P58 The Novel Tyrosine Kinase Inhibitor Nilotinib is Equipotent with Imatinib and Does Not Induce Apoptosis in Chronic Myeloid Leukaemia Stem Cells

The novel tyrosine kinase inhibitor nilotinib is equipotent with imatinib and does not induce apoptosis in CML stem cells Chronic myeloid leukaemia (CML) is a myeloproliferative disorder of stem cell origin characterised by a shortened ‘Philadelphia’ (Ph+) chromosome 22. A novel oncoprotein BcrAbl is translated from the fusion oncogene, bcrabl, that arises from the t 9;22 reciprocal translocation. BcrAbl is a constitutively active tyrosine kinase that upregulates diverse intracellular signal transduction pathways resulting in multiple adverse effects on cell processes including proliferation, differentiation and adhesion. The rationally designed tyrosine kinase inhibitor, imatinib mesylate (IM; Glivec®, Novartis Pharma, Switzerland) has proven highly effective in the treatment of CML with the majority of early chronic phase patients achieving complete cytogenetic remisssion and in some cases molecular remission. However, many advanced phase patients have developed clinical resistance. Previous in vitro studies by our group have highlighted a population of quiescent Ph+ stem cells that persist and accumulate following IM exposure demonstrating the anti-proliferative effects of this drug. Nilotinib, (AMN 107; Novartis Pharma) is a novel Abl tyrosine kinase inhibitor specifically designed to be more selective for BcrAbl with activity against IM- resistant mutations. In the Ph+ K562 cell line, nilotinib's IC50 was 30 ± 10 nm versus 600 ± 60 nM for IM consistent with its reported 20-fold increased potency. However, in primary CD34+ CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, with 5 μM of either compound producing equal but incomplete reduction in Crkl phosphorylation. CD 34+ cells were able to expand over 72 h with either drug up to 5 μM although there was a concentration dependent restriction of amplification. These results confirmed an anti-proliferative rather than pro-apoptotic effect of nilotinib. Morever, the most primitive cells persisted and accumulated over 72 h with nilotinib and remained caspase-3 negative. Combination of IM with nilotinib led to increased accumulation of this population suggesting at least additive anti-proliferative effects. Thus despite activity against IM resistant mutants, nilotinib may not improve clinical responses at the stem cell level with respect to IM.