Insights on the mechanisms involved in acquired resistance to
dasatinib in lymphoma cell lines
Introduction
Lymphoma cells (type DLBCL and CLL cells), as it has been recently reported,
are sensitive to the dual c-abl/src kinase inhibitor dasatinib in vitro in the clinical
attainable range, and de novo resistance to the drug is associated with SRC independent
calcium mobilization induced by IgG/IgM stimulation. As well, acquired resistance to
dasatinib is associated with abrogation of IgG/IgM-induced signalling downstream the Bcell receptor (BCR) (Hollmann, 2009).
We hypothesize that lymphoma cells with acquire resistance to dasatinib are able
to activate survival pathways that are downstream the BCR receptor by activating
alternative autocrine/paracrine pathways. This leads us to test the cell’s response to
dasatinib treatment while being maintained in conditioned media versus fresh media.
Further explanations on procedures will be given in the materials and methods step.
Preliminary results using mRNA microarray expression analysis has suggested
that in lymphoma cells with acquired resistance to dasatinib there is an activation of proinflammatory pathways. This leads to the hypothesis that chronic exposure to dasatinib
will result in the selection of cells with alternative autocrine / paracrine survival pathways
mediated by cytokines. There have already been experiments associated with a cell’s
conditioned media. According the paper published by Infection and Innunity (Jan. 1988),
it was reported that there are factors released from the cells into the supernatants after
being suspended for a period of time. This brings us to the topic of cytokines. Cytokines,
which are extremely important for cell communication, is often associated with the
extracellular matrix of a cell.
In diffuse large B-cell lymphoma (DLBCL), dasatinib cytotoxicity is mediated by
the inhibition of signaling downstream the BCR receptor (Hollmann et a.. 2009). This
prompted us to investigate the signaling events associated with acquired resistance to
dasatinib in a panel of DLBCL cell lines. We obtained independent clones (2) of 3
DLBCL cell lines sensitive to dasatinib. Sensitivity defined as an IC50 which falls in the
attainable range (<200nM). We find that selection in non toxic concentrations of
dasatinib resulted in resistance to dasatinib (2 to 1000 fold) and abrogation of signaling
downstream the BCR, suggesting that the selected cells are not dependent on the BCR
receptor activation for survival (Hollmann et al., submitted manuscript, March 2010).
The hypothesis to be tested is that acquired dasatinib resistance is mediated by a
shift on the phenotype of DLBCL cells where the survival signals mediated by activation
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of the BCR receptor are replaced by paracrine/autocrine mechanisms that sustain the
survival of DLBCL.
Materials and Methods:
Cell Line Culture
DHL4 and OCI-Ly1 (sensitive cell lines) and DHL4B1 and OCI-Ly1B1 (cells with
acquired resistance to dasatinib) were maintained in RPMI1640, 10% fetal calf serum,
0.05 mM mercaptoethanol, 10 U/mL of penicillin and 10 µg/mL of streptomycin at 37˚C
in 5% CO2. The passage of the cells was kept under 30 and the cells were passage every
three days.
MTT Assay
Cells were seeded in 96-welled plates, and depending on the cell line, exposed to
different concentrations of dasatinib at 37˚C in 5% CO2 for 72 hours. The different
concentrations of dasatinib were diluted with PBS and the number of cells used for drug
treatment was 60 000 cells per well. Plating efficiencies were also plated and measured to
ensure that the number of cells used was sufficient. For control, certain columns of cells
were left untreated, and certain cells were treated with DMSO, the solvent for dasatinib.
After 72 hours of drug treatment, 10 uL of MTT (2-(3,5-diphenyltetrazol-2-ium-2-yl)4,5-dimethyl-1,3-thiazole bromide) was added to each well, and the plates were again
incubated for an hour. After one hour, the plates were centrifuged for 10 minutes at 1500
rpm and the supernatants were then removed. The cells were then dissolved in 25uL of
Sorenson’s Buffer and 100 uL of DMSO on a rocker, and the MTT absorption data were
collected one hour later using an ELISA reader at 570nm. These data were then evaluated
using Microsoft Excel.
IC50 Calculations
The optical density readings were plotted on excel against the number of cells plated, and
the IC50s were calculated based on a logarithmic scale; this is the concentration of drug
that will kill 50% of the cells relative to the vehicle cells.
Working With Supernatants
Conditioned media (72 hours) from the different clones of DHL4 and OCI-Ly1 cells with
acquired resistance to dasatinib were collected. The parental sensitive cell lines were
harvested and resuspended in the different conditioned media and exposed to different
concentrations of dasatinib. Survival (IC50) was assessed using the MTT assay after 3
days. The hypothesis was that the conditioned media of cells with acquired resistance will
antagonize dasatinib cytotoxicity.
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Calcium Mobilization:
2×106 OCI-Ly1 sensitive cells were suspended in 500uL of either RPMI media or DHL4
B1 conditioned supernatants. 30 minutes into the incubation, 2.5uL of Fluo-4 AM and
Fura-Red AM respectively were added to the tubes, and the cells were allowed to
incubate for another 30 minutes for a total of one hour of media exposure. Samples were
then centrifuged for 5 minutes at 1500 rpm, and the pellets were resuspended in 500 uL
of HBBS. We then used anti-IgG antibody (10ug/mL) to induce the calcium flux, and the
samples were analyzed on the BD LSRII flow cytometer with the BD FACSDiva
software.
Results:
The IC50 of all the cell lines were determined in RPMI1640, 10% fetal calf serum, 0.05
mM mercaptoethanol, 10 U/mL of penicillin and 10 µg/mL of streptomycin at 37˚C in
5% CO2. The DHL4 cells were determined experimentally while the DHL4B1, OCI-Ly1,
and OCI-Ly1B1 IC50s were obtained previously (Hollmann et al., submitted manuscript,
March 2010)
DHL4 IC50s:
Trial
1
2
3
4
5
6
7
AVG
SD
SE
IC50 (nM)
18.2
27.0
13.9
30.6
13.5
11.7
21.0
19.4
7.21
2.72
Table 1 – DHL4 IC50s treated with dasatinib in RPMI media
DHL4 B1(with acquire resistance to dasatinib derived from DHL4, Table 1) IC50:
4.7 ± 0.5uM (4700nM) (SD)
OCI-Ly1 repeated IC50:
70± 10nM (SD)
OCI-Ly1B1 repeated IC50:
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2300 ± 500nM (SD)
With the IC50s of the cell lines in mind, we then proceeded to treat the sensitive cell lines
with different medias (RPMI Media and resistant supernatants respectively) to compare
their responses to dasatinib. As stated, we hypothesize that the conditioned media of cells
with acquired resistance will antagonize dasatinib cytotoxicity. The results are listed
below:
DHL4 Treatments with different Supernatants:
Treated in:
Jun-20
IC50
(nM)
Jul-2
IC50
(nM)
Jul-15
IC50
(nM)
Jul-23
IC50
(nM)
AVG
IC50
(nM)
RPMI
39.2
18.3
7.12
19.0
20.9
With DHL4B1
Supernatant
12.0
1.80
6.8
With OCI-Ly1B1
Supernatant
Fold Difference
3.25
SD IC50
(nM)
10.0
3.30
5.30
4.3
2.11
3.59
4.74
3.56
Table 2 – DHL4 IC50s treated with dasatinib in RPMI media, DHL4B1, and OCILy1B1 supernatants respectively
Example of Curves:
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
-20.00%
0
20
40
60
80
100
120
-40.00%
Figure 1 – DLH4 treatment with dasatinib with both RPMI media (pink series) and DHL4 B1
resistant supernatant (Blue series)
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OCI-Ly1 Treatments with different Supernatants:
From our data in table 1 and table 2, the results suggest that the resistant media lowers the
IC50 of the sensitive cell lines in comparison to the RPMI media. However, this brings
up another question: We do not know whether the resistant media simply too old (72
hours) and lacks nutrients for the cells to grow, or there really is an extracellular factor
that is causing the sensitive cell liens to be hypersensitive to dasatinib. Therefore for
control, we treated OCI-Ly1B1 (resistant cell lines) with conditioned media from both
DHL4 (sensitive) DHL4-B11 (resistant) cells, and the results are shown below.
OCI-Ly1B1 treatments in different supernatants - Control (Jul-23):
Treated in:
Jul-23 IC50 (nM)
RPMI
1680
With DHL4B1 Supernatant
1730
Fold Difference
1
Calcium Mobilization:
Because of time constraints, calcium mobilization was only performed once. Due to this
reason, and the fact that less than enough cells was used to perform the experiment, the
results were not reliable, and will therefore not be included until further trials take place.
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Discussion:
Our experiment dealt with two different cell lines, sensitive and resistant
respectively: Su-DHL4, Su-DHL4B1, OCI-ly1, and OCI-Ly1B1. Both cell lines share
certain fundamental similarities and differences. OCI-Ly1 has IgM receptors while SuDHL4 has IgG receptors. Even though they have this phenotype difference, they still
behave similarly and fall under the same classification. In all our experiments, the
sensitive cells suspended in the conditioned resistant media (in comparison to RPMI
regular media) all experienced a decrease in their IC50 after treatment with dasatinib.
This means that all the resistant cell lines (and hence resistant supernatants) must share a
similar characteristic in order to produce this effect. Therefore, a further improvement to
this experiment in the future would be to discover the characterization of the resistant
supernatants.
Previous results from the laboratory indicated that the supernatant from Su-DHL4
cells with acquired resistance to dasatinib increased the survival of OCI-Ly1 and OCILy8 to the tyrosine kinase inhibitor. These results lead us to hypothesize that the resistant
cells activate an alternative pathways for survival, independent of BCR receptor
activation. And that this/these pathways involve the release of survival factors from cells
with acquired resistance to dasatinib. During my summer student scholarship, I assessed
this hypothesis using Su-DHL4 parental cell lines and the supernatant from Su-DHL4
cells with acquired resistance to dasatinib. Surprisingly, I find is that the supernatant from
resistance cells increase the sensitivity of the parental Su-DHL4 cells to dasatinib. These
results taken together with our previous results suggests that although the mechanisms of
resistance (de novo and acquired) to dasatinib in Su-DHL4 and OCI-Ly cells are
associated with altered BCR signaling, the alternative survival/antiapoptotic pathways
activated in cells with acquired resistance are different.
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References:
Therapeutic implications of Src independent calcium mobilization in diffuse large
B-cell lymphoma. Hollmann CA, Tzankov A, Martínez-Marignac VL, Baker K,
Grygorczyk C, Grygorczyk R, Foulkes W, Nadeau J, Dirnhofer S, Aloyz R. Leuk Res,
2009 PMID: 19758698
Dasatinib sensitizes primary chronic lymphocytic leukaemia lymphocytes to
chlorambucil and fludarabine in vitro. Amrein L, Hernandez TA, Ferrario C, Johnston
J, Gibson SB, Panasci L, Aloyz R. Br J Haematoly, 2008 PMID: 19062342
Molecular Mechanisms of acquired resistance to tyrosine kinase targeted therapy. J
Rafael Sierra, Virna Cepero, and Silvia Grodano. Molecular Cancer 2010
Suppression of Cytotoxic Responses by a Supernatant Factor derived from
Mycoplasma hyorhinis-infected mammalian cell lines. Hung-sia Teh, Margaret Ho,
and Laura D. Williams , 1987 Infection and Immunity. Vol. 56, No.1
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