The seminal discovery of the Bcr-Abl tyrosine kinase inhibitor, imatinib, has revolutionized the therapy of a clonal myeloproliferative disorder, chronic myeloid leukemia (CML). Polycythemia vera (PV) is also a clonal myeloproliferative disorder due to an acquired mutation of hematopoietic stem cells. The majority of PV patients have a somatic mutation of the Janus 2 tyrosine kinase (JAK2 V617F). This provides an excellent clinical opportunity for development of novel tyrosine kinase inhibitors. We studied the efficacy of tyrosine kinase inhibitors in PV and showed a limited effect of imatinib in vitro and in vivo (

Gaikwad et al, Blood 106: 2601, 2005
). Here, we report on the effect of two tyrosine kinase inhibitors, AEE788 and AMN107 (Novartis Pharma) on PV. AEE788 causes growth arrest and apoptosis via inhibition of multiple receptor kinases, including EGFR, HER-2 and VEGFR, which are known to stimulate tumor cell growth and angiogenesis. AMN107 targets Bcr-Abl, KIT, and PDGFR and has potent activity (30 times) even against mutated Bcr-Abl variants that are resistant to imatinib. We used mouse FDCP reporter cells expressing either the wild-type JAK2 (JAK2 cells) or the mutant JAK2 V617F protein (V617F cells) to examine the growth inhibitory activity of these drugs. AEE788 specifically inhibited the growth of V617F cells at an IC50 of 1μM with no effect on wild type JAK2 cells. We also tested the effect of AEE788 on human erythroleukemic cells (HEL) that carry the JAK2 V617F mutation. AEE788 markedly inhibited the growth of HEL cells with an IC50 ~0.8μM. In contrast, AMN107 showed only marginal growth inhibition on V617F cells (IC50 of 12μM) and on HEL cells (IC50 of 8μM). The effect of AMN107 was similar to imatinib activity, wherein only high concentrations of imatinib (IC50 of 5μM) would inhibit the V617F cells, in contrast to imatinib’s known growth inhibitory effect at lower concentrations on the Bcr-Abl carrying cells (Sun X et al Blood 97, 2008Sun X et al Blood 97, 2001). This data suggest that the tyrosine kinase inhibitors that target Bcr-Abl kinase may not be suitable for treating PV. We then tested the combination of AMN107 and AEE788 but did not observe synergistic effect at varying concentrations on reporter cells. The potent activity of AEE788 on JAK2 V617F suggests that AEE788 may act on unique target(s). We observed a decrease in Heat Shock Protein70 (HSP70) upon AEE788 treatment in HEL cells. Elevated levels of HSP70 are associated with drug resistance and poor prognosis (
Creagh EM et al, Leukemia 14: 1161, 2000
). Down-regulation of HSP70 by anti-sense (
Nylandsted et al, PNAS 97:7871, 2000
) and other interventions (Gaikwad A et al, Clin. Cancer Res 11: 1953Gaikwad A et al, Clin. Cancer Res 11: 2005) show the ability to overcome apoptotic resistance. Furthermore, AEE788 showed a specific inhibitory effect on native PV cells when we used in vitro expanded erythroid progenitors from PV patients. These treated PV expanded cells also showed down-regulation of HSP70. We conclude that AEE788 should be explored for the therapeutic trials of PV.

Topics:
polycythemia vera, protein-tyrosine kinase inhibitor, bcr-abl tyrosine kinase, imatinib mesylate, nilotinib, heat-shock proteins 70, cancer, myeloproliferative disease, phosphotransferases, epidermal growth factor receptors

Disclosure: No relevant conflicts of interest to declare.

2006, The American Society of Hematology
2006
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