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Combination of imatinib with rapamycin or RAD001 acts synergistically only in Bcr-Abl-positive cells with moderate resistance to imatinib

TO THE EDITOR

Chronic myelogenous leukemia (CML) is caused by the constitutively active Bcr-Abl tyrosine kinase, which can be effectively inhibited by imatinib mesylate (Gleevec, STI571). Imatinib has become the standard treatment for newly diagnosed CML.1 However, resistance to imatinib due to point mutations in the Bcr-Abl kinase domain is a frequent problem in advanced phase CML therapy.2, 3 This has led to the development of alternative Abl kinase inhibitors and efforts to combine imatinib with inhibitors of pathways downstream of Bcr-Abl. The serine/threonine kinase m-TOR is believed to be important for Bcr-Abl-mediated leukemogenesis, since m-TOR is activated by the PI3-Kinase/Akt-pathway, which in turn is induced by Bcr-Abl.4, 5 The m-TOR inhibitors rapamycin (sirolimus) and its derivative RAD001 (everolimus) have been shown to inhibit the growth of a variety of transformed cells including CML-derived cell lines, and it was recently shown that imatinib and rapamycin can act in a synergistic manner on Bcr-Abl expressing cells.5, 6 However, conflicting data have been reported on the combined effects of imatinib and rapamycin on cells expressing imatinib resistant Bcr-Abl mutations such as Bcr-AblT315I, a mutation mediating strong imatinib resistance.2, 3 Mohi et al6 found imatinib and rapamycin to act synergistically both on Bcr-Abl wild type and Bcr-AblT315I expressing Ba/F3 cells, whereas Ly et al5 observed increased inhibition of Bcr-Ablwt cells, but in contrast to the work by Mohi et al6 resistance to both imatinib and rapamycin in Bcr-AblT315I cells. We therefore examined the effects of combining imatinib with rapamycin using Bcr-Abl mutations mediating different degrees of imatinib resistance and extended our work to RAD001, a derivative of rapamycin with improved oral bioavailability.

We first examined the effects of rapamycin and its more bioavailable derivative RAD001 on IL-3-dependent parental Ba/F3 cells and Ba/F3 cells transformed with p185 Bcr-Abl wild type (Bcr-AblWT). Both cell lines were exposed to escalating doses of rapamycin and RAD001. Rapamycin (Figure 1a) and RAD001 (Figure 1b) inhibited growth of both parental, and wild type Bcr-Abl-positive Ba/F3 cells (Bcr-AblWT) with equal effectivity, suggesting that m-TOR inhibitors suppressed cell growth independent of Bcr-Abl expression. At concentrations above 5 nM rapamycin and 3.2 nM RAD001, a plateau of about 60% inhibition was reached.

Figure 1
figure1

Growth inhibition of parental Ba/F3 cells and Bcr-AblWT expressing Ba/F3 cells by rapamycin (a) and RAD001 (b). Proliferation was measured using an MTS-based method by absorption of formazam at 490 nm. Measures were taken as triplicates after 24 and 48 h. Two independent experiments were performed. Values are expressed as mean of triplicates. Bars: ±s.e. Representative results of one experiment after 48 h of incubation are shown. OD indicates optical density.

Next, we used different concentrations of rapamycin or imatinib and combinations thereof to treat Ba/F3 cells expressing Bcr-Abl WT. As expected, in Bcr-AblWT cells imatinib alone was sufficient to inhibit the cell growth by more than 80% at a concentration of 1 μm (Figure 2a). Rapamycin alone at 1 nM led to 59% growth inhibition, and a combination of 1 nM rapamycin and 500 nM imatinib increased inhibition to 84% (Figure 2a). The combination index (CI) values for imatinib and rapamycin for Bcr-AblWT expressing cells indicated synergistic effects (Figure 2a, lower right), with a CI of 0.55 at 75% growth inhibition (ED75). There have been conflicting data on the effects of imatinib/rapamycin combination treatment of Bcr-Abl mutations that cause strong resistance towards imatinib, such as T315I and E255K. When Ba/F3 cells expressing Bcr-Abl mutants mediating strong imatinib resistance were examined, imatinib alone did not inhibit growth of Bcr-AblT315I or Bcr-AblE255K cells at concentrations up to 2 μ M as expected (Figure 2b and c). In contrast, rapamycin alone clearly inhibited proliferation of these cells, albeit to the same extent as in cells expressing Bcr-AblWT or in parental Ba/F3 cells (compare Figures 1a, 2a–c). Combining rapamycin and imatinib led to inhibition levels that corresponded to those of rapamycin as a single agent (Figure 2b and c). We next examined imatinib and rapamycin on Bcr-AblF317V expressing cells, a mutant that mediates a moderate resistance to imatinib.8 In these cells, synergistic growth inhibition in the presence of various combinations of imatinib and rapamycin could be achieved (Figure 3a, CIED75 of 0.46). Thus, when m-TOR inhibition is employed together with an Abl kinase inhibitor, synergistic inhibition may require significant activity of the kinase inhibitor when used as a single agent. This hypothesis was further strengthened by the observation of synergistic growth inhibition of Bcr-AblE255K cells, when rapamycin was used in combination with PD166326, an Abl kinase inhibitor which is known to suppress Bcr-AblE255K.8 As a single agent, both PD166326 (20 nM) or rapamycin (1 nM) led to a growth inhibition of 60 and 61%, respectively (Figure 3b), their combination however resulted in 85% inhibition. At inhibition levels greater than 60%, synergistic activity could be seen, with a CIED75 of 0.84 (Figure 3b, lower right).

Figure 2
figure2

Growth inhibition of Bcr-AblWT (a) Bcr-AblT315I (b) and Bcr-AblE255K (c) cells by a combination of rapamycin and imatinib after 48 h. Measures were taken as triplicates after 24 and 48 h. Two independent experiments were performed. Values are expressed as mean of triplicates. Representative results of one experiment after 48 h of incubation are shown. In Figure 2a, CalcuSyn software (Biosoft, Ferguson, MO, USA) was used to calculate corresponding combination index (CI) values (lower right), using the median effect method with a constant ratio drug dilution setup to ensure equitoxic effects of two drugs combined, according to Chou and Talalay.7 CI values greater than 1.1 indicate antagonism, between 0.9 and 1.1 additivism, and values below 0.9 synergism.

Figure 3
figure3figure3

Growth inhibition of Bcr-AblF317V cells by a combination of rapamycin and imatinib (a), Bcr-AblE255K cells by a combination of rapamycin and PD166326 (b), and Bcr-AblWT cells (c) and Bcr-AblE255K cells (d) by a combination of RAD001 and imatinib. Representative results of one out of two experiments after 48 h incubation are shown. Values are expressed as mean of triplicates. Corresponding CI values (lower right) were calculated as stated in Figure 2.

Similar results were obtained using the rapamycin derivative RAD001, which was active when used as a single agent, and synergistically active in combination with imatinib in Bcr-AblWT cells (Figure 3c, lower right, CIED75 value 0.74). Like rapamycin, RAD001 effectively suppressed cell growth in strongly imatinib resistant Bcr-AblE255K cells (Figure 3d), and the addition of imatinib did not further increase inhibition. In contrast, combinations of RAD001 with imatinib in Bcr-AblF317V cells (imatinib sensitive, PD166326 resistant, Figure 4a, CIED75 of 0.72) or with PD166326 in Bcr-AblE255K cells (imatinib resistant, PD166326 sensitive, Figure 4b, CIED75 of 0.57) again clearly lead to synergistic action. Thus, RAD001, like rapamycin is a potent combination partner of imatinib or PD166326.

Figure 4
figure4

Growth inhibition of Bcr-AblF317V cells by a combination of RAD001 and imatinib (a) and inhibition of Bcr-AblE255K cells by a combination of RAD001 and PD166326. (b) Representative results of one out of two experiments after 48 h incubation are shown. Values are expressed as mean of triplicates. Corresponding CI values (lower right) were calculated as stated in Figure 2.

Our data suggest that inhibitors of m-TOR, when used in combination with Abl tyrosine kinase inhibitors may increase chances of preventing the emergence of inhibitor resistance. However, synergistic activity of both inhibitor classes was strictly dependent on at least some residual activity of the Abl kinase inhibitor on a particular Bcr-Abl mutant. Mohi et al6 recently reported single activity of imatinib in Bcr-AblT315I cells. Consequently, these authors achieved synergistic inhibition using combinations of imatinib and rapamycin in Ba/F3 cells expressing Bcr-AblT315I.6 This synergistic effect was not seen in our study or in the study by Ly et al5 as imatinib reportedly is not capable of suppressing Bcr-AblT315I.2, 3 Therefore, combination treatment with imatinib should be most effective in cases where Bcr-Abl mutants cause moderate resistance to imatinib but not in cells harbouring a Bcr-Abl mutant mediating complete resistance to Abl inhibition. In this case, novel Abl kinase inhibitors may be employed in combination with m-TOR inhibitors.

RAD001 displays superior bioavailability after oral administration and showed equal effectivity to rapamycin. RAD001 can therefore be viewed as a reasonable alternative to rapamycin in CML therapy and has already entered clinical trials for imatinib resistant CML patients.

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Correspondence to J Duyster.

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Dengler, J., von Bubnoff, N., Decker, T. et al. Combination of imatinib with rapamycin or RAD001 acts synergistically only in Bcr-Abl-positive cells with moderate resistance to imatinib. Leukemia 19, 1835–1838 (2005). https://doi.org/10.1038/sj.leu.2403848

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