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Blood, 15 December 2001, Vol. 98, No. 13, pp. 3864-3867
BRIEF REPORT
Restoration of sensitivity to STI571 in
STI571-resistant chronic myeloid leukemia cells
Alex J. Tipping,
François X. Mahon,
Valerie Lagarde,
John M. Goldman, and
Junia V. Melo
From the Department of Haematology, Imperial College
School of Medicine, Hammersmith Hospital, London, United Kingdom; and
Laboratoire de Greffe de Moelle, Université Victor Segalen,
Bordeaux, France.
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Abstract |
STI571 induces sustained hematologic remission in patients with
chronic myeloid leukemia (CML) in chronic phase. However, in advanced
phases, especially blast crisis, the leukemia usually becomes resistant
within months. It has been investigated whether resistance to
STI571 is stable and immutable or whether it can be reversed in
selected CML cell lines. Withdrawal of STI571 for varying lengths of
time from cultures of 3 resistant lines (K562-r, KCL22-r, and
Baf/BCR-ABL-r1) did not restore sensitivity to the inhibitor. In contrast, LAMA84-resistant cells experienced a sharp reduction in survival and proliferation during the first week of STI571
withdrawal but recovered thereafter. Moreover, when left off the
inhibitor for 2 months or longer, this cell line reacquired sensitivity
to STI571. It is hypothesized, therefore, that patients who
have become resistant to the drug may respond again if STI571
therapy is temporarily interrupted.
(Blood. 2001;98:3864-3867)
© 2001 by The American Society of Hematology.
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Introduction |
Chronic myeloid leukemia (CML) results from
the oncogenic transformation of hematopoietic stem cells by the
BCR-ABL fusion gene generated by the t(9;22)(q34;q11)
translocation that produces the Philadelphia chromosome. The disease
usually evolves in 3 stages, starting in a relatively "benign"
chronic phase and progressing through an accelerated phase to an
ultimately fatal blast crisis. Conventional treatment includes
allogeneic stem cell transplantation, hydroxyurea, interferon- , and
cytosine arabinoside.1 Presently, an inhibitor of the
deregulated Bcr-Abl tyrosine kinase named STI571 is being tested in
clinical trials for CML. Preliminary results for patients treated in
chronic phase are very encouraging.2 When given at
accelerated phase or blast crisis, STI571 can induce frequent
hematologic responses, but these are nearly always followed by
relapse,3 suggesting development of de novo resistance to the drug or selection of a resistant clone.
We recently generated a series of STI571-sensitive and -resistant
clones from BCR-ABL-positive cell lines in order to model the phenomenon of acquired resistance to this inhibitor.4
In some of these lines the mechanism of resistance was attributable to
BCR-ABL amplification and overexpression, multidrug
resistance gene (MDR1) overexpression, or ineffective
inhibition of Bcr-Abl tyrosine phosphorylation. An outstanding question
from that study was the fate of resistant cell lines upon STI571
withdrawal. This is obviously an important question in the analogous
context of STI571-resistant patients who are temporarily taken off
therapy. We show here that whereas the resistance of some cell lines is unaltered even after various months in culture without STI571, in other
lines the molecular mechanism of resistance is reversed and sensitivity
to STI571 is restored.
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Study design |
The STI571-sensitive (s) and -resistant (r) cell lines
Baf/BCR-ABL-s, Baf/BCR-ABL-r1, K562-s, K562-r,
KCL22-s, KCL22-r, LAMA84-s, and LAMA84-r have been described
previously.4 Baf/BCR-ABL was derived from the
Ba/F3 murine hemopoietic line, whereas K562, KCL22, and LAMA84 were
established from patients with CML in blast crisis.5 The
sensitive lines were grown in RPMI 1640 supplemented with penicillin,
streptomycin, L-glutamine, and 10% fetal calf serum
(complete medium referred to as RF-10) and subcultured every 2 to 3 days as indicated by acidification of the supernatant. The resistant
counterparts were routinely kept in RF-10 supplemented with 1 µM
STI571 unless stated otherwise.
For each experiment, exponentially growing cells were
centrifuged, washed 3 times in phosphate-buffered saline without
Ca++ and Mg2+, and resuspended at
2 × 105/mL in media with or without 1 µM STI571. After
variable time periods, resistant cells cultured in the absence of
STI571 were reexposed to 1µM of the inhibitor. Throughout the
experiment, 5 mL aliquots were harvested daily or at longer intervals
for (1) cell counts on a hemocytometer, (2) assessment of viability by
trypan blue exclusion on duplicate aliquots, and (3) analysis of
proliferation by the MTS assay performed in quadruplicate wells according to the manufacturer's instructions (CellTiter 96 AQueous One Solution Cell Proliferation Assay,
Promega, Southampton, United Kingdom). Each complete experiment was
repeated 2 or 3 times.
The number of BCR-ABL gene copies was investigated by
fluorescence in situ hybridization as previously
reported.6 Analysis of Bcr-Abl protein expression was done
by Western blotting as previously described4 and by
intracytoplasmic staining with an anti-Abl monoclonal antibody (24-11;
Santa Cruz Biotechnology, Santa Cruz, CA) using a
fluorescein-conjugated goat antimouse immunoglobulin G (Becton
Dickinson, Basingstoke, United Kingdom) as secondary reagent and
reading on a FACScan flow cytometer (Becton Dickinson) as described
elsewhere.7 Total phosphotyrosine content was assessed by
Western blotting4 using an anti-pY monoclonal antibody
(PY-99; Santa Cruz Biotechnology). Apoptosis was assessed by measuring
the level of caspase-3 as previously reported.4
| |
Results and discussion |
The 4 STI571-resistant cell lines used in this study had been
growing continuously in 1 µM STI571 for longer than 1 year, with a
baseline viability of 80% to 98%. When K562-r and KCL22-r were washed
free of STI571 and grown in RF-10 only for short (7-15 days) or longer
(4-6 months) periods, no change in their behavior was observed.
Moreover, reexposure of the 2 lines to STI571 after various periods of
withdrawal did not result in significant cell death or reduction in
proliferation as compared with resistant cells grown continuously with
the inhibitor (data not shown). This was entirely expected for KCL22-r,
because the parental KCL22 is itself exceptionally resistant to
STI571.4,6 Neither cell line overexpresses
BCR-ABL, and K562-r but not KCL22-r was shown to require
larger concentrations of STI571 to inhibit the same proportion of
Bcr-Abl autophosphorylation as its sensitive counterpart.4 Although the exact mechanism of resistance in both lines is still unknown, it appears to be stable in the absence of STI571.
In the case of the Baf/BCR-ABL-r1 line, a 7-day withdrawal
of STI571 led to a small but reproducible drop in viability and cell
proliferation. The effect peaked around days 4 to 8 of withdrawal, after which the cultures began to recover with or without reexposure to
the compound (Figure 1A). Likewise,
resistant cells kept in the absence of STI571 for 14, 28, 35, or 125 days showed no restoration of sensitivity upon reinitiation of
treatment with the inhibitor (not shown).
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| Figure 1.
The effect of STI571 withdrawal.
(A) Viability of Baf/BCR-ABL-r1 and LAMA84-r cells
upon withdrawal of STI571 from the culture. On day 0, exponentially
growing cells were washed off the compound and replated in RF-10 media
(R /), whereas control cells were maintained in media supplemented
with 1 µM STI571 (R+/+). Subculturing with the respective media was
done whenever necessary for each cell line. On day 7 (indicated by the
black arrow), one half of the RF-10 culture was reexposed to 1 µM
STI571 (R/+) and the 3 cultures were observed for 1 additional week.
Results are shown as the mean and range percentage of trypan
blue-negative cells out of 200 cell counts from duplicate
culture aliquots, in 1 of 3 representative experiments. (B) Caspase-3
activity in LAMA84-s and LAMA84-r cells after 3 days in culture with
(+) and without () 1 µM STI571. Results represent the mean ± SD
fluorescent units of quadruplicate cultures.
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The profile of LAMA84-r cells was markedly different from the
other lines. When washed thoroughly and replated in media without STI571, LAMA84-r cells showed a significant loss of viability and
reduction in proliferation, starting around day 3 and peaking at days 5 to 9. Similarly to Baf/BCR-ABL-r1, these cells also recovered from the short (< 15 days) withdrawal interval and, with
some additional delay (average 15 days vs 10 days for
Baf/BCR-ABL-r1), returned to the viability and proliferation
pattern exhibited when kept continuously in the presence of STI571
(Figure 1A). Nevertheless, when LAMA84-r cells were grown in RF-10
without the inhibitor for 2 weeks, a slight increase in cell death was observed upon reexposure to STI571. This effect was further accentuated if the period of withdrawal was extended to 4 weeks, and it culminated in a nearly complete restoration of sensitivity after 4 months without
STI571 (Figure 2A). As expected, these
cells became again prone to inhibition of Bcr-Abl autophosphorylation
and tyrosine kinase activity by STI571, similarly to the original
LAMA84-s cells and unlike the LAMA84-r clone kept continuously in
STI571 (Figure 3).
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| Figure 2.
Restoration of sensitivity to STI571.
(A) Viability (3 upper panels) and proliferation (bottom panel)
of LAMA84-r cells in the presence (gray line) or absence (black line)
of 1 µM STI571 after the indicated periods of withdrawal of the drug.
Results are shown as the mean and range percentage of viable cells or
absolute number of cells, respectively. (B) Flow cytometric histograms
of LAMA84-s (S), LAMA84-r cells grown continuously in STI571 (R), and
LAMA84-r grown in the absence of STI571 for the indicated periods
(thick dark line) and stained with an anti-Abl antibody. One
representative histogram of the 3 cell populations stained with an
isotype control (IC) is shown. Note that the anti-Abl staining is due
entirely to the Bcr-Abl protein, because LAMA84 does not express the
normal ABL gene (no normal chromosome
9).8
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| Figure 3.
Restoration of sensitivity to STI571.
Phosphotyrosine (p-Tyr) immunoblot of LAMA84-s (S), LAMA84-r (R), and
resistant cells grown in the absence of STI571 for 234 days
(R234d) exposed to 1 µ µM STI571 for 2 hours, as
compared with nonexposed aliquots. Densitometric analysis of the p210
Bcr-Abl band corrected for the actin control showed a 2.1-fold decrease
in phosphorylation of LAMA84-s and a 3.0-fold for the
LAMA84-R234d line upon treatment with STI571. As previously
shown,4 LAMA84-r cells kept continuously in the presence
of STI571 have an elevated phosphotyrosine content.
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Because BCR-ABL gene amplification and overexpression
were shown to underlie the STI571 resistance in both
Baf/BCR-ABL-r and LAMA84-r, we investigated these phenomena
in culture aliquots harvested at the various withdrawal time points. No
reduction in Bcr-Abl overexpression was observed in
Baf/BCR-ABL-r grown for up to 3 months in the absence of
the inhibitor, in agreement with the fact that these cells retain their
resistant phenotype under these conditions. In contrast, LAMA84-r cells
experienced a progressive decrease in the level of Bcr-Abl protein with
time after STI571 withdrawal (Figures 2B and
4): The reduction was already noticeable
at 4 days and became prominent at around 2 months. By 4 to 6 months,
virtually all the cells from the original LAMA84-r culture had the same
level of Bcr-Abl expression as the parental LAMA84-s line, coinciding
with restoration of sensitivity to STI571. Fluorescence in situ
hybridization analysis of these cells confirmed that the average number
of BCR-ABL gene copies per cell was similar to that of
LAMA84-s cells and approximately 5-fold lower than the number in
LAMA84-r kept continuously in the presence of STI571 (not shown).
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| Figure 4.
Reduction in Bcr-Abl protein expression upon withdrawal
of STI571 from LAMA84-r cultures.
Total cellular protein was harvested from parallel cultures growing in
the presence or absence of 1 µM STI571 for the periods indicated and
subjected to Western analysis with anti-Abl (Bcr-Abl band shown) and
antiactin antibodies. Densitometric analysis of the Bcr-Abl signal
relative to that of actin is presented for each lane.
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Our study reveals 2 interesting points. First, both resistant
cell lines that overexpress BCR-ABL experience "withdrawal
symptoms" when abruptly deprived of STI571. This surprising result
suggests that the "acute" accumulation of a vast excess of
kinase-active Bcr-Abl protein is in fact deleterious to cell viability
and growth and may have a proapoptotic effect, as illustrated by the
increase in caspase-3 activity in LAMA84-r cells after 3 days in the
absence of STI571 (Figure 1B). Certainly, withdrawal of STI571 from
LAMA84-r leads to a rapid and marked increase in Bcr-Abl
autophosphorylation and total phosphotyrosine content (Figure
5). This phenomenon may be analogous to
the recent demonstration by Vigneri and Wang9 that forced
nuclear entrapment of Bcr-Abl by treatment with STI571 and leptomycin-B
followed by sudden withdrawal of the kinase inhibitor leads to
apoptosis. We are presently investigating whether the "surplus"
Bcr-Abl protein in the STI571-resistant lines has an altered cellular
localization that could account for the early response of these lines
to STI571 withdrawal. In any case, the effect in both
Baf/BCR-ABL-r1 and LAMA84-r is transient, suggesting that the postulated imbalance of Bcr-Abl accumulation and localization may be rapidly "normalized" to allow survival in the absence of the inhibitor.
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| Figure 5.
Increased phosphotyrosine content on withdrawal of
STI571 from LAMA84-r cells.
Total cellular protein was harvested from cells grown continuously in
the presence of 1 µM STI571 (time zero) and then hourly after
thorough washing and reculture in media without STI571. The Western
blot membrane was probed with an antiphosphotyrosine antibody (upper
panel) and reprobed with an antiactin antisera (lower panel). The
densitometric ratio of the phospho-Bcr-Abl (p-p210)/actin bands is
shown for each time point and demonstrates a 41-fold increase in
Bcr-Abl autophosphorylation after 5 hours of STI571 withdrawal.
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The second noteworthy result was the nearly complete restoration
of sensitivity to STI571 in LAMA84-r cells maintained long-term off the
drug. This clearly correlated with a loss of Bcr-Abl overexpression, probably due to absence of selection pressure for high
BCR-ABL copy number and/or positive selection for lower
expression. In Baf/BCR-ABL-r1 none of these phenotypic
changes were observed, which may be attributed to the fact that this is
not a true CML but, rather, a murine cell line whose transfected
BCR-ABL gene is under the control of an exogenous promoter
that may allow cells with the amplified copies to be more stable in the
absence of selective pressure.
Similarly to the situation in some cell lines, BCR-ABL
amplification and overexpression have now been documented as a cause of
acquired STI571 resistance in patients treated with this
drug.10,11 In fact, in one patient recently described by
Gorre et al10 in whom resistance to STI571 therapy was
associated with BCR-ABL gene amplification, this phenomenon
was no longer detectable 4 weeks after STI571 was discontinued and the
patient was placed under alternative treatment for her leukemia. We
conclude that, if the results from the present study are equally
applicable to primary CML cells, it is possible that selected patients
who become refractory to STI571 may benefit from a second course of
therapy after an interval off this inhibitor.
| |
Acknowledgments |
We are grateful to Dr Elisabeth Buchdunger (Novartis Pharma, Basel,
Switzerland) for the generous gift of STI571.
| |
Footnotes |
Submitted March 30, 2001; accepted August 9, 2001.
Supported by grants from the Leukaemia Research Fund, United Kingdom
(A.J.T., J.M.G., J.V.M.), and the Association pour la Recherche sur le
Cancer and Fondation de la Recherche Medicale, France (F.X.M., V.L.).
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 U.S.C.
section 1734.
Reprints: Junia V. Melo, Dept of Haematology ICSM,
Hammersmith Hospital, Ducane Rd, London W12 0NN, United Kingdom;
e-mail: j.melo{at}ic.ac.uk.
| |
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Abstract
Introduction