BCR-ABL nuclear entrapment kills human CML cells: ex vivo study on 35 patients with the combination of imatinib mesylate and leptomycin B
Blood Aloisi et al.
107: 1591
Supplemental materials for: Aloisi et al
METHODS
Cell lines and Pharmacological associations
Experiments were carried out on four human and four murine BCR-ABL-positive cell lines. Specifically, three of the four human CML cells (K562, KCL22 and LAMA84) are sensitive to IM, while LAMA84r are resistant to the drug because of BCR-ABL gene amplification1.
Mouse pro-B BaF cells expressing either p210 BCR-ABL (BaF/BCR-ABL) or BCR-ABL mutants Y253F (Ba/F3p210Y253F), D276G (Ba/F3p210D276G) or T315I (Ba/F3p210T315I) have been reported previously2,3. BaF/BCR-ABL are sensitive to IM. Ba/F3p210D276G show a 3 fold increase in the IC50 for IM, while both Ba/F3p210Y253F and Ba/F3p210T315I display a >10 fold increase in the IC50 for IM.
All cells were maintained in RPMI 1640 medium (Sigma-Aldrich) additioned with 10% heat-inactivated fetal bovine serum, 100 Units/mL Penicillin, 100 µg/mL Streptomycin and 2 mM Glutamine (all from Sigma-Aldrich).
Antileukemic agents associated with IM were: Cytosine Arabinoside (AraC); Arsenic Trioxide (As2O3); Hydroxyurea (HU) and Etoposide (VP-16). All drugs were purchased from Sigma-Aldrich. AraC was dissolved in sterile water as a 200mM stock and kept at –20°C. In our experiments the drug was used at a final concentration of 100nM. As2O3 was prepared in 1N NaOH as a 1mM stock, stored at room temperature and used at a final concentration of 1µM. HU was dissolved in sterile water to obtain a stock solution of 200mM and used at a final concentration of 200µM. Because of the instability of the solubilized compound, fresh stocks were prepared prior to each experiment. VP-16 was prepared in 100% DMSO as a 16mM stock that was kept at –20°C. The working concentration of the drug used in our experiments was 16µM. Final concentrations for each drug were obtained by serial dilutions of the stock with medium and prepared fresh before each experiment.
Treatment regimens and MTT assays
Treatments with IM and LMB, alone or in combination, followed the scheme depicted in Figure 1. For all other pharmacological associations, cells were exposed for 24 hours to 10µM IM plus the indicated antileukemic agent at the concentration specified above.
For Methyl-thiazol-tetrazolium (MTT) assays, 100µL of cell suspensions (at a concentration of 1×106/mL) were dispensed into individual wells of a 96-well plate (BD Falcon, Franklin Lakes, NJ), with each experimental condition being plated in an 8-well column. In experiments comparing the efficacy of IM or LMB alone versus their combination, one 8-well control column contained cells exposed for 12 hours to 70% Methanol (LMB diluent). In experiments studying the growth inhibition of the IM-LMB combination compared to other pharmacological associations, we plated three 8-well control columns: one with cells treated for 12 hours with Methanol, one with cells additioned with NaOH (As2O3 diluent) for 24 hours, and a further one with cells exposed for 24 hours to DMSO (VP-16 diluent). At the end of treatment, the antiproliferative effect of each drug combination was ascertained by measuring the uptake of the MTT reagent (Calbiochem, San Diego, CA) with a spectrophotometer at 570 nm. Growth inhibition rates were determined using average and standard deviation of four or five independent experiments and calculating percentage viable cells versus control cells arbitrarily set at 100%. As previously reported, for each octuplicate representative of an experimental condition we discarded the two highest and the two lowest numbers and used the remaining four values4.
Statistical analysis
Unpaired, single-tail t tests were used to evaluate the efficacy of the combination of IM and LMB versus each drug alone.
RESULTS
Effect of the combination of IM and LMB on IM-resistant cells
Despite the clinical success of IM, an increasing amount of evidence suggests that a relevant number of CML patients treated with IM will develop resistance to the drug. To establish the efficacy of the combination of IM and LMB on cells that have become unresponsive to IM, we used two different cell models.
We initially employed murine pro-B cells engineered to express the p210 BCR-ABL oncoprotein (BaF/BCR-ABL), or BCR-ABL mutants that have partially (Ba/F3p210D276G) or completely (Ba/F3p210Y253F and Ba/F3p210T315I) lost sensitivity to IM2,3. Control BaF/BCR-ABL cells exhibited a good response to IM, unlike the other cells tested (Figure S1A). LMB displayed a modest growth inhibition on every cell line. Compared to IM or LMB monotherapy, the combination of the two agents produced the highest proliferative reduction in BaF/BCR-ABL and Ba/F3p210D276G cells (p<0.001) that are either completely or partially responsive to IM (Figure S1A). However, the two-drug combination was ineffective on cells expressing BCR-ABL mutants that could no longer be inactivated by IM.
We then repeated these experiments on LAMA84s (IM sensitive) and LAMA84r cells that are resistant to IM because of BCR-ABL gene amplification1. Proliferation of LAMA84s was reduced by both IM and LMB, albeit at different levels (Figure S1B). As expected, the IM-LMB combination produced a more significant cell growth reduction than either IM or LMB alone (p<0.001) (Figure S1B). Interestingly, the combination of IM and LMB generated similar results on LAMA84r cells, with a highly significant increase in growth inhibition compared to IM or LMB used as single agents (p<0.001) (Figure S1B).
Our data suggested that cells expressing BCR-ABL mutants that have completely lost their sensitivity to IM will not be killed by the combination of IM and LMB. However, leukemic cells harboring BCR-ABL mutants that have a lower affinity for IM, or expressing an increased number of IM-sensitive BCR-ABL molecules (due to gene or chromosome amplifications), are still likely to respond to the two-drug combination.
Comparison of the efficacy of IM plus LMB versus the association of IM with other antileukemic agents
We next wanted to determine how the antiproliferative effect of IM and LMB would compare to previously reported associations of IM with antileukemic agents5-7. We therefore selected a concentration of each drug that had shown significant growth inhibitory activity on BCR-ABL-positive cells5-7 and compared the efficacy of IM and LMB to that of the combinations of IM with AraC, As2O3, HU and VP-16. Using three human CML lines sensitive to IM (K562, KCL22, LAMA84) and murine BaF/BCR-ABL cells, we observed that the combination of IM with LMB generated the highest growth inhibition (Figure S2). All other drug associations were less effective.
Comparison of the efficacy of IM plus LMB versus other IM-based associations in IM-resistant cells
In order to compare the efficacy of the combination of IM and LMB with that of other IM-based regimens in cells that had become unresponsive to IM, we treated BaF/BCR-ABL, Ba/F3p210Y253F, Ba/F3p210D276G, and Ba/F3p210T315I cells with IM and LMB or with the pharmacological associations described in Figure S2. The combination of IM and LMB was the most effective treatment for BaF/BCR-ABL and Ba/F3p210D276G cells (Figure S3A). However, this treatment was ineffective on cells expressing BCR-ABL mutants totally insensitive to IM. When we repeated these experiments on LAMA84s (control) cells, we found the association of IM with As2O3 to produce the highest growth inhibition, with the combination of IM and LMB producing similar results (Figure S3B). However, the IM-LMB combination was by far the most effective treatment on LAMA84r cells over-expressing BCR-ABL because of gene amplification (Figure S3B).
REFERENCES
1. le Coutre P, Tassi E, Varella-Garcia M, et al. Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification. Blood. 2000;95:1758-1766.
2. Corbin AS, La Rosee P, Stoffregen EP, Druker BJ, Deininger MW. Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib. Blood. 2003;101:4611-4614.
3. Piazza RG, Magistroni V, Gasser M, et al. Evidence for D276G and L364I Bcr-Abl mutations in Ph+ leukaemic cells obtained from patients resistant to Imatinib. Leukemia. 2005;19:132-134.
4. Kano Y, Akutsu M, Tsunoda S, et al. In vitro cytotoxic effects of a tyrosine kinase inhibitor STI571 in combination with commonly used antileukemic agents. Blood. 2001;97:1999-2007.
5. Thiesing JT, Ohno-Jones S, Kolibaba KS, Druker BJ. Efficacy of STI571, an abl tyrosine kinase inhibitor, in conjunction with other antileukemic agents against bcr-abl-positive cells. Blood. 2000;96:3195-3199.
6. Topaly J, Zeller WJ, Fruehauf S. Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells. Leukemia. 2001;15:342-347.
7. La Rosee P, Johnson K, Corbin AS, et al. In vitro efficacy of combined treatment depends on the underlying mechanism of resistance in imatinib-resistant Bcr-Abl-positive cell lines. Blood. 2004;103:208-215.
