Elsevier

Leukemia Research

Volume 30, Issue 11, November 2006, Pages 1365-1370
Leukemia Research

Effects of AMN107, a novel aminopyrimidine tyrosine kinase inhibitor, on human mast cells bearing wild-type or mutated codon 816 c-kit

https://doi.org/10.1016/j.leukres.2006.04.005Get rights and content

Abstract

Most adults with systemic mastocytosis (SM) carry an activating mutation in the codon 816 of c-kit. We investigated the activity of the new tyrosine kinase inhibitor AMN107 on c-kit mutated mast cell lines and bone marrow samples from patients with SM and compared it to that of imatinib mesylate, a tyrosine kinase inhibitor effective in some patients with SM. In HMC-1560 mast cells carrying wild-type codon 816 c-kit, AMN107 was very effective and as potent as imatinib in inhibiting cellular proliferation and inducing apoptosis (P < 0.0823). By contrast, in HMC-1560,816 cells bearing a c-kit mutation in codon 816, neither drug exerted a significant effect (P < 0.0015). AMN107 was also as effective as imatinib in inhibiting phosphorylation of c-kit in HMC-1560 cells. However, AMN107 had little effect on ex vivo survival of bone marrow mast cells with 816 c-kit mutation obtained from patients with SM. Based upon our results, AMN107 and imatinib are equipotent against mast cells with wild-type c-kit and those harboring the juxtamembrane D560G c-kit mutant but have no significant activity over the dose range tested against cells expressing the c-kit D816V mutant tyrosine kinase.

Introduction

Systemic mastocytosis (SM) is characterized by a clonal accumulation of mast cells (MC) in bone marrow and other tissues [1], [2]. Malignant MC are abnormally spindle-shaped, express aberrant surface markers (CD2 and CD25), and carry, in most cases, a mutation involving codon 816 of the c-kit gene (D816V) [1], [2]. This mutation results in constitutively activated c-kit receptor tyrosine kinase activity that is believed to be important for disease progression. Thus, agents that antagonize this mutated form of c-kit may have clinical benefit in SM [3]. MC lines with D816V mutation may serve as effective screening models to test for such agents. Imatinib mesylate (imatinib, Gleevec®), a highly effective inhibitor of the Bcr-Abl tyrosine kinase, is associated with high complete cytogenetic remission rates in patients with chronic myelogenous leukemia (CML) resistant or intolerant to imatinib [4]. Because of its potential activity on c-kit tyrosine kinase, imatinib was evaluated in vitro against MC expressing the D816V tyrosine kinase mutant but proved ineffective [5]. AMN107 is a novel, potent, orally bioavailable Bcr-Abl inhibitor, rationally designed based on the crystallographic structure of the imatinib–Bcr-Abl complex. Against CML cell lines, AMN107 exhibited 20- to 50-fold higher potency as a Bcr-Abl inhibitor than imatinib [6], [7]. We have recently reported on the activity of AMN107 in both imatinib-sensitive and -resistant p190 Bcr-Abl-expressing lymphoid cells and p210 Bcr-Abl-expressing myeloid leukemic cells [8], [9].

In the current study, we report on the preclinical activity of AMN107 against MC lines harboring juxtamembrane and activation loop mutations and compared it to that of imatinib. Moreover, we compared the activity of both tyrosine kinase inhibitors against primary MC from patients with SM harboring D816V c-kit. Our results suggest equipotency of both tyrosine kinase inhibitors against the above-mentioned cellular targets.

Section snippets

Drugs

Imatinib and AMN107 (NVP-AMN107-NX; 4-methyl-N-[3-(4-methyl-1H-imidazol)-5-(trifluoromethyl) phenyl]-3-(3-pyridinyl)-2pyridinyl]amino] benz amide) were kindly provided by Novartis Pharma AG, Basel, Switzerland. Drug 10 mM stock dilutions were prepared in dimethylsulfoxide and stored at −20 °C (imatinib) or +4 °C (AMN107). Working dilutions were prepared in 10% media just before use.

Cell lines

MC lines were kindly provided by Dr. Joseph Butterfield (Mayo Clinic, Rochester, MN): HMC-1560 carrying juxtamembrane

Results and discussion

SM is a rare hematological malignancy with variable clinical outcome [1], [2]. Most patients present with indolent SM and exhibit signs and symptoms related to MC activation. In these cases, empirical therapy aiming to improve quality of life, rather than cytoreductive therapy, is customarily employed [1], [2]. On the other hand, patients presenting with aggressive SM (ASM) experience organ dysfunction due to infiltration of neoplastic MC and their prognosis is dismal [1], [2]. Current

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