In vitro validation of γγ -secretase inhibitors alone or in ...

Original Article
In vitro validation of γ-secretase inhibitors alone or
in combination with other anti-cancer drugs for
the treatment of T-cell acute lymphoblastic leukemia
Kim De Keersmaecker,1,2 Idoya Lahortiga,1,2,3 Nicole Mentens,1,2 Cedric Folens,1,2
Leander Van Neste,4 Sofie Bekaert,4 Peter Vandenberghe,5 Maria D. Odero,3 Peter Marynen,1,2
and Jan Cools1,2
1Human Genome Laboratory, Department of Molecular and Developmental Genetics, VIB, Leuven, Belgium; 2Human
Genome Laboratory, Center for Human Genetics, K.U. Leuven, Leuven, Belgium; 3Division of Oncology, Center for
Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain; 4Department of Molecular Biotechnology,
Faculty for Bioscience Engineering, Ghent University, Belgium; 5Department of Human Genetics, K.U. Leuven,
Leuven, Belgium
Funding: this work was supported by
ABSTRACT
grant SCIE2006-34 from the Belgian
Federation against Cancer (JC),
grants G.0287.07 (JC) and
Background
G.0507.04 (PM) of the ‘Fonds voor
Activating NOTCH1 mutations are common in T-cell acute lymphoblastic leukemia. Inhibition of
Wetenschappelijk Onderzoek-
NOTCH1 signaling with γ-secretase inhibitors causes cell cycle block, but only after treatment for
Vlaanderen’, a concerted action
several days. We further documented the effects of γ-secretase inhibitor treatment on T-cell acute
grant from the K.U.Leuven (PM, JC,
lymphoblastic leukemia cell lines and tested whether combining γ-secretase inhibitors with other
PV), and the Interuniversity Attraction
anti-cancer drugs offers a therapeutic advantage.
Poles (IAP) granted by the Federal
Office for Scientific, Technical and
Design and Methods
Cultural Affairs, Belgium
(JC, PM, PV, MO). KDK is an
The effect of γ-secretase inhibitor treatment and combinations of γ-secretase inhibitors with
‘Aspirant’, JC a postdoctoral
chemotherapy or glucocorticoids was assessed on T-cell acute lymphoblastic leukemia cell lines.
researcher, and PV a senior clinical
We sequenced NOTCH1 in T-cell acute lymphoblastic leukemia cases with ABL1 fusions and test-
researcher of the ‘Fonds voor
ed combinations of γ-secretase inhibitors and the ABL1 inhibitor imatinib in a T-cell acute lym-
Wetenschappelijk Onderzoek-
phoblastic leukemia cell line.
Vlaanderen’. IL is a postdoctoral
researcher of the KU Leuven.
Results
γ-secretase inhibitor treatment for 5-7 days reversibly inhibited cell proliferation, caused cell
Manuscript received June 26, 2007.
cycle block in sensitive T-cell acute lymphoblastic leukemia cell lines, and caused differentiation
Revised version arrived on November
of some T-cell acute lymphoblastic leukemia cell lines. Treatment for 14 days or longer was
9, 2007. Manuscript accepted
required to induce significant apoptosis. The cytotoxic effects of the chemotherapeutic agent vin-
December 5, 2007.
cristine were not significantly enhanced by addition of γ-secretase inhibitors to T-cell acute lym-
phoblastic leukemia cell lines, but γ-secretase inhibitor treatment sensitized cells to the effect
Correspondence:
of dexamethasone. NOTCH1 mutations were identified in all T-cell acute lymphoblastic leukemia
Jan Cools, Campus Gasthuisberg
patients with ABL1 fusions and in a T-cell acute lymphoblastic leukemia cell line expressing
O&N1, Herestraat 49 (box 602),
NUP214-ABL1. In this cell line, the anti-proliferative effect of imatinib was increased by pre-treat-
B-3000 Leuven, Belgium.
ment with γ-secretase inhibitors.
E-mail: jan.cools@med.kuleuven.be
Conclusions
The online version of this article con-
tains a supplemental appendix.
Short-term treatment of T-cell acute lymphoblastic leukemia cell lines with γ-secretase inhibitors
had limited effects on cell proliferation and survival. Combinations of γ-secretase inhibitors with
other drugs may be required to obtain efficient therapeutic effects in T-cell acute lymphoblastic
leukemia, and not all combinations may be useful.
Key words: NOTCH1, γ-secretase, inhibitor, T-cell, oncogene.
Citation: De Keersmaecker K, Lahortiga I, Mentens N, Folens C, Van Neste L, Bekaert S,
Vandenberghe P, Odero MD, Marynen P, and Cools J. In vitro validation of γ-secretase inhibitors
alone or in combination with other anti-cancer drugs for treatment of T-cell acute lymphoblastic
leukemia. Haematologica 2008 Apr;93(4):533-542. doi: 10.3324/haematol.11894
©2008 Ferrata Storti Foundation. This is an open-access paper.
haematologica | 2008; 93(4) | 533 |

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K. De Keersmaecker et al.
NOTCH1 still requires γ-secretase activity to generate
Introduction
critical downstream signals.8 Because of the involve-
ment of γ-secretase in the production of amyloidogenic
T-cell acute lymphoblastic leukemia (T-ALL) is associ-
peptides in Alzheimer’s disease, efforts have already
ated with several molecular defects including deregulat-
been made to develop potent and selective γ-secretase
ed expression of a variety of transcription factors such
inhibitors. Unfortunately, long-term therapy of
as TLX1, TLX3, TAL1, LYL1, and LMO1, deletion of
Alzheimer’s disease with -secretase inhibitors (GSI)
CDKN2A (p16), the episomal NUP214-ABL1 fusion,
does not seem appropriate because of the side effects of
duplication of the MYB gene and mutation of PTEN and
the current inhibitors, such as disturbance of lympho-
FBXW7 genes.1-7 In addition, activating mutations in
cyte development and gut epithelial cell differentia-
NOTCH1 were identified in more than 50% of T-ALL
tion.18 In T-ALL these compounds could provide a
patients.8-10
rational, molecularly targeted therapy with an accept-
The mature NOTCH1 receptor consists of an extra-
able level of toxicity, but this still needs to be investigat-
cellular and a transmembrane subunit, which are non-
ed in clinical trials.18
covalently kept together by the heterodimerization
The first proof of principle of the potential of GSI in the
domain.11,12 Binding of delta-serrate-lag2 (DSL) family
context of T-ALL was provided by the observation that
ligands to the extracellular subunit results in activation
five out of 30 tested T-ALL cell lines went into G0/G1 cell
of the NOTCH1 receptor by removal of the extracellu-
cycle arrest after 4 to 8 days of γ-secretase inhibition.8
lar subunit and by initiating a cascade of proteolytic
Several studies confirmed these initial observations and
cleavages of the transmembrane subunit. The final
described induction of cell cycle arrest and reduced cell
cleavage is catalyzed by the γ-secretase complex and
proliferation after treating sensitive NOTCH1 mutation-
generates active intracellular NOTCH1. This intracellu-
positive T-ALL cell lines for several days with GSI.15,19-21 In
lar NOTCH1 subsequently translocates to the nucleus
this work, we further document the short- and long-term
where it associates with other proteins to form a tran-
effects of GSI on the growth of T-ALL cell lines and test-
scription activator complex. Actived intracellular
ed whether combinations of GSI with other anti-cancer
NOTCH1 has a short half-life, being subject to ubiqui-
agents could offer any therapeutic advantage over single
tination and degradation via mechanisms involving the
agent therapy.
C-terminal proline, glutamate, serine and threonine
(PEST) domain.13
The heterodimerization domain of NOTCH1 is
Design and Methods
mutated in 29-44% of T-ALL samples, causing destabi-
lization of the association between the NOTCH1 extra-
NOTCH1 mutation detection
cellular and transmembrane subunits, resulting in
Genomic DNA was prepared from T-ALL cell lines
increased NOTCH1 rates of activated intracellular
and samples from patients with ABL1 fusion positive T-
NOTCH1 production in the absence of ligand stimula-
ALL using the Wizard genomic DNA purification kit
tion.5-7 In addition, 5% to 30% of the tumors display
(Promega, Madison, WI, USA). The detection of mutat-
PEST domain mutations, which prolongs the half-life of
ed NOTCH1 sequences encoding the heterodimezation
the intracellular NOTCH1-containing transcriptional
domain and PEST domains was described previously.22
activation complex. Combined hetero-dimerization
This study was approved by the Ethical Committee of
domain and PEST domain mutations cause synergistic
the Medical Faculty of the University of Leuven.
activation of NOTCH1 signaling pathways and are
Informed consent was obtained from all subjects.
observed in 1 to 18% of analyzed tumors.5-7 Apart from
mutations in the heterodimerization and PEST domains
Cell cultures
of NOTCH1, rare mutations in the NOTCH1 transacti-
DND-41, HSB-2, RPMI-8402, KARPAS-45, ALL-SIL,
vation domain and in its ankyrin repeat regions have
MOLT-4, LOUCY (DSMZ, Braunschweig, Germany)
been described.10
and JURKAT (ATCC CRL 8163) cells were cultured in
The discovery of these very frequent NOTCH1 muta-
RPMI-1640 medium supplemented with 20% fetal calf
tions in T-ALL instigated several research groups to elu-
serum. In order to assess cell growth, 0.4×106 cells/mL
cidate the signaling network downstream of NOTCH1
were seeded in triplicate and different concentrations of
in the context of T-ALL. These studies revealed impor-
GSI were added. After 2 to 3 days, the cells were sub-
tant connections between NOTCH1 and MYC, NFKB
cultured by centrifugation and resuspension in fresh
and the PI3K/AKT signaling pathways.5,14-16
medium containing a GSI. Viable cells were counted at
At the moment, up to 75% of T-ALL patients are
various time points on a Vi-cell XR cell viability analyz-
cured by very intensive chemotherapy;17 however, less
er (Beckman Coulter, Fullerton, CA, USA). In order to
toxic, more efficacious drug combinations are desirable.
determine dose-response curves, 0.4×106 cells were
The discovery of frequent NOTCH1 mutations in T-
seeded in 1 mL medium containing inhibitors and were
ALL has important therapeutic implications. Mutant
incubated for 24 to 72 h, depending on the doubling
| 534 | haematologica | 2008; 93(4)

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γ-secretase inhibitors for T-ALL treatment
time of the cell lines. Compound E (γ-secretase inhibitor
oxidase-labeled antibodies (AP Biotech, Uppsala,
XXI), L-685458 (γ-secretase inhibitor X), vincristine,
Sweden).
daunorubicin, dexamethasone and corticosterone were
obtained from Calbiochem (San Diego, CA, USA).
Fluorescence activated cell sorting analysis
Quantification of apoptosis, analysis of CD3, CD4
Western blotting
and CD8 expression and cell cycle analysis were per-
Total cell lysates were analyzed by standard proce-
formed on 0.5-1×106 cells after 2-3, 7 and 14 days of
dures using the following antibodies: anti-cleaved
treatment with 0.1 or 1 µM of Compound E, using the
NOTCH1 (Val1744), (Cell Signaling, Beverly, MA,
Annexin-V-FLUOS Staining kit (Roche, Penzberg,
USA), anti-NOTCH1 (C-20) (Santa Cruz Biotechnology,
Germany), the TriTEST CD4 FITC/CD8 PE/CD3 PerCP
Santa Cruz, CA, USA) and anti-mouse/anti-rabbit per-
Reagent kit (Becton Dickinson, San Jose, CA, USA) and
A
B
T
T
T + E
AS-45
AS-45AS-45 + E
T-4
T-4 T-4 + E
kDa
DND-41HSB-2RPMI-8402
JURKA
KARP ALL-SIL
LOUCY
kDa
DND-41
DND-41 + E
HSB-2HSB-2 + E
RPMI-8402
RPMI-8402 + E
JURKAJURKAKARP KARPALL-SILALL-SIL + E
MOL
MOL MOL LOUCYLOUCY + E
116
116
97
97
anticleaved NOTCH1 (Val1744)
anticleaved NOTCH1 (Val1744)
290
290
240
240
160
160
116
116
97
97
anti-NOTCH1
anti-NOTCH1
C
DND-41
ALL-SIL
RPMI-8402
200
50
0 µM Compound E
300
160
0,1 µM Compound E
40
)
)
)
6
1 µM Compound E
6
6
120
30
200
80
20
cells (x 10
cells (x 10
cells (x 10 100
40
10
Viable
Viable
Viable
0
0
0
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
Period of Compound E treatment (Days)
Period of Compound E treatment (Days)
Period of Compound E treatment (Days)
JURKAT
KARPAS-45
HSB-2
800
)
)
)
6
6000
6
80
6
600
60
4000
cells (x 10
cells (x 10
cells (x 10 400
40
2000
Viable
Viable
Viable 200
20
0
0
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
Period of Compound E treatment (Days)
Period of Compound E treatment (Days)
Period of Compound E treatment (Days)
400
MOLT-4
50
LOUCY
)
)
40
6
300
6
30
200
cells (x 10
cells (x 10
20
100
Viable
Viable
10
0
0
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
Period of Compound E treatment (Days)
Period of Compound E treatment (Days)
Figure 1. Inhibition of hyperactive NOTCH1 signaling in T-ALL cell lines by γ-secretase inhibition affects cell proliferation. (A) The activity
of the NOTCH1 pathway in T-ALL cell lines was evaluated by western blot detection on whole cell lysates with anti-cleaved NOTCH1
(Val1744) antibody followed by stripping and reprobing the blot with anti-NOTCH1 antibody. (B) Western blot detection of active NOTCH1
(anti-cleaved NOTCH1 (Val1744) antibody) in T-ALL cell lines that were treated for 2 days with DMSO or with 1 µM Compound E. The blot
was stripped and reprobed with anti-NOTCH1. (C) The indicated T-ALL cell lines were grown in the presence of DMSO, 0.1 or 1 µM
Compound E and the total number of viable cells in the culture was measured on the Vi-cell XR cell viability analyzer at the indicated
time points.
haematologica | 2008; 93(4) | 535 |

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K. De Keersmaecker et al.
sion of CD4, CD8 and CD3 was evaluated in the gated
population of viable cells.
Statistics
The t-test was used to evaluate the differences in effect
between GSI treated and untreated samples for each test-
ed concentration of imatinib, vincristine or glucocorti-
coid. A p-value of 0.05 was used as the cut-off below
which results were considered statistically significant.
Results
γ-secretase inhibitor treatment reversibly inhibits the
growth of T-ALL cell lines and induces cell cycle block
We monitored the effect of continuous treatment
with a GSI as a single agent on the proliferation of eight
T-ALL cell lines: DND-41, HSB-2, RPMI-8402, JURKAT,
KARPAS-45, ALL-SIL, MOLT-4 and LOUCY. DNA
sequencing confirmed the presence of NOTCH1 muta-
tions in DND-41, RPMI-8402, KARPAS-45, ALL-SIL,
and MOLT-4 (Online Supplementary Table S1). No muta-
tions were found in HSB-2, JURKAT and LOUCY.
Western blot analysis showed detectable levels of γ-sec-
retase cleaved, activated intracellular NOTCH1 in the
cell lines with NOTCH1 mutations (Figure 1A). In addi-
tion, we detected activate intracellular NOTCH1 in
JURKAT cells, suggesting that the JURKAT cells harbor
a NOTCH1 mutation outside the domains we
sequenced or in another gene that would result in aber-
rant NOTCH1 activation.10 Treatment of the cells with
1 µM Compound E, a non-transition state analog GSI,
resulted in a strong reduction of detected levels of acti-
vated intracellular NOTCH1, confirming that NOTCH1
Figure 2. γ-secretase inhibitor-induced inhibition of proliferation and
cleavage was efficiently inhibited by Compound E
cell cycle block is reversible. A. ALL-SIL, DND-41, RPMI-8402 and
LOUCY cells were pre-treated for 7 or 14 days with 1 µM of
(Figure 1B).
Compound E. On day 7 (left column) or 14 (right column) cells were
We next determined the effect of continuous
washed; for half of the cells the treatment with 1 µM Compound E
was continued, while the other half was treated with DMSO for the
Compound E treatment of the cell lines during 14 days
remaining days of the experiment. The graphs represent the num-
(Figure 1C). At concentrations of 0.1 µM and 1 µM
ber of cells in each treatment group, day 0 on the graphs corre-
sponds to the day Compound E was washed away after the pre-
Compound E, inhibition of proliferation was clearly
treatment period. B. The reversibility of the G0/G1 cell cycle block
observed in four of the six cell lines with detectable
after 7 days of pre-treatment with 1 µM Compound E was evaluat-
ed by propidium iodide staining of DNA content of the cells. The
NOTCH1 activation (DND-41, RPMI-8402, KARPAS-45
experiment was performed 9 days after stopping the pre-treatment
and ALL-SIL) (Figure 1C). The period required to obtain
with Compound E. The upper row of graphs correspond to the cells
that were treated with DMSO after the Compound E pre-treatment,
a 50% inhibition of growth varied between 7 to 18 days
the lower row corresponds to the cells that continued to be exposed
with 0.1 µM Compound E and between 7 to 12 days
to Compound E after the pre-treatment period. The percentages of
cells in G0+G1 phase (left peak in the graphs) and in S + M + G2
with 1 µM Compound E. Although the HSB-2 cell line
phase are indicated in the graphs.
did not have a mutation in NOTCH1, nor did it have
detectable NOTCH1 activation by western blot analy-
the CycleTEST™ PLUS DNA Reagent Kit (Becton
sis, the proliferation of this cell line was also inhibited
Dickinson), respectively. For some cell lines staining to
by GSI treatment (Figure 1C). MOLT-4 cells and
assess apoptosis was also done after 20 and 28 days of
JURKAT cells were not sensitive to GSI treatment
treatment, as indicated in Online Supplementary Figure
although activated NOTCH1 was detectable in these
S1. After staining, cells were detected on a FACSCanto
cell lines. We conclude that MOLT-4 and JURKAT cell
Flow Cytometer (Becton Dickinson) and the data were
lines have become independent of mutant NOTCH1 for
analyzed with the BD FACSDiva software (Becton
their proliferation and cell cycle progression.
Dickinson). Unstained cells and cells treated with
The Compound E-induced inhibition of cell prolifera-
dimethyl sulfoxide were used as controls. The expres-
tion was not immediately associated with increased cell
| 536 | haematologica | 2008; 93(4)

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γ-secretase inhibitors for T-ALL treatment
death, as trypan blue staining and annexin V/propidium
transcriptase (TERT) expression (Online Supplementary
iodide staining did not reveal significant differences in
Figure S2C) or to a shortening of telomeres (Online
the number of dead cells between treated and untreated
Supplementary Figure S2D).
cultures after 7 days of treatment (Online Supplementary
Figure S1). We found that continuous GSI treatment for
Gamma-secretase inhibitor treatment can induce
14 days or longer was required to induce significant
differentiation of T-ALL cell lines
apoptosis in the GSI-sensitive cell lines. In RPMI-8402
Aberrant activation of NOTCH1 is known to affect T-
cultures, 30% apoptotic cells and 15% dead cells were
cell differentiation.23 The effect of GSI treatment on dif-
detected after 14 days of gamma-secretase inhibition
ferentiation of the T-ALL cell lines was tested by deter-
and this effect became even more pronounced after
mining expression of the cell-surface antigens CD3,
longer treatment. The other GSI-sensitive cell lines had
CD4 and CD8. GSI treatment induced differentiation of
to be treated for more than 14 days before apoptosis
the MOLT-4 cell line from CD4–/CD8+/– to CD4+/CD8+
became evident (Online Supplementary Figure S1).
(Figure 3). Changes in CD3 expression became apparent
It was previously reported that γ-secretase inhibition
for the DND-41 and RPMI-8402 cells, which showed a
results in G0/G1 cell cycle arrest in some T-ALL cell lines
change from CD3+/- to CD3– and from CD3– to CD3+,
after 4 to 8 days of treatment.8,15,20,21 By flow cytometric
respectively (Figure 3). For the ALL-SIL cell line, GSI
analysis of the DNA content of propidium iodide stained
cell populations, we confirmed induction of G0/G1 cell
DMSO
1µM Compound E
cycle arrest after 7 days of GSI treatment in all cell lines
6%
80%
that expressed mutant NOTCH1 and displayed a GSI
induced proliferation defect (data not shown).
MOLT-4
To investigate whether the GSI-induced proliferation
and cell cycle defects were reversible, Compound E was
removed after 7 or 14 days of treatment for the GSI-sen-
CD8
CD8
CD4
CD4
sitive cell lines ALL-SIL, DND-41 and RPMI-8402. For
ALL-SIL and DND-41, the inhibition of proliferation
44%
13%
was reversible when the GSI-treatment was stopped
(Figure 2A). For the RPMI-8402 cell line, the effect of
DND-41
treatment for 7 days was reversible, but cell growth did
not recover when the GSI was removed after 14 days
(Figure 2A). As we found that induction of apoptosis
Counts
Counts
CD3
CD3
started in RPMI-8402 when the cells were treated for 14
days (Online supplementary Figure S1), these results indi-
2%
79%
cate that GSI treatment does not cause irreversible
changes in the cells as long as apoptosis is not induced.
RPMI-8402
Recovery of proliferation upon removal of the GSI was
associated with restoration of the cell cycle (Figure 2B).
Counts
Counts
Telomerase activity of untreated and GSI-treated cells
CD3
CD3
was compared in a telomeric repeat amplification proto-
col (TRAP) assay. In this assay, active telomerase in a
98%
86%
cell lysate causes extension of a synthetic primer with
the TTAGGG telomere repeat sequence after which this
ALL-SIL
mixture of elongated products is amplified by poly-
merase chain reaction. The amount of generated poly-
Counts
Counts
merase chain reaction product can then be used as a
CD8
CD8
measure for telomerase activity and can be compared
between samples by gel electrophoresis or by ELISA.
47%
65%
We found that GSI treatment for 12 days reduced
telomerase activity in DND-41, ALL-SIL and RPMI-
KARPAS-45
8402, three cell lines showing proliferation defects upon
GSI treatment (Online Supplementary Figures S2A-B).
Counts
Counts
This decrease was, to a lesser extent, also detectable
CD4
CD4
after 2 and 6 days of treatment (data not shown). In the
remaining cell lines, telomerase activity was absent
Figure 3. Treatment with γ-secretase inhibitors can cause changes
in CD4/CD8/CD3 expression in T-ALL cell lines. FACS comparison
(KARPAS-45) or not significantly changed by GSI treat-
of cell surface staining for CD4/CD8 (MOLT-4), CD3 (DND-41 and
ment. However, we could not correlate this reduction in
RPMI-8402), CD8 (ALL-SIL) or CD4 (KARPAS-45) in cells treated
for 14 days with DMSO or with 1 µM of Compound E.
telomerase activity to a reduction in telomerase reverse
haematologica | 2008; 93(4) | 537 |

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K. De Keersmaecker et al.
treatment decreased CD8 expression and for KARPAS-
erentially associated with NOTCH1 mutations. This
45 cells we detected increased CD4 expression upon
observation raised the question of whether the com-
GSI treatment. GSI treatment did not result in
bined inhibition of the ABL1 fusion protein with ima-
detectable changes in the CD3/CD4/CD8 immunophe-
tinib, and activated NOTCH1 with a GSI could offer a
notypes of the HSB-2, JURKAT and LOUCY cells (data
therapeutic advantage over single agent therapy.
not shown).
We previously showed that the ALL-SIL cell line
expresses, apart from mutant NOTCH1, the NUP214-
Combinations of -secretase inhibitors and kinase
ABL1 fusion tyrosine kinase and that it is sensitive to
inhibitors can offer a therapeutic advantage
imatinib.25 This cell line, therefore, represented an
Results presented in previous reports and our results
experimental model to study the effect of combinations
described above indicate that a GSI as a single agent
of imatinib and GSI. When imatinib and Compound E
may only have modest effects on T-ALL growth and
were added at the same time to ALL-SIL cell cultures,
survival, except when long-term treatment is possi-
the inhibitory effect of imatinib on cell proliferation was
ble.8,15,19-21 We, therefore, investigated whether combin-
antagonized by Compound E (Figure 4A). This antago-
ing GSI with other anti-cancer agents used for T-ALL
nism was also observed for the combination of imatinib
treatment could offer a therapeutic advantage over sin-
with L-685458, a transition state analog inhibitor of γ-
gle agent therapy.
secretase with a different structure from that of
ABL1 fusions are present in 8% of T-ALL cases and
Compound E (data not shown). These data indicate that
are currently the most frequent known mutations pro-
this antagonism is not restricted to a combination of
viding the cells with proliferation and survival advan-
imatinib and Compound E, but that it is also observed
tages.1 We sequenced NOTCH1 in one T-ALL patient
with a structurally different GSI. It was shown that γ-
with an EML1-ABL1 fusion gene, and in five T-ALL
secretase can modulate the activity and trafficking of
patients with the NUP214-ABL1 episomal fusion.24,25
transporter molecules.26,27 We hypothesized that the
NOTCH1 was mutated in all six patients (two patients
antagonism between imatinib and the GSI could be due
with a truncating PEST domain mutation, four patients
to a decreased influx of imatinib into the cells. However,
with a hetero-dimerization mutation, Online Supple-
imatinib potently inhibited phosphorylation of
mentary Table S1), indicating that ABL1 fusions are pref-
NUP214-ABL1 in the presence of Compound E, demon-
A
no pre-treatment
B
D
9 days Compund E pre-treatment
+ DMSO
+ 1 µM Compound E
120
120
0 µM Compound E
0 µM Compound E
100
0.1 µM Compound E
imatinib (µM)
0 0.05 0.1 0.5 1 0 0.05 0.1 0.5 1
100
0.1 µM Compound E
1 µM Compound E
anti-P-ABL1
1 µM Compound E
80
80
60
anti-ABL1
60
40
40
relative to confront (%)
Compound E (µM)
0 0.1 1
C
20
relative to confront (%) 20
anti-P-ABL1
0
0
Growth
0
0.05
0.1
0.5
anti-ABL1
Growth
0
0.05
0.1
Imatinib (µM)
Imatinib (µM)
E
12 days Compund E pre-treatment
F
1 day imatinib pre-treatment
G
3 day imatinib pre-treatment
120
100
100
0 µM Compound E
0 µM Compound E
0 µM Compound E
0.1 µM Compound E
0.1 µM Compound E
0.1 µM Compound E
100
80
80
1 µM Compound E
1 µM Compound E
1 µM Compound E
80
60
60
60
40
40
40
relative to confront (%) 20
relative to confront (%) 20
20
0
0
Growth relative to confront (%)
0
Growth
0
0.05
0.1
0.5
0
0.05
0.1
0.5
0
0.05
0.1
Growth
Imatinib (µM)
Imatinib (µM)
Imatinib (µM)
Figure 4. Kinase inhibitors and γ-secretase inhibition can act synergistically or antagonistically, depending on the sequence in which they
are administered. All experiments were performed on the ALL-SIL cell line. The concentrationsof the GSI are indicated by the different col-
ors of the bars. Kinase inhibitor concentrations are indicated on the X-axis. The Y-axis represents the growth relative to DMSO-treated cells.
After the pre-treatment conditions indicated above the graphs, cells were incubated for 3 additional days with indicated combinations of
imatinib and Compound E. After these 3 days, the amount of viable cells was determined on a Vi-cell XR cell viability analyzer and prolif-
eration of treated cells relative to untreated cells was calculated. An asterisk indicates a statistically significant difference between the
groups connected by the line underneath the asterisk. (A) ALL-SIL cells were treated for 3 days with imatinib and Compound E, without pre-
treament. (B) Western blot showing the effect of addition of Compound E on the inhibition of NUP214-ABL1 phosphorylation by imatinib in
the ALL-SIL cell line. The blot was stripped and reprobed with anti-ABL1. (C) The effect of Compound E treatment on NUP214-ABL1 activi-
ty was assessed by detecting NUP214-ABL1 phosphorylation on western blot with anti-phospho-ABL1 antibody after treatment with 0, 0.1µM
or 1 µM Compound E for 2 days. The blot was stripped and reprobed with anti-ABL1. (D, E) ALL-SIL cells were pre-treated for 9 or 12 days
with 0, 0.1µM or 1 µM Compound E. After the pre-treatment, cells were treated for 3 more days with the indicated combinations of ima-
tinib and Compound E. (F, G) ALL-SIL cells were pre-treated for 1 or 3 days with 0.025 µM imatinib. After this pre-treatment, cells were
treated for 3 more days with the indicated combinations of imatinib and Compound E.
| 538 | haematologica | 2008; 93(4)

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γ-secretase inhibitors for T-ALL treatment
strating that intracellular imatinib levels were not
Combining γ-secretase inhibitors with vincristine does
reduced (Figure 4B). The observed antagonism between
not offer a common therapeutic advantage
Compound E and imatinib in ALL-SIL cells was also not
We next tested combinations of the chemotherapeu-
due to a direct increase of NUP214-ABL1 activation by
tic agent vincristine and a GSI. Treatment of T-ALL cell
Compound E, as we could not detect an increase in
lines with vincristine alone resulted in potent inhibition
NUP214-ABL1 phosphorylation upon Compound E
of proliferation at concentrations below 10 nM. In the
treatment (Figure 4C).
ALL-SIL cell line, we tested concomitant treatment with
We next tested whether pre-treatment of the cell lines
Compound E and vincristine without pre-treatment of
for 9 or 12 days with a GSI could sensitize the cells to
the cells (Figure 5A), after pre-treatment with the GSI
imatinib. After 9 days of pre-treatment with Compound
(Figure 5B) and after pre-treatment with vincristine
E, imatinib was added to the Compound E treatment
(Figure 5C). No significant interaction or antagonism
and cells were incubated with this combination of drugs
occurred in ALL-SIL cells, independently of the admin-
for 3 more days. No significant interaction was
istration scheme of the compounds. Combinations of
observed at a concentration of 0.1 µM Compound E,
vincristine and Compound E after pre-treatment with
but 1 µM Compound E sensitized the cells to the effect
the GSI were also tested in DND-41, RPMI-8402, HSB-
of imatinib (Figure 4D). After 12 days of pre-treatment,
2 and KARPAS-45, the four other cell lines that showed
this sensitization was also observed for 0.1 µM
a proliferation defect upon GSI treatment. For RPMI-
Compound E (Figure 4E). This can probably be
8402, significant synergy was observed when combin-
explained by the fact that inhibition of cell proliferation
ing vincristine with 1 µM Compound E, but not when
by 0.1 µM Compound E treatment occurs with a slight
combining it with the lower dose of 0.1 µM. No signif-
delay compared to treatment with the 1 µM concentra-
icant therapeutic advantage of combining the GSI and
tion. We also tested pre-treatment with the kinase
vincristine could be documented in the other cell lines
inhibitor instead of with the GSI. ALL-SIL was pre-
(Figures 5 D-G).
treated for 1 day or 3 days with a low dose of imatinib.
As can be seen in Figure 4 F and G, kinase inhibitor pre-
-secretase inhibitor treatment can sensitize T-ALL
treatment did not abolish the antagonism between the
cells to the effect of dexamethasone
kinase inhibitor and the GSI.
Glucocorticoids have been used for a long time for the
ALL-SIL, no pre-treatment
All-SIL, 9 days Compound E pre-treatment
A
B
C
All-SIL, 3 days Vincristine pre-treatment
0 µM Compound E
0 µM Compound E
0.1 µM Compound E
0.1 µM Compound E
120
120
1 µM Compound E
120
1 µM Compound E
0 µM Compound E
0.1 µM Compound E
100
100
100
1 µM Compound E
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0
0
1
2
0
1
2
0
1
2
Growth relative to control (%)
Growth relative to control (%)
Growth relative to control (%)
Vincristine (nM)
Vincristine (nM)
Vincristine (nM)
D
DND-41, 6 days Compound E pre-treatment
E
DRPMI-8402, 6 days Compound E pre-treatment
F
HSB-2, 5 days Compound E pre-treatment
120
0 µM Compound E
0 µM Compound E
120
120
0 µM Compound E
0.1 µM Compound E
0.1 µM Compound E
100
0.1 µM Compound E
100
100
1 µM Compound E
1 µM Compound E
1 µM Compound E
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0
0
1
2
4
0
1
5
10
0
5
10
20
Growth relative to control (%)
Vincristine (nM)
Growth relative to control (%)
Vincristine (nM)
Growth relative to control (%)
Vincristine (nM)
G
KARPAS-45, 7 days Compound E pre-treatment
Figure 5. Combinations of vincristine chemotherapy and a GSI do not offer any therapeu-
tic advantage. The concentrations of Compound E are indicated by the different colors of
0 µM Compound E
100
0.1 µM Compound E
the bars. Vincristine concentrations are indicated on the X-axis. The asterisk indicates a
1 µM Compound E
statistically significant difference between the groups connected by the line underneath
80
the asterisk. (A) Vincristine and Compound E were added at the same time to ALL-SIL
60
cells, without any pre-treatment of the cells. (B) ALL-SIL cells were pre-treated for 9 days
40
with 0, 0.1 µM or 1 µM Compound E. After this pre-treatment, the cells were treated for 3
20
more days with the indicated combinations of vincristine and Compound E. (C) ALL-SIL
cells were pre-treated for 3 days with 1 nM of vincristine. The pre-treatment was followed
0
by treatment for 3 days with the indicated combinations of vincristine and Compound E.
Growth relative to control (%)
0
1
2,5
(D, E, F, G) DND-41, RPMI-8402, HSB-2 or KARPAS-45 cells were pre-treated for 5, 6 or 7
Vincristine (nM)
days with 0, 0.1µM or 1 µM Compound E. After this period of pre-treatment the cells were
treated for 2 more days with the indicated combinations of vincristine and Compound E.
haematologica | 2008; 93(4) | 539 |

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K. De Keersmaecker et al.
treatment of ALL.28 We, therefore, tested the interaction
Differentiation in T-ALL is also believed to be affected by
between a GSI and the glucocorticoids dexamethasone
the abnormal expression of transcription factors such as
and corticosterone. In ALL-SIL and DND-41, dexam-
TLX1, TLX3, HOXA10, TAL1 and MYB.1,4,30 It is, there-
ethasone treatment showed a weak, but significant
fore, unlikely that GSI treatment alone can completely
inhibitory effect on the proliferation of the cells, which
release the block of differentiation, but this aspect may
was significantly enhanced when cells were pre-treated
also contribute to the therapeutic potential of GSI treat-
for 7 days or 14 days with 1 µM Compound E (Figure
ment. As these GSI-induced differentiation changes also
6). The RPMI-8402 and LOUCY cell lines showed the
occurred in MOLT-4, a cell line expressing mutant
weakest sensitivity to dexamethasone as a single agent,
NOTCH1, but not showing any inhibition of prolifera-
but GSI pre-treatment also made these cell lines more
tion upon GSI treatment, there seems to be no clear cor-
sensitive to the effect of dexamethasone (Figure 6).
relation between differentiation and proliferation effects
Maximal inhibition of proliferation of ALL-SIL, DND-
of GSI treatment.
41, RPMI-8402 and LOUCY cells was already obtained
Similar to previous findings, we observed that not all
with 1 µM of dexamethasone alone or in combination
cell lines with NOTCH1 mutations were sensitive to
with 1 µM Compound E (data not shown). No clear inter-
GSI treatment.8 We observed that JURKAT and MOLT-
actions could be documented for corticosterone combi-
nations (data not shown).
7 Days pre-treatment
14 Days pre-treatment
0 µM Compound E
Discussion
120
120
1 µM Compound E
100
100
The identification and validation of novel molecular
80
80
ALL-SIL
60
60
targets for cancer therapy are important objectives of
40
40
cancer research. The aims are to reduce the toxicity of
20
20
current treatments and to further improve treatment out-
0
0
Growth relative to control (%)
Growth relative to control (%)
0
1
0
1
comes. Mutant NOTCH1 could represent an important
Dexamethasone (µM)
Dexamethasone (µM)
new target for therapy of T-ALL patients, since NOTCH1
120
120
is frequently mutated in T-ALL and the generation of
100
100
80
80
activated NOTCH1 can be inhibited by γ-secretase
DND-41
60
60
inhibitors.2,8 Treatment of T-ALL cell lines with GSI was
40
40
shown to induce a G0/G1 cell cycle block, but the exact
20
20
therapeutic consequences of γ-secretase inhibition in the
0
0
Growth relative to control (%)
0
1
Growth relative to control (%)
0
1
context of T-ALL treatment have not been fully estab-
Dexamethasone (µM)
Dexamethasone (µM)
lished.8,15,19-21 In this study, we have further evaluated the
120
120
100
consequences of GSI treatment of T-ALL cell lines, and
100
80
80
investigated possible synergism between GSI and other
RPMI-8402
60
60
anti-cancer agents.
40
40
In agreement with previous reports, we observed that
20
20
0
the generation of activated NOTCH1 was completely
0
Growth relative to control (%)
0
1
Growth relative to control (%)
0
1
inhibited after 2 days of GSI treatment in all cell lines, but
Dexamethasone (µM)
Dexamethasone (µM)
120
120
that the effect on proliferation only became evident 5 to
100
100
7 days later, and correlated with a G0/G1 block in the cell
80
80
cycle. Our study demonstrates that these effects are
LOUCY
60
60
reversible and that treatment periods of 14 days or longer
40
40
20
20
are required to induce apoptosis. We also observed
0
0
decreased telomerase activity in cell lines with GSI
Growth relative to control (%)
0
1
Growth relative to control (%)
0
1
Dexamethasone (µM)
Dexamethasone (µM)
induced proliferation and cell cycle defects. As telom-
erase activity is known to vary depending on the stage in
the cell cycle and is minimal during the G0/G1 phase,29
Figure 6. Pre-treatment with a GSI sensitizes dexamethasone-
this observation could be explained by the G
responding cells. ALL-SIL, DND-41, RPMI-8402 and LOUCY cells
0/G1 cell
were treated with the glucocorticoid dexamethasone after DMSO
cycle block induced by GSI treatment in these cell lines.
pre-treatment (white bars), or with a combination of 1 µM
In addition, we observed GSI-induced differentiation of
Compound E and dexamethasone, after pre-treatment with 1 µM
Compound E (dark bars). The pre-treatment period was either 7
some T-ALL cell lines with NOTCH1 mutations, indicat-
days (left part of figure) or 14 days (right part of figure). After
ing that mutant NOTCH1 could be partially responsible
the pre-treatment period, cells were treated for 2 days (DND-41,
RPMI-8402 and LOUCY) or 3 days (ALL-SIL) and cell growth rela-
for the differentiation defect observed in T-ALL. Indeed,
tive to DMSO-treated cells was calculated. The asterisk indicates
expression of activated NOTCH1 has been shown to
a statistically significant difference between the groups connect-
ed by the line underneath the asterisk.
block differentiation of CD4+CD8+
T cells.23
| 540 | haematologica | 2008; 93(4)

---

γ-secretase inhibitors for T-ALL treatment
4, two cell lines displaying detectable levels of activated
combined NOTCH1- and ABL-targeted therapy for the
intracellular NOCH1 on western blot, were resistant to
treatment of these cases. Our results indicate, however,
the GSI-induced proliferation and cell cycle defects
that GSI and imatinib cannot unconditionally be com-
observed in the GSI-sensitive cell lines. It was recently
bined with each other, and that the exact timing and
reported that loss of the tumor suppressor gene PTEN is
order of administration could determine whether the
associated with resistance to GSI in cell lines with
combinations have synergistic/additive or antagonistic
NOTCH1 mutations.5 From the collection of T-ALL cell
effects. In addition, the finding that ABL1 fusions occur
lines we tested, MOLT-4 and JURKAT lack PTEN
together with NOTCH1 mutations provides genetic evi-
expression,31 which likely explains why these cell lines
dence that these mutations are co-operating events in the
are resistant to GSI treatment. Our results also confirm
pathogenesis of T-ALL.
that NOTCH1 mutation-negative cell lines can be sensi-
In contrast to imatinib, pre-treatment of T-ALL cell
tive to GSI treatment. The proliferation of HSB-2 cells
lines with a GSI did not make the cells more sensitive to
was found to be weakly inhibited by GSI treatment in
the cytotoxic chemotherapeutic vincristine. This may be
the absence of detectable NOTCH1 activation. Weng et
explained by the fact that GSI induces a cell cycle block,
al. observed that the TALL-1 cell line displayed cell cycle
and that cytotoxic chemotherapy is dependent on cell
block upon GSI treatment although this cell line also
division to induce its effect. We also tested whether com-
lacked NOTCH1 mutations.8 In addition, murine GSI-
binations of a GSI and glucocorticoids (dexamethasone
sensitive cell lines without mutant NOTCH1 have been
or corticosterone) offer any therapeutic advantage over
described.19 Possible mechanisms for the observed GSI
glucocorticoids as single agent therapy. For dexametha-
sensitivity in these cell lines could be the presence of
sone, pre-treatment with the GSI enhanced the weak
activating mutations in NOTCH2, NOTCH3 or
inhibitory effects of dexamethasone as a single agent; we
NOTCH4 or aberrant expression of NOTCH ligands.32
found the strongest synergy between the GSI and dex-
Alternatively, these observed effects could be due to
amethasone in the cell lines that were most sensitive to
aspecific effects of the GSI on another target. Our find-
dexamethasone. It remains to be determined to what
ings differ in some points from the results reported by
extent the observed in vitro interactions could translate
Weng et al., as we found that RPMI-8402 and KARPAS-
into responses of T-ALL patients to these drug combina-
45 cells are sensitive to GSI treatment. For RPMI-8402,
tions. Our data indicate that GSI pre-treatment of T-ALL
the difference is likely to be caused by the different
cells can make the cells more susceptible to other
assays, and different time-points that we used for the
inhibitors, and this could provide important therapeutic
read-outs, so that we were able to observe that RPMI-
advantages.
8402 is in fact sensitive to GSI treatment. This is also in
The clinical application of small molecule tyrosine
agreement with the fact that RPMI-8402 does not have
kinase inhibitors for the treatment of chronic myeloid
a complete loss of PTEN.5 For KARPAS-45, the
leukemia has demonstrated the efficacy and low toxicity
NOTCH1 DNA sequencing and western blot analysis
of targeted therapies.33 The introduction of GSI in the
revealed that the difference is due to the fact that our
treatment protocols for NOTCH1 mutation-positive T-
KARPAS-45 is different from the cell line reported by
ALL could further reduce the toxicity of current treat-
Weng et al. Such differences between cell lines have
ments, and improve long-term outcomes. To date, how-
been reported before, and warrant a full analysis by
ever, exclusive targeting of mutant NOTCH1 is not pos-
sequencing and western blot of each cell line used in
sible, and the currently used GSI generally inhibit γ-sec-
functional studies.
retase with associated effects on wild type NOTCH1, the
The fact that GSI only induce apoptosis after long peri-
three other NOTCH members, as well as a variety of
od of treatment, and the fact that the effects of gamma-
other proteins that are cleaved by the γ-secretase com-
secretase inhibition are reversible, suggests that the use
plex. Our data show that combining GSI with kinase
of a GSI as a single agent for the treatment of T-ALL may
inhibitors or dexamethasone could potentially enhance
be limited. In addition, it was previously shown that GSI
the anti-proliferative effects of GSI on leukemic cells,
treatment for 15 days is associated with toxic effects on
which may improve their application as agents for the
the gastrointestinal tract and on normal T-cell develop-
treatment of T-ALL.
ment.18 To investigate possible synergistic effects with
other currently used therapeutic agents, we tested
whether the addition of GSI could enhance the inhibito-
Authorship and Disclosures
ry effect of tyrosine kinase inhibitors or other cytotoxic
drugs. We recently identified NUP214-ABL1 and EML1-
KDK and IL designed and performed research, ana-
ABL1 fusions in T-ALL, and showed that these are sensi-
lyzed data and wrote the paper; NM and CF performed
tive to the small molecule kinase inhibitor imatinib.24,25
research; LVN and SB provided analytical tools and per-
Here we show that these ABL1 fusions are frequently
formed research; PV, MDO, PM and JC designed the
associated with the presence of NOTCH1 mutations (six
study, analyzed data and wrote the paper.
out of six cases), supporting the possible application of
The authors reported no potential conflicts of interest.
haematologica | 2008; 93(4) | 541 |

---

K. De Keersmaecker et al.
notch receptors. Mol Cell Biol 2000;
121-8.
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