1 Leukemia 2005 Vol: 19(10):1835-1838. DOI: 10.1038/sj.leu.2403848

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

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Figures
Figure 1: 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. Figure 2: 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 a, 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: 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 4: 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
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References
  1. O'Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M & Cervantes F et al.. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: 994−1004 , .
    • . . . 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 . . .
  2. Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R & Rao PN et al.. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001; 293: 876−880 , .
    • . . . 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 . . .
    • . . . 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 . . .
  3. von Bubnoff N, Schneller F, Peschel C & Duyster J. BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: a prospective study. Lancet 2002; 359: 487−491 , .
    • . . . 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 . . .
    • . . . 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 . . .
  4. Deininger MW, Goldman JM & Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000; 96: 3343−3356 , .
    • . . . 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 . . .
  5. Ly C, Arechiga AF, Melo JV, Walsh CM & Ong ST. Bcr-Abl kinase modulates the translation regulators ribosomal protein S6 and 4E-BP1 in chronic myelogenous leukemia cells via the mammalian target of rapamycin. Cancer Res 2003; 63: 5716−5722 , .
    • . . . 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 . . .
    • . . . 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 . . .
  6. Mohi MG, Boulton C, Gu TL, Sternberg DW, Neuberg D & Griffin JD et al.. Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs. Proc Natl Acad Sci USA 2004; 101: 3130−3135 , .
    • . . . 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 . . .
    • . . . 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 . . .
    • . . . 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 . . .
  7. Chou TC & Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27−55 , .
    • . . . 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. . . .
  8. von Bubnoff N, Veach DR, van der Kuip H, Aulitzky WE, Sanger J & Seipel P et al.. A cell-based screen for resistance of Bcr-Abl-positive leukemia identifies the mutation pattern for PD166326, an alternative Abl kinase inhibitor. Blood 2005; 105: 1652−1659 , .
    • . . . 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) . . .
    • . . . 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 . . .
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