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 IC 50 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.