BMS-354825 Potently Inhibits the Kinase Activity of KIT Activation Loop Mutations Associated with Systemic Mastocytosis and Induces Apoptosis of Mastocytosis Cell Lines.

Activating mutations of the activation loop (AL) of KIT are reported in several human neoplasms including a subset of patients with AML or systemic mast cell disorders. The small molecule tyrosine kinase inhibitor, imatinib, is a potent inhibitor of wild type KIT and certain mutant KIT isoforms and has become the standard of care for treating patients with metastatic GI stromal tumors. However, KIT AL mutations such as D816V that are typically found in AML and systemic mast cell disease are insensitive to imatinib (IC₅₀ > 5–10 micromolar). In addition, acquired AL mutations are associated with imatinib resistance in GI stromal tumors. Experimental models of these diseases suggests that targeted inhibition of AL-mutant KIT in such cells would result in decreased proliferation and induction of apoptosis. BMS-354825 is a synthetic small-molecule ATP-competitive inhibitor of SRC and ABL tyrosine kinases with potency in the low nanomoloar range. In addition, BMS-354825 also inhibits many of the acquired BCR-ABL mutations associated with imatinib-resistant CML. Notably, BMS-354825 binds to the ATP-binding site in ABL regardless of the conformation of the AL and is therefore able to potently inhibit the kinase activity of AL-mutants of BCR-ABL. BMS-354825 is currently being tested in phase I studies for safety and efficacy in the treatment of imatinib-resistant CML as well as for various solid tumors. We investigated the inhibitory potential of BMS-354825 against KIT AL-mutants expressed by different mastocytosis/mast cell leukemia cell lines. We first examined the activity of BMS-354825 against the kinase activity of wild-type KIT kinase expressed by M07e cells. BMS-354825 potently inhibited the kinase activity of SLF-dependent M07E cells with an IC₅₀ of 1–10 nM for autophosphorylation and cellular proliferation. In contrast, the IC₅₀ for inhibition of GM-CSF-dependent proliferation was ~5 micromolar. BMS-354825 also potently inhibited the proliferation of the human mast cell leukemia cell line HMC-1 that expresses a juxtamembrane KIT V560G mutant kinase (IC₅₀ 1–10 nM). Notably, BMS-354825 inhibited autophosphorylation of KIT D816V with an IC50 of ~100 nM. Similar potency for inhibition of proliferation and induction of apoptosis (IC₅₀ 150–400 nM) was seen using three different cell lines expressing human D816V or the homologous murine D814Y KIT kinase. We conclude that BMS-354825 potently inhibits the kinase activity of wild-type KIT and imatinib-resistant KIT AL-mutant kinases associated with AML and systemic mast cell disorders. If BMS-354825 proves to be safe and tolerable in ongoing phase I trials, it should be further tested in clinical studies targeting patients with human malignancies associated with imatinib-resistant KIT AL-mutations.