO-inhibitor
treatment, showed no response to the Ab treatment, but an adverse
reaction with a dose-dependent increase in viable cells (Figure 3D).
Those results strongly support the theory that patients with trisomy
12 in contrast to other genetic subgroups have an autocrine active
HH-signaling pathway, which is essential for their survival.
Altogether, elevated GLI1 and PTCH1 transcript levels, presence of trisomy 12, and elevated expression of DHH are valuable
parameters to discriminate between SMO inhibitor-responsive and
-resistant samples and might be useful biomarkers for future
clinical trials.
Stromal cells reduce SMO-inhibitor sensitivity of CLL cells
To investigate the effect of the microenvironment on the activation
status of the HH-signaling pathway in CLL cells, we performed
stroma-CLL coculture assays with the use of the murine stroma cell
line M2-10B4. We examined the transcription of the HH target
genes GLI1 and PTCH1 in CLL cells (n ⫽ 8) either cultivated
alone or in coculture with the stromal cell line M2-10B4, which
produces high amounts of DHH. PTCH1 was up-regulated in all
(8 of 8) and GLI1 in 7 of 8 CLL cells in the presence of the stromal
cells (Figure 4A). Mean GLI1 transcript levels were increased
1.9-fold compared with control, and mean PTCH1 transcript levels
were increased ⬎ 30.7-fold, indicating induction of HH signaling
in CLL cells by stromal cells. This activation of HH target genes
was seen in trisomy 12 patient samples (n ⫽ 4) and in 13q14 patient
samples (n ⫽ 4; Figure 4A), which indicates that the paracrine
ligand stimulation is dominant over the autocrine ligand stimulation present in patients with trisomy 12. Comparison of transcript
levels of Dhh in the murine stromal cell line M2-10B4 versus DHH
in primary human trisomy 12 CLLs showed a ⬎ 20-fold increase of
Dhh transcript levels in the stroma. In contrast to our expectations,
the presence of murine (M2-10B4) or human (HS-5) stromal cells
reduced the sensitivity of CLL cells to all 3 SMO inhibitors and in
some cases even resulted in complete resistance to SMO inhibition
(Figure 4B). This stroma-induced resistance was seen in both
patients with trisomy 12 and patients with del 13q14. To identify
the mechanism of apoptosis resistance induced by stromal cells, we
examined the HH-signaling pathway in the stromal cell line
M2-10B4 and in primary mouse stroma from Cdkn2a⫺/⫺ mice.
SMO inhibition with NVP-LDE225 for 24 hours resulted in a
reduction of Gli1 in both M2-10B4 and primary mouse stromal
cells and interestingly in a simultaneous up-regulation of the HH
ligand Dhh in the stromal cells (Figure 4C). Those results indicate
that SMO inhibition induces a negative autoregulatory loop with
enhanced secretion of Dhh by stromal cells on SMO inhibition,
which results in an at least continuous or even increased stimulation of the PTCH1 receptor in the CLL cells.
Noncanonical HH signaling in CLL cells is partially mediated by
ERK phosphorylation
In previous reports, Chang et al described a non-canonical HHsignaling pathway in mammary gland cells with direct ERK
activation by the PTCH1 receptor without involvement of SMO.33
To investigate whether ERK activation might be involved in
noncanonical HH signaling in CLL cells, we investigated the effect
of the presence of stromal cells on ERK phosphorylation in CLL
cells with the use of intracellular p-ERK staining. Interestingly,
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
BIOMARKERS FOR HH-INHIBITOR RESPONSIVENESS IN CLL
1003
Figure 4. Stromal cells activate HH signaling in CLL cells and induce SMO inhibitor resistance. (A) GLI1 and PTCH1 transcript levels compared with GAPDH with (right)
and without (left) stromal (M2-10B4) support in 8 CLL samples (trisomy 12, n ⫽ 4; 13q14, n ⫽ 4). Figure shows up-regulation of the HH target genes GLI (n ⫽ 8; P ⫽ .0391,
Student t test) in 7 of 8 samples and PTCH1 (n ⫽ 8; P ⫽ .0072, Student t test) in 8 of 8 samples (analyzed patients 1, 7, 18, 19, 61, 62, 63, and 64). (B) Percentage of viable
cells measured by annexin V/7-AAD staining after treatment with different concentrations of cyclopamine compared with the DMSO control. CLL cells were either cultivated on
murine (M2-10B4) or human (HS-5) stroma or without stroma. Presence of stromal cells reduces sensitivity of CLL cells toward SMO inhibitors (trisomy 12, n ⫽ 3; responsive
del 13q14, n ⫽ 3). (C) Inhibition of SMO in M2-10B4 cells and primary mouse stromal cells reduces transcript levels for Gli1 measured by qPCR and relative to Gapdh and
results in a simultaneous up-regulation of Dhh.
baseline levels for ERK phosphorylation measured by intracellular
p-ERK staining were significantly higher in patients with trisomy
12, which have an autocrine HH-signaling pathway, compared with
the biggest other CLL cohort with del 13q14 (Figure 5A).
Coculture of CLL cells with stromal cells or treatment with stroma
cell supernatant fluid further induced ERK phosphorylation in both
CLL subtypes (Figure 5B; supplemental Figure 4). Although SMO
inhibitor treatment with cyclopamine could not significantly reduce
stroma-induced ERK phosphorylation in CLL cells (Figure 5B),
blocking HH ligand binding with the HH-blocking Ab 5E1 could
completely abolish stroma-induced ERK phosphorylation (Figure
5C) and could reduce the phosphorylation status in patients with
trisomy 12 even below the baseline levels. Stroma-induced ERK
phosphorylation could also be reduced by the addition of the ERK
inhibitor U0126 (Figure 5B), which verifies the specificity of our
staining method for p-ERK. Figure 5D shows a diagram displaying
the interaction of stromal cells with CLL cells mediated by HH
ligands in the presence or absence of SMO inhibitors.
Stroma-induced SMO inhibitor resistance mediate by
noncanonical HH signaling can be overcome by the
HH-blocking Ab 5E1 or ERK inhibitors
To investigate whether noncanonical HH signaling mediated by
high levels of stroma-secreted Dhh might be one of the reasons for
stroma-induced SMO inhibitor resistance of otherwise SMO
inhibitor–sensitive CLLs, we treated CLL cells with or without
stromal support with the HH ligand–blocking Ab 5E1. The 5E1 Ab
blocks the interaction of all HH ligands (Shh, Ihh, and Dhh) with
the PTCH1 receptor. In contrast to SMO inhibitor treatment, the
5E1 Ab could completely overcome stroma-mediated resistance
and induced apoptosis rates ⬎ 50% within 48 hours even in the
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1004
DECKER et al
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
Figure 5. Noncanonical HH signaling in CLL cells is partially mediated by ERK phosphorylation. (A) Intracellular p-ERK staining and measurement by flow cytometry in
4 patients with trisomy 12 and 4 patients with del 13q14 in the absence of stromal cells. Comparison of pERK baseline levels show a statistically significant increase in trisomy
12 patient samples compared with del 13q14 (n ⫽ 8; P ⫽ .0331, Student t test; analyzed patients 1, 7, 44, 50, 60, 66, 68, 69). (B) Intracellular pERK staining shows induction of
ERK phosphorylation in CLL cells on coculture with stromal cells measured by the mean fluorescence intensity (n ⫽ 7; P ⫽ .0029, Student t test). Although 4 hours of treatment
with 5␮M cyclopamine could not significantly reduce ERK phosphorylation (n ⫽ 7; P ⫽ .2173, Student t test), the ERK inhibitor U0126 could significantly reduce
stroma-induced ERK phosphorylation (n ⫽ 7; P ⫽ .0043, Student t test; patients 3, 7, 44, 50, 60, 64, 65). (C) Treatment of stromally cocultured CLL cells with the HH-blocking
Ab 5E1 could block stroma-induced ERK phosphorylation in CLL cells (n ⫽ 8; P ⫽ .0285, Student t test; patients 1, 7, 18, 50, 60, 66, 68, 69). (D) Diagram shows the interaction
in between stromal cells and CLL cells mediated by HH ligands with and without SMO inhibitor treatment.
presence of stromal cells (Figure 6A, 1 patient with trisomy 12). In
addition, the protective effect induced by the human stromal cell
line HS-5 could be completely overcome by the 5E1 Ab (Figure
6A, 1 patient with trisomy 12). Trisomy 12 patient samples
were more sensitive to 5E1 treatment than were patients with del
13q14 in the presence of stromal cells (Figure 6B). Comparing the
effect of the 5E1 Ab in patients with trisomy 12 with and without
stroma support shows that we need ⬃ 6-fold concentrations to
reach the same effect (50% reduction of viable cells) in the
presence of stromal cells, indicating that higher amounts of ligands
have to be blocked in the presence of the stromal cells (Figure 3D;
Figure 6B). In addition, treatment of CLL cells with stromal
supernatant fluid significantly enhanced their survival, and this
effect could again be blocked by the HH-blocking Ab 5E1,
indicating soluble HH ligands to be responsible for the prosurvival
effect of stromal supernatant fluid (supplemental Figure 5). In
addition, a combination of SMO and ERK inhibitors could
overcome stroma-mediated resistance, and both compounds showed
additive effects, indicating that it is necessary to block both
canonical and noncanonical HH signaling for apoptosis induction
in the presence of stromal cells (Figure 6C). Figure 6D shows the
effect of the HH ligand–binding Ab 5E1 and a combination of SMO
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
BIOMARKERS FOR HH-INHIBITOR RESPONSIVENESS IN CLL
1005
Figure 6. Stroma-induced SMO inhibitor resistance mediated by noncanonical HH signaling can be overcome by the HH-blocking Ab 5E1 or ERK inhibitors.
(A) Percentage of viable CLL cells compared with the DMSO control after 48 hours of treatment with the SMO inhibitor cyclopamine or the HH ligand–blocking Ab 5E1 or a
combination of cyclopamine and the 5E1 Ab. The HH-blocking Ab 5E1 overcomes SMO inhibitor resistance induced by stromal cells (patient 1). (B) Percentage of viable cells
after treatment of primary CLL cells with the SMO inhibitor cyclopamine and the HH-blocking Ab for 48 hours compared with control. Although in both karyotypes SMO inhibitor
treatment with 10␮M cyclopamine cannot significantly reduce viable cells (trisomy 12: n ⫽ 4; P ⫽ .1539, Student t test; del 13q14: n ⫽ 3; P ⫽ .0799, Student t test), the
HH-blocking Ab 5E1 can significantly reduce the percentage of viable cells (trisomy 12: n ⫽ 4; 20 ␮g/mL 5E1 P ⫽ .0131, Student t test; Del 13q14: 40 ␮g/mL; n ⫽ 3;
P ⫽ .0075, Student t test; patients 1, 7, 18, 50, 60, 66, 68, 69). (C) Numbers of viable CLL cells compared with the DMSO control in cells treated with cyclopamine or the ERK
inhibitor U0126 or a combination of both for 48 hours and measured by flow cytometry after annexin V/7-AAD staining. Combination of SMO and ERK inhibitors shows a
significant increase in apoptosis compared with single treatment alone (n ⫽ 7; P ⫽ .0007, Student t test). (D) Diagram shows the interaction between stromal cells and
CLL cells and changes in canonical and noncanonical HH signaling on treatment with the HH ligand–blocking Ab 5E1 (left) or a combination of SMO and ERK inhibitors (right).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1006
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
DECKER et al
and ERK inhibitors on noncanonical and canonical HH signaling in
CLL cells and stromal cells.
Discussion
Previous publications showed contrary results about the role of HH
signaling in CLL. Hedge et al reported dependency of all CLL cells
on the presence of the HH-signaling pathway on the level of
SMO.42 In contrast to those results, Desch et al described no
dependency on the SMO receptor but dependency on the GLI1
transcription factor.43 Therefore, the aim of our study was to
identify biomarkers or clinical parameters that can discriminate
between SMO inhibitor responsive and nonresponsive CLLs.
Because the SMO inhibitor cyclopamine is not completely specific
for the HH-signaling pathway, we added 2 more specific and more
potent SMO inhibitors, NVP-LDE22547 and IPI-92648,49 to our
assays, which are already in clinical trials and therefore might be
more relevant for future implications. We identified 3 different
response groups in our CLL cohort with 40% of CLL cells showing
no response to any of the inhibitors and 36.7% of the samples with
good response to SMO inhibitors. The intermediate response group
showed only responses to 1 or 2 of the SMO inhibitors, and,
because this group does not have elevated GLI1 and PTCH1
transcript levels, this might be unspecific effects.
We identified several biomarkers, which can discriminate
between the good response group and the intermediate/nonresponder group. An increase in GLI1 and PTCH1 transcript levels
⬎ 3-fold over the median of all samples is highly predictive for a
good response to SMO inhibitors. Furthermore, all patients with
trisomy 12 showed HH pathway activation measured by GLI1
transcript levels and showed high responsiveness to SMO inhibitor
treatment. In addition, DHH was overexpressed in all patients with
trisomy 12, another biomarker for SMO inhibitor responsiveness.
Treatment of trisomy 12 patient samples in the absence of stromal
cells with the HH ligand–blocking Ab 5E1 induced apoptosis rates
up to 90%, indicating the importance of this autocrine HH pathway
activation loop for the survival of this CLL subtype. However,
treatment of del 13q14 patient samples with the 5E1 Ab did not
induce any apoptosis in contrast to SMO inhibitor treatment,
indicating a nonautocrine HH pathway activation mechanism in
those samples. Interestingly, the gene locus for DHH and GLI1 is
present on chromosome 12. The pathomechanism by which
trisomy 12 influences CLL development is currently not understood, but according to our results overactivation of HH signaling
might be one of the mechanisms involved in this process.
Another interesting finding is the SMO inhibitor protective
effect of murine or human stromal cells on CLL cells (Figure 5A).
In addition, stromal cells possess an intact HH-signaling pathway
and SMO inhibitor treatment reduces stromal Gli1 levels but
simultaneously increases Dhh levels. Therefore, high amounts of
paracrine Dhh continue to stimulate the PTCH1 receptor in
CLL cells and support noncanonical activation of ERK directly
downstream of the PTCH1 receptor. The fact that SMO inhibitors
cannot reduce the stroma-induced ERK activation indicates that
ERK activation is not dependent on SMO signaling. In addition, the
finding that the DHH-blocking Ab 5E1 can completely overcome
stroma-mediated resistance to SMO inhibitors and abolishes stromainduced ERK phosphorylation argues for the stroma-dependent
Dhh production to be responsible for the resistance process. In
addition, the fact that trisomy 12 patient samples with autocrine
DHH secretion show a higher baseline ERK phosphorylation
compared with patients with del 13q14 without autocrine DHH
production indicate the ERK pathway to be downstream of the
PTCH1 receptor.
Our results in CLL cells are the first to show the existence of
noncanonical HH signaling in cancer cells. Furthermore, they show
a potential resistance mechanism of cancer cells toward SMO
inhibitors induced by stromal cells. The in vitro simulation of
stroma-CLL interaction by cocultivation of high amounts of
stromal cells with CLL cells might overestimate the protective
effect of stromal cells on CLL cells, but still gives insights into
important interaction mechanisms. The protective effect might not
be relevant for circulating CLL cells, because they are not in direct
contact with BM stromal cells and might therefore still be
responsive to the treatment with SMO inhibitors alone.
In general, our results argue for further clinical development of
HH ligand–blocking Abs, because they can effectively block
canonical and noncanonical HH signaling and might therefore be
more effective than SMO inhibitors for the treatment of human
CLL or other cancer types with ligand-mediated HH pathway
activation. Regarding CLL, our results argue for 2 different
treatment strategies of patients with CLL according to their genetic
background: patients with trisomy 12 with autocrine HH pathway
activation should be treated with the HH ligand–blocking Abs to
overcome both the autocrine- and stroma-mediated paracrine HH
pathway activation. Other patients with CLL with high GLI1 and
PTCH1 transcript levels, but intrinsic HH pathway activation,
would need a combination treatment of HH-blocking Abs to
overcome the stroma-protective effect and in addition SMO
inhibitors for the intrinsic canonical HH pathway activation.
Further experiments need to prove those concepts in xenograft
models in vivo and could be tested in future clinical trials.
Taken together, our results show that the HH-signaling pathway
is an interesting therapeutic target for a subset of patients with
CLL, characterized by high GLI1 and PTCH1 transcript levels
and/or the presence of trisomy 12 and indicate HH-blocking Abs to
be favorable over SMO inhibitors in overcoming stroma-mediated
protective effects.
Acknowledgments
The authors thank Prof Roland Mertelsmann for helpful advice,
Marie Follo for help with flow cytometry, Novartis for providing
NVP-LDE225, and Infinity Pharmaceuticals for providing IPI-926.
The monoclonal Ab 5E1 developed by Thomas M. Jessell and
Susan Brenner-Morton was obtained from the Developmental
Studies Hybridoma Bank developed under the auspices of the
National Institute of Child Health and Human Development and
maintained by The Department of Biology, University of Iowa.
This work was supported by the Deutsche Krebshilfe Verbund
“Receptor signaling and comparative genomics in CLL” (grant
108935 TP03) and by the “BIOSS Center for Biologic Signaling
Studies” at the University of Freiburg.
Authorship
Contribution: S.D. performed research and wrote the manuscript;
K.Z., L.D., D. Hartmann, and D. Herchenbach performed experiments; G.I. performed the statistical analysis; M.W., H.J., A.S., and
H.V. designed experiments; and C.D. performed and designed
experiments and wrote the paper.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 26 JANUARY 2012 䡠 VOLUME 119, NUMBER 4
BIOMARKERS FOR HH-INHIBITOR RESPONSIVENESS IN CLL
Conflict-of-interest disclosure: M.W. was employed by and
owns stock in a company (Novartis) whose product was studied in
the present work. The remaining authors declare no competing
financial interests.
1007
Correspondence: Christine Dierks, Department of Hematology/
Oncology, University Medical Center Freiburg, Hugstetter Str
55, 79106 Freiburg, Germany; e-mail: christine.dierks@
uniklinik-freiburg.de.
References
1. Munk Pedersen I, Reed J. Microenvironmental
interactions and survival of CLL B-cells. Leuk
Lymphoma. 2004;45(12):2365-2372.
2. Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med. 2005;352(8):
804-815.
3. Rai KR, Sawitsky A, Cronkite EP, et al. Clinical
staging of chronic lymphocytic leukemia. Blood.
1975;46(2):219-234.
4. Binet JL, Auquier A, Dighiero G, et al. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis.
Cancer. 1981;48(1):198-206.
5. Damle RN, Wasil T, Fais F, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.
Blood. 1999;94(6):1840-1847.
6. Wiestner A, Rosenwald A, Barry TS, et al.
Zap-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood. 2003;
101(12):4944-4951.
7. Rassenti LZ, Huynh L, Toy TL, et al. Zap-70 compared with immunoglobulin heavy-chain gene
mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl
J Med. 2004;351(9):893-901.
8. Döhner H, Stilgenbauer S, Benner A, et al.
Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343(26):
1910-1916.
9. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic
lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer InstituteWorking Group 1996 guidelines. Blood. 2008;
111(12):5446-5456.
10. Hallek M. Therapy of chronic lymphocytic leukaemia. Best Pract Res Clin Haematol. 2010;23(1):
85-96.
11. Gribben JG, Zahrieh D, Stephans K, et al. Autologous and allogeneic stem cell transplantations for
poor-risk chronic lymphocytic leukemia. Blood.
2005;106(13):4389-4396.
12. Brandt L. Environmental factors and leukaemia.
Med Oncol Tumor Pharmacother. 1985;2(1):7-10.
13. Burger M, Hartmann T, Krome M, et al. Small
peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in
chronic lymphocytic leukemia B cells. Blood.
2005;106(5):1824-1830.
14. Pleyer L, Egle A, Hartmann TN, Greil R. Molecular and cellular mechanisms of CLL: novel therapeutic approaches. Nat Rev Clin Oncol. 2009;
6(7):405-418.
15. Burger JA, Tsukada N, Burger M, et al. Bloodderived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood.
2000;96(8):2655-2663.
16. Dierks C, Grbic J, Zirlik K, et al. Essential role of
stromally induced hedgehog signaling in B-cell
malignancies. Nat Med. 2007;13(8):944-951.
17. Ingham PW, McMahon AP. Hedgehog signaling in
animal development: paradigms and principles.
Genes Dev. 2001;15(23):3059-3087.
18. Humke EW, Dorn KV, Milenkovic L, Scott MP,
Rohatgi R. The output of hedgehog signaling is
controlled by the dynamic association between
suppressor of fused and the Gli proteins. Genes
Dev. 2010;24(7):670-682.
19. Duman-Scheel M, Weng L, Xin S, Du W. Hedgehog regulates cell growth and proliferation by inducing cyclin D and cyclin E. Nature. 2002;
417(6886):299-304.
20. Regl G, Neill GW, Eichberger T, et al. Human
GLI2 and GLI1 are part of a positive feedback
mechanism in basal cell carcinoma. Oncogene.
2002;21(36):5529-5539.
21. Ikram MS, Neill GW, Regl G, et al. GLI2 is expressed in normal human epidermis and BCC
and induces GLI1 expression by binding to its
promoter. J Invest Dermatol. 2004;122(6):15031509.
22. Goodrich LV, Scott MP. Hedgehog and patched in
neural development and disease. Neuron. 1998;
21(6):1243-1257.
23. Berman DM, Karhadkar SS, Hallahan AR, et al.
Medulloblastoma growth inhibition by hedgehog
pathway blockade. Science. 2002;297(5586):
1559-1561.
24. Oro AE, Higgins KM, Hu Z, et al. Basal cell carcinomas in mice overexpressing sonic hedgehog.
Science. 1997;276(5313):817-821.
25. Xie J, Murone M, Luoh S, et al. Activating
smoothened mutations in sporadic basal-cell
carcinoma. Nature. 1998;391(6662):90-92.
26. Aszterbaum M, Beech J, Epstein EH. Ultraviolet
radiation mutagenesis of hedgehog pathway
genes in basal cell carcinomas. J Investig Dermatol Symp Proc. 1999;4(1):41-45.
27. Berman DM, Karhadkar SS, Maitra A, et al. Widespread requirement for hedgehog ligand stimulation in growth of digestive tract tumours. Nature.
2003;425(6960):846-851.
28. Karhadkar SS, Bova GS, Abdallah N, et al.
Hedgehog signalling in prostate regeneration,
neoplasia and metastasis. Nature. 2004;
431(7009):707-712.
29. Sheng T, Li C, Zhang X, et al. Activation of the
hedgehog pathway in advanced prostate cancer.
Mol Cancer. 2004;3:29.
30. Thayer SP, di Magliano MP, Heiser PW, et al.
Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature. 2003;
425(6960):851-856.
31. Feldmann G, Dhara S, Fendrich V, et al. Blockade of hedgehog signaling inhibits pancreatic
cancer invasion and metastases: a new paradigm
for combination therapy in solid cancers. Cancer
Res. 2007;67(5):2187-2196.
32. Watkins DN, Berman DM, Burkholder SG, et al.
Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature.
2003;422(6929):313-317.
33. Chang H, Li Q, Moraes RC, Lewis MT, Hamel PA.
Activation of Erk by sonic hedgehog independent
of canonical hedgehog signalling. Int J Biochem
Cell Biol. 2010;42(9):1462-1471.
34. Dierks C, Beigi R, Guo G, et al. Expansion of BcrAbl-positive leukemic stem cells is dependent on
hedgehog pathway activation. Cancer Cell. 2008;
14(3):238-249.
35. Zhao C, Chen A, Jamieson CH, et al. Hedgehog
signalling is essential for maintenance of cancer
stem cells in myeloid leukaemia. Nature. 2009;
458(7239):776-779.
36. Gao J, Graves S, Koch U, et al. Hedgehog signaling is dispensable for adult hematopoietic stem
cell function. Cell Stem Cell. 2009;4(6):548-558.
37. Hofmann I, Stover EH, Cullen DE, et al. Hedgehog signaling is dispensable for adult murine
hematopoietic stem cell function and hematopoiesis. Cell Stem Cell. 2009;4(6):559-567.
38. Singh RR, Kim JE, Davuluri Y, et al. Hedgehog
signaling pathway is activated in diffuse large
B-cell lymphoma and contributes to tumor cell
survival and proliferation. Leukemia. 2010;24(5):
1025-1036.
39. Singh RR, Cho-Vega JH, Davuluri Y, et al. Sonic
hedgehog signaling pathway is activated in ALKpositive anaplastic large cell lymphoma. Cancer
Res. 2009;69(6):2550-2558.
40. Hegde GV, Munger CM, Emanuel K, et al. Targeting of sonic hedgehog-gli signaling: a potential
strategy to improve therapy for mantle cell lymphoma. Mol Cancer Ther. 2008;7(6):1450-1460.
41. Peacock CD, Wang Q, Gesell GS, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc Natl Acad Sci
U S A. 2007;104(10):4048-4053.
42. Hegde GV, Peterson KJ, Emanuel K, et al.
Hedgehog-induced survival of B-cell chronic lymphocytic leukemia cells in a stromal cell microenvironment: a potential new therapeutic target. Mol
Cancer Res. 2008;6(12):1928-1936.
43. Desch P, Asslaber D, Kern D, et al. Inhibition of
GLI, but not smoothened, induces apoptosis in
chronic lymphocytic leukemia cells. Oncogene.
2010;29(35):4885-4895.
44. Pfeifer D, Pantic M, Skatulla I, et al. Genomewide analysis of DNA copy number changes and
LOH in CLL using high-density SNP arrays.
Blood. 2007;109(3):1202-1210.
45. Incardona JP, Gaffield W, Kapur RP, Roelink H.
The teratogenic veratrum alkaloid cyclopamine
inhibits sonic hedgehog signal transduction.
Development. 1998;125(18):3553-3562.
46. Taipale J, Chen JK, Cooper MK, et al. Effects of
oncogenic mutations in smoothened and patched
can be reversed by cyclopamine. Nature. 2000;
406(6799):1005-1009.
47. Buonamici S, Williams J, Morrissey M, et al. Interfering with resistance to smoothened antagonists
by inhibition of the PI3K pathway in medulloblastoma. Sci Transl Med. 2010;2(51):51ra70.
48. Tremblay MR, Lescarbeau A, Grogan MJ, et al.
Discovery of a potent and orally active hedgehog
pathway antagonist (IPI-926). J Med Chem.
2009;52(14):4400-4418.
49. Lin TL, Wang QH, Brown P, et al. Self-renewal of
acute lymphocytic leukemia cells is limited by the
hedgehog pathway inhibitors cyclopamine and
IPI-926. PLoS One. 2010;5(12):e15262.
50. Ericson J, Morton S, Kawakami A, Roelink H,
Jessell TM. Two critical periods of sonic hedgehog signaling required for the specification of motor neuron identity. Cell. 1996;87(4):661-673.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2012 119: 997-1007
doi:10.1182/blood-2011-06-359075 originally published
online November 30, 2011
Trisomy 12 and elevated GLI1 and PTCH1 transcript levels are
biomarkers for Hedgehog-inhibitor responsiveness in CLL
Sarah Decker, Katja Zirlik, Lauritte Djebatchie, David Hartmann, Gabriele Ihorst, Annette
Schmitt-Graeff, Dieter Herchenbach, Hassan Jumaa, Markus Warmuth, Hendrik Veelken and
Christine Dierks
Updated information and services can be found at:
http://www.bloodjournal.org/content/119/4/997.full.html
Articles on similar topics can be found in the following Blood collections
Lymphoid Neoplasia (2562 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.