CHIR258: A Multitargeted Growth Factor Receptor Tyrosine Kinase (RTK) Inhibitor Induces Significant Apoptosis of Human FLT3 ITD Acute Myelogenous Leukemia Cells In Vitro and in Vivo.

Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. CHIR258, a novel, orally active, small molecule exhibits potent activity against FLT3 kinase and other RTKs involved in myeloid leukemias as well as endothelial and solid tumor cell proliferation (biochemical IC₅₀ for FLT3 = 1 nM; cKIT = 2 nM; VEGFR1/2/3 ~ 8 nM, FGFR1/3 ~ 10nM, PDGFRβ = 27 nM, CSF-R1 = 36 nM). CHIR258 was tested in two human leukemic cell lines with differing FLT3 mutational status (MV4;11, FLT3 ITD vs. RS4;11, FLT3 WT). Cell cycle analysis of leukemic cells incubated with CHIR258 (0.1 μM, 48 h) showed a significant increase in the sub-Go/G1 cell population in MV4;11cells compared to the RS4;11 cells. The pro-apoptotic activity of CHIR258 against MV4;11 cells in culture was confirmed by AnnexinV staining, and appearance of cleaved poly ADP-ribose polymerase (PARP) after 24 h. Interestingly, cleaved PARP fragments were detected with either pulsed (1 h) or continuous incubations of MV4;11 cells with CHIR258. In addition, cell cycle arrest and apoptosis was consistent with degradation of survivin and loss of phosphorylation of Retinoblastoma (Rb) protein observed at CHIR258 concentrations ≥ 0.1 μM. The mechanism of action of CHIR258 was examined in a subcutaneous (s.c.) xenograft model of leukemia in SCID-NOD mice. In s.c. xenografts, daily oral doses of CHIR258 resulted in dose-dependent increased efficacy against FLT3-ITD MV4;11 tumors compared to RS4;11 WT tumors. Modulation of FLT3 downstream signaling molecules (p-FLT3, p-ERK) was achieved in MV4;11 tumors at biologically active doses in vivo, confirming molecular mechanism of action. Target modulation (p-FLT3) was maintained for up to 24 h post-dose. MV4;11 tumor responses to CHIR258 were evidenced within 1–2 days of initiating drug treatment. Immunohistochemical analyses of MV4;11 tumors revealed that early responses to CHIR258 correlated well with decreased tumor cellularity, and proliferation. Tumors stained positive for active caspase-3 and cleaved PARP suggesting cell death was mainly mediated via apoptosis. Efficacy was also demonstrated in an intravenous MV4;11 bone marrow (BM) engraftment model, wherein daily CHIR258 treatments substantially increased the life span of mice, with 33% long term survivors or “cures”. Strikingly, CHIR258-treated mice were devoid of MV4;11 tumor cells in BM as confirmed by flow cytometry and immunohistochemical analyses. In conclusion, our data indicates that CHIR258 might become a highly effective therapy in AML associated with FLT3 expression and warrants clinical trials with AML patients.