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The systemic mastocytosis-specific activating cKit mutation D816V can be inhibited by the tyrosine kinase inhibitor AMN107


Activating mutations of the Kit receptor tyrosine kinase have been identified in several neoplastic diseases, including gastrointestinal stromal tumors (GIST),1 acute myelogenous leukemia2 and systemic mast cell disease (SMCD).3 In systemic mastocytosis, an exchange from an aspartate to a valine at position 816 can be identified in the majority of cases.3 Imatinib (Glivec, STI571) is a tyrosine kinase inhibitor capable of inhibiting mutant cKit and thus induces responses in patients with GIST4 and cKit-positive AML.5 Unfortunately, imatinib is not active in mast cell disease, since the D816V activating cKit mutation is located within the activation loop and causes a strong resistance to imatinib by virtue of a structural change at the entrance of the enzymatic pocket.6 AMN107 is a novel tyrosine kinase inhibitor displaying promising activity in patients with imatinib-resistant chronic and advanced-phase CML.7 In cKit activating mutations other than cKitD816V such as cKitK642E or cKitdel560−561, AMN107 displays equipotent activity to imatinib.8 We therefore aimed to determine whether AMN107 might be useful in the treatment of systemic mastocytosis. We examined the effect of AMN107 on Ba/F3 cells, which were transformed with murine cKit harbouring cKitD814V corresponding to human cKitD816V. As shown in Figure 1, imatinib did not inhibit cell proliferation in Ba/F3 cKitD814V cells as expected. In contrast, AMN107 effectively suppressed the growth of Ba/F3 cKitD814V cells at inhibitor concentrations of 1–2 μ M (Figure 1). Similar results were obtained with Ba/F3 cells expressing the human cKitD816V mutant. We then examined whether treatment of cKitD814V cells with AMN107 gives rise to apoptotic cell death. AMN107 initiated apoptosis in cKitD814V cells with 83 and 96 per cent Annexin V-positive cells after 48 hours of culture in the presence of 2 and 4 μ M AMN107, respectively (Figure 2).

Figure 1

Growth inhibition of Ba/F3 cells expressing cKitD814V by AMN107 and imatinib. Proliferation was measured using an MTS-based method by absorption of formazam at 490 nm. Measures were taken as triplicates after 24 h of culture without and in the presence of inhibitor at the indicated concentrations. Values are expressed as mean values of growth inhibition from three independent experiments. For each experiment, measures were taken in triplicates. Bars: ±s.e. OD indicates optical density.

Figure 2

Induction of apoptosis by AMN107 in Ba/F3 cells expressing cKitD814V. Annexin V-positive cells were measured at the indicated time points in the presence of AMN107 at different concentrations. Two experiments were performed. Results of one representative experiment are shown.

We conclude that AMN107 constitutes a promising candidate for the treatment of systemic mastocytosis, a disease which is typically driven by the imatinib-resistant, activating cKit mutation D816V.


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Correspondence to J Duyster.

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von Bubnoff, N., Gorantla, S., Kancha, R. et al. The systemic mastocytosis-specific activating cKit mutation D816V can be inhibited by the tyrosine kinase inhibitor AMN107. Leukemia 19, 1670–1671 (2005).

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