Abstract

Imatinib mesylate (Novartis Pharma), an inhibitor of the bcr/abl tyrosine kinase, has rapidly become the first-line therapy for CML. Imatinib has proved remarkably effective at reducing the number of leukaemia cells in individual CML patients and promises to prolong life substantially in comparison with earlier treatments. However, in patients in advanced phases of the disease, the development of resistance to this drug is a frequent setback. Therefore, new inhibitors of bcr/abl are needed. Very recently, a new bcr/abl inhibitor, AMN107 (Novartis Pharma), has been developed. We have tested AMN107 on human leukaemia cell lines and on blasts isolated from imatinib-resistant CML patients. After a 24 h incubation, AMN107 (10 nM) blocked K562 cells in the G1 phase of the cell cycle. To obtain the same effect with imatinib, a 200 nM concentration was required. AMN107 had no affect on cell cycle progression of bcr/abl-negative cell lines such as HL60 and NB4, even if the concentration was raised to 500 nM. After 48 h incubation, AMN107 (10 nM) was capable of inducing a massive apoptosis of K562 cells whereas, once again, 200 nM imatinib was required to obtain the same effect.

Western blot analysis with phosphospecific antibodies revealed that in K562 cells AMN107 (50 nM) markedly down-regulated autophosphorylation of bcr/abl Tyr177 and Tyr412, whereas autophosphorylation of Thr735 was unaffected. In contrast, imatinib even if used at 200 nM, did not diminish phosphorylation of either bcr/abl Tyr177 or Tyr412. This finding seems particularly important because recent evidence has demonstrated that the signalling pathway emanating from Tyr177 plays a major role in the pathogenesis of CML. Indeed, phosphorylated Tyr177 forms a high-affinity binding site for the SH2 domain of the adapter Grb2. The main effectors of Grb2 are Sos and Ras, however Grb2 also recruits the scaffolding adapter protein Gab2 to bcr/abl via a Grb2-Gab2 complex, which results in activation of phosphoinositide 3-kinase (PI3K)/Akt and Erk signalling networks. Consistently, we found by immunoprecipitation decreased levels of bcr/abl-associated Gab2, Grab2, and p85 regulatory subunit of PI3K in AMN107-treated cells. AMN107 treatment of K562 cells also caused a reduction of STAT5, cCBL, CRKL, and Akt phosphorylation levels, as well as Bcl-XL expression. AMN107 (5 μM for 24h) significantly increased the apoptosis rate of CML blasts isolated from patients resistant to imatinib. Therefore, AMN107 might represent a new bcr/abl selective inhibitor useful for overcoming imatinib resistance.

COFIN 2003 (Molecular therapy of Ph+ leukemias), by FIRB 2001, by the University of Bologna (60%), by the Italian Association for Cancer Research (A.I.R.C.), by the Italian National Research Council (C.N.R), by Fondazione Del Monte of Bologna and Ravenna (Italy) and A.I.L. grants.

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