ALBERT

Description: Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myelogenous Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Imatinib is a selective inhibitor of Bcr-Abl tyrosine kinase and is now used in frontline therapy for CML. However clinical resistance is the main concern using this treatment, mediated by mutations within the kinase domain of Bcr-Abl, amplification of the BCR-ABL genomic locus or other as yet unknown mechanisms. AMN107 (Novartis Pharma AG, Basel, Switzerland) is a synthetic, second generation inhibitor of Bcr-Abl tyrosine kinase. In the current study, we tested AMN107 against different Bcr-Abl positive cell lines such as K562, LAMA84, AR230 or murine Ba/F3 cells transfected with BCR-ABL (BaF/BCR-ABL). In a 4 day proliferation assay (MTS) the dose of AMN107 that inhibited 90% of the cells (IC90) was 0.01 μM which was 100-fold lower than the IC90 of imatinib. In addition, proliferation of imatinib resistant cell lines which exhibited amplification of BCR-ABL was inhibited by 75% in the presence of 0.01 μM AMN107. However, Ba/F3 cells expressing the imatinib resistant BCR-ABL T315I mutant were only inhibited with 10μM of AMN107. Furthermore, K562-R, an imatinib-resistant cell line exhibiting a new mechanism of imatinib resistance (modification of chaperone proteins such as heat shock proteins) was also insensitive to AMN107; the IC90 for AMN107 at day 4 was 1μM versus 4μM for imatinib. Finally, we investigated potential resistance to AMN107 in Bcr-Abl positive cells. Resistant cell lines were generated after long-term (2 month) gradual dose-escalation exposure to the inhibitor. Up to now, we have obtained four cell lines, AR230-ra, K562-ra, LAMA 84-ra, and BaF/BCR-ABL-ra resistant to 8, 10, 10, and 100 nM of AMN107, respectively. Resistance was defined as the capacity to survive in the continuous presence of doses of AMN107 that kill in three days more than 90% of the parental cells in liquid culture. Preliminary investigations of AMN107 resistance using western blot and cytometry have shown that only BaF/BCR-ABL-ra overexpressed wildtype Bcr-Abl as we have already reported for imatinib resistance. Studies with these resistant cell lines investigating cross resistance with imatinib and looking for additional mechanisms of resistance are in progress.We conclude that in vitro, AMN107 is more powerful than imatinib in inhibiting the proliferation of BCR-ABL positive cell lines. In addition, we have demonstrated that it is possible to develop resistant cell lines to this new inhibitor of Bcr-Abl.

Description: Targeting the tyrosine kinase activity of Bcr-Abl by imatinib mesylate is an attractive therapeutic strategy in chronic myelogenous leukemia (CML) and in Bcr-Abl positive acute lymphoblastic leukemia. However, resistance to imatinib monotherapy is currently a major issue preventing the successful treatment of CML patients. It may be mainly mediated by mutations within the kinase domain of Bcr-Abl and/or amplification of the BCR-ABL genomic locus. The K562-r imatinib-resistant cell line, derived in our laboratories from the sensitive parental K562-s has neither mechanism of resistance, nor overexpression of Src-kinases such as Lyn and Hck, as described for other cell lines. In the current study we used two-dimensional (2D) difference gel electrophoresis (DIGE) and MALDI-TOF-TOF mass spectrometry to compare the proteome of K562-r and K562-s. With the aid of the Image Master TM 2D platinium software, we detected 31 different proteins in K562-r and K562-s. These proteins were classified in 3 different groups. The first includes proteins involved in the synthesis and stability of RNA (hnRNP K, hnRNP H, CstF , transcription elongation factor A protein 1, PCBP2, TCP1), the second encompasses structural proteins (CAPG, fascin, tubulin, vimentin, laminA, C tubulin beta-1 chain, actin cytoplasmic 1, keratin type I type II), and the third was represented by different enzymes participating in general metabolic pathways (glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, mitochondrial precursor, glutamate dehydrogenase 2, pyruvate kinase, PURH protein). Furthermore, chaperone proteins such as heat-shock protein Hsp60, P60HOP or STI-1, Hsp105 and Hsp70 were differentially expressed in the sensitive and resistant cell lines. Since these proteins complex with Hsp90 and this complex has been reported to interact with the Bcr-Abl protein, we focused on these molecular chaperones. Hsp70 family proteins such as Hsc70 and Hsp74 were found to be more expressed 2.5-fold higher in K562-r than in K562-s, and/or exhibited post translational modifications (phosphorylation and acetylation) confirmed by Western blotting. Hsp70 was recently described as an inhibitor of apoptosis (Ray S et al., JBC 2004) and its overexpression in K562-r could thus contribute to its imatinib-resistant phenotype. Preliminary functional studies showed that whereas K562-s and K562-r were equally sensitive to the apoptotic effect of geldanamycin (an inhibitor of Hsp90), the combination of geldanamycin and a proteasome inhibitor (MG132) was more efficient in K562-r than in K562-s (viability of 16% and 40% respectively after 4 days in culture). Ongoing experiments utilizing siRNA against Hsp70 will help understand the link between the expression profile of Hsp proteins and the imatinib-resistant phenotype of this cell line. In conclusion, the use of a new experimental strategy, i.e. proteomic analysis by DIGE and mass spectrometry, allowed us to identify selected proteins whose patterns of expression and post-translational modification may underlie a new mechanism of resistance to imatinib in Bcr-Abl positive cells.