Impact of Hypomethylating Agents on hTERT Expression and Synergistic Effect in Combination
With Imetelstat, a Telomerase Inhibitor, in Acute Myeloid Leukemia Cell Lines
2731
Joshua Rusbuldt, Jacqueline Bussolari, Aleksandra Rizo, Fei Huang
Janssen Research & Development, LLC
Imetelstat
Imetelstat
1.0
CellTiter-Glo®
Substrate
CellTiter-Glo®
Buffer
CellTiter-Glo®
Reagent
Mixer
Luminometer
• Flow cytometry performed with BioLegend AnnexinV/
Propidium Iodide Apoptosis Kit.
EL
0.2
0.4
0.6
DAC, µM
H
KG
-1
-1
SK
M
i-1
um
L5
I-A
M
DAC
DAC
DAC
DAC
DAC
DAC
to
to
to
to
to
to
OCI-AML3
OCI-AML3, then 2 wks media
OCI-AML3, then 4 wks media
OCI-AML5
OCI-AML5, then 2 wks media
OCI-AML5, then 4 wks media
100
50
0.25 µM
1 µM
OCI-AML3
150
hrs
hrs
hrs
hrs
hrs
hrs
AZA
AZA
AZA
AZA
AZA
AZA
to
to
to
to
to
to
OCI-AML3
OCI-AML3, then 2 wks media
OCI-AML3, then 4 wks media
OCI-AML5
OCI-AML5, then 2 wks media
OCI-AML5, then 4 wks media
100
50
0.5 µM
1 µM
OCI-AML3
0.5 µM
1 µM
OCI-AML5
Cells were treated with DAC (top) or AZA (bottom) every 24 hours for 72 hours (solid bars),
followed by removal of drug, or washout. Cells were then continually cultured in the absence
of imetelstat for 2 weeks (diagonally striped bars) or 4 weeks (dotted bars) in parallel with
the formal combinations with imetelstat detailed in Figure 4. Both cell lines had reduced
viability at 72 hours post dose (solid bars) with the 1 μM concentrations. OCI-AML5 (orange)
cells had greater viability reductions in response to either DAC or AZA, as expected based on
results in Figure 2. Viability of cells under all conditions had completely recovered by 4 weeks,
and as early as 2 weeks in OCI-AML3 (teal).
0.4
0.2
0
0.25
0.5
0.75
AZA, μM
1
1.25
AML cell lines OCI-AML3 (high hTERT, teal) and OCI-AML5 (low hTERT, orange) were treated with a single dose of DAC or AZA for 72 hours with
no washout period and assessed for cell viability (dashed) and hTERT expression (solid) compared with vehicle (DMSO in PBS). Both lines appear
susceptible to AZA at concentrations > 1 μM; however, only OCI-AML5 appears to be sensitive to DAC. High doses of AZA seem to induce expression of
hTERT in OCI-AML5. Black arrows highlight concentration points utilized in combinations for experiments demonstrated in Figures 3 and 4. However, it
should be noted that daily treatment with DNMTi for 72 hours in experiments in Figures 3 and 4 differed from the single dose in this experiment.
DAC
100
80
60
40
20
0.25 μM
0.25 μM
1 μM
0
0 μM
Imet
0.25 µM
1 µM
OCI-AML5
72
72
72
72
72
72
200
1
B
25 μM Imetelstat 50 μM Imetelstat
250 nM DAC
10 μM Imetelstat
Figure 2. Effect of single-agent DNMTi on viability and hTERT expression of AML cell lines in vitro 72 hours following a single dose.
5 μM
Imet
AZA
250
0.8
0.6
25 μM
Imet
72
72
72
72
50 μM
Imet
80
60
40
20
0.50 μM
0.50 μM
0
5 μM
Imet
1 μM
25 μM
Imet
60
20
72
72
72
72
hrs
hrs
hrs
hrs
0.25 μM
0.25 μM
1 μM
0
0.25 μM DAC, then 2 wks imetelstat
0.25 μM DAC, then 4 wks imetelstat
1.0 μM DAC, then 2 wks imetelstat
1.0 μM DAC, then 4 wks imetelstat
5 μM
Imet
25 μM
Imet
1 μM
50 μM
Imet
• Upon removal of drugs, growth inhibition by both DAC and AZA was not sustained,
and cell proliferation had recovered by 2 weeks in cells with high hTERT expression
(OCI‑AML3) and by 4 weeks in cells with lower hTERT expression (OCI-AML5).
• Pretreatment with DAC (noting that DAC is more potent than AZA, and a lower initial
concentration of DAC was used in all experiments) followed by imetelstat treatment
reduced cell viability more than either agent administered alone, and administration of
imetelstat after AZA pretreatment prevented or slowed recovery.
OCI-AML5
• Since treatment with AZA or DAC for 3 days in this study may not generate optimal
hypomethylation, a follow-up study with an optimal dosing schedule was conducted.
100
80
—— Apoptosis increased in a dose-dependent manner with imetelstat treatment as well
as DAC (Figure 5) and AZA (data not shown).
60
40
20
0.50 μM
0.50 μM
0
0 μM
Imet
1 μM
50 μM
Imet
• Both DAC and AZA caused dose-dependent decreases in hTERT expression and
differential growth inhibition on OCI-AML3 and OCI-AML5 cell lines.
40
AZA
100
OCI-AML3 cells were pretreated once every 24 hours for 5 days (Panel A) with either dose
of DAC or DMSO/PBS (0 nM DAC). Following washout after the fifth day of exposure, cells
were cultured in the presence of biweekly imetelstat treatment as a single agent for 2 weeks
(Panel B). Populations of dying and dead cells increased in a dose-dependent manner for
both DAC and imetelstat, with greatest effect observed at the combination of highest DAC
with highest imetelstat doses. Similar experiments were performed with AZA as well as in
OCI-AML5 (not shown).
CONCLUSIONS
80
0 μM
Imet
hrs
hrs
hrs
hrs
Figure 5. Flow cytometric analysis of apoptosis in OCI-AML3 in vitro post DAC
treatment (5 days) followed by imetelstat (in the absence of DAC) for 2 weeks.
100
1 μM
OCI-AML3
0 μM
Imet
OCI-AML5
Viability Relative to
Vehicle Control (%)
2
C
C
O
SK
N
O
I-M
-1
4
-B
N
L3
M
0
I-A
K
as
hrs
hrs
hrs
hrs
hrs
hrs
Figure 3. Recovery time of AML cell lines following treatment with DNMTi.
Epigenetic
modifiers
0
0.8
0
OCI-AML3
72
72
72
72
72
72
150
0
0.2
DAC
200
0
0.4
Viability Relative to
Vehicle Control (%)
• Cells were monitored for viability with CellTiter-Glo®
(Promega) assay immediately after DNMTi (DAC or AZA)
dosing for 72 hours, and again after treatment with
imetelstat for 2 weeks and 4 weeks.
0.6
0
0.0
250
• Cells were passaged weekly and dosed twice weekly with
imetelstat (50 μM, 25 μM, 5 μM) or fresh media.
0.8
1
2 weeks (imetelstat only, post-DAC)
0 μM
25 nM DAC
2.0
1
5 day
(DAC only)
0 nM DAC
3.0
Viability Relative
to Vehicle Control (%)
Viability Relative
to Vehicle Control (%)
4.0
Figure 1. Determination of hTERT RNA expression in AML cell lines.
DAC,
AZA, or
vehicle for 72 hours
• Post DNMTi dose
• 2 weeks imetelstat
• 4 weeks imetelstat
hTERT
5.0
O
OCI-AML5
(Low hTERT)
Viability readouts:
Epigenetic
modifiers
6.0
A
OCI-AML3 Viability
OCI-AML3 hTERT
OCI-AML5 Viability
OCI-AML5 hTERT
Viability Relative to
Vehicle Control (%)
Better
than
1.2
Viability Relative to
Vehicle Control (%)
• To determine whether the combination of a DNMTi and
imetelstat enhances inhibition of cell viability in vitro
compared with either agent alone.
1.2
A panel of AML cell lines was measured for hTERT RNA expression using an RT-qPCR
method. Levels of RNA expression varied across the lines investigated. Cell lines OCI-AML3
and OCI-AML5 were investigated in single-agent and combination experiments as they
represented the bounds of the observed expression range.
• Decitabine (DAC) and 5-azacitidine (AZA) are both DNA
methyltransferase inhibitors (DNMTis) that are currently
used for the treatment of AML.
EXPERIMENTAL OBJECTIVE
7.0
C
OCI-AML3
(High hTERT)
• Imetelstat has limited single-agent activity in the AML cell
lines tested up to 4 weeks on treatment (internal data not
shown).
• As it has been reported that hTERT expression is modulated
by DAC, 5 the combination of imetelstat with DAC or AZA
is hypothesized to improve treatment benefit in AML by
modulating hTERT expression.
1.4
O
• Imetelstat is currently being investigated in clinical trials
as a single agent, and recently reported clinical results
show activity in patients with essential thrombocythemia
or primary, post-essential thrombocythemia, and postpolycythemia vera myelofibrosis.3,4
• The effects of DAC or AZA treatment for 72 hours (daily
dosing) followed by media alone (Figure 3) or imetelstat
(Figure 4) for 2 or 4 weeks were then examined as follows:
1.4
L6
• Imetelstat is a 13-mer oligonucleotide that specifically
targets the RNA template of human telomerase and is a
potent first-in-class competitive inhibitor of telomerase
activity.
• AML cell lines expressing high hTERT (OCI-AML3) or low
hTERT (OCI-AML5) were treated with a single dose of DAC
or AZA or dimethylsulfoxide/phosphate-buffered saline
(DMSO/PBS) vehicle for 72 hours and assessed for cell
viability and hTERT expression (Figure 2).
8.0
H
• hTERT expression is highly regulated (eg, by epigenetic
modification), and reports have suggested correlative
links between overexpression and hypermethylation of the
hTERT promoter1; conversely, normal tissues are largely
hypomethylated in this region. 2
Viability Relative to
Vehicle Control (%)
• Telomerase is not expressed in normal tissues, and is only
transiently activated in hematopoietic progenitor cells.
• Levels of hTERT RNA expression were investigated across
a panel of AML cell lines using a reverse transcriptasequantitative polymerase chain reaction (RT-qPCR) method
(Figure 1) in order to identify those with high and low hTERT
expression levels.
Viability Relative to
Vehicle Control (%)
• Acute myeloid leukemia (AML) cells express high levels of
the catalytic unit of human telomerase reverse transcriptase
(hTERT).
RESULTS
hTERT
Mean Normalized Expression
MATERIALS AND METHODS
BACKGROUND
0.50 μM AZA, then 2 wks imetelstat
0.50 μM AZA, then 4 wks imetelstat
1.0 μM AZA, then 2 wks imetelstat
1.0 μM AZA, then 4 wks imetelstat
5 μM
Imet
1 μM
25 μM
Imet
1 μM
50 μM
Imet
Figure 4. Viability of AML cell lines after 72 hours treatment with DNMTi (DAC or AZA) followed by long-term treatment with imetelstat
for 2 or 4 weeks.
Cells were pretreated once every 24 hours for 72 hours with 1 of 2 doses of DAC (top) or AZA (bottom). Following washout removal of DNMTi at 72
hours, the cells were then cultured in the presence of biweekly imetelstat treatment as a single agent for up to 4 weeks. Cells generally recovered by
2 weeks, though the highest doses of imetelstat (50 μM) suppressed recovery for both lines in conjunction with DAC. With AZA in combination with
imetelstat, reduced viability was noted in OCI-AML3, particularly with the lower (0.5 μM) dose of AZA, but not in OCI-AML5.
REFERENCES
1. Sui X, et al. Oncol Lett. 2013;6:317-322.
2. Renaud S, et al. Nucleic Acids Res. 2007;35:1245-1256.
3. Baerlocher GM, et al. N Engl J Med. 2015;373:920-928.
4. Tefferi A, et al. N Engl J Med. 2015;373:908-919.
5. Zhang X, et al. Oncotarget. 2015;6:4888-4900.
ACKNOWLEDGMENTS
This study was funded by Janssen Research & Development, LLC (Raritan, NJ). Writing assistance was provided by Efy Leonardi, PhD, and
Tamara Fink, PhD, of PAREXEL (Hackensack, NJ), with funding from Janssen Global Services, LLC (Raritan, NJ).
Poster presented at the American Association for Cancer Research Annual Meeting 2016, April 16-20, 2016, New Orleans, LA.
An electronic version of the poster can be viewed by scanning the QR code.
The QR code is intended to provide scientific information for individual reference.
The PDF should not be altered or reproduced in any way.
Sponsored by Janssen Global Services, LLC