KX-01, a Novel Src Kinase Inhibitor Directed towards the Peptide Substrate Site, Induces Robust Apoptosis and Synergizes with Tamoxifen and Chemotherapy in Breast Cancer.

Abstract

Abstract New therapeutic regimens that increase efficacy or reduce onset of resistance for endocrine therapy and chemotherapy are needed for better clinical management of breast cancer. c-Src is an oncogenic non-receptor tyrosine kinase that is up-regulated in approximately half of all breast cancer. However the efficacy of existing multi-kinase src inhibitors in breast cancer has been limited. KX-01 (Kinex Pharmaceuticals) is a novel class of non-ATP src inhibitor that targets the peptide binding site of src and is currently completing Phase-1 testing for solid tumors. In a panel of breast cancer cell lines, KX-01 resulted in dose dependent inhibition of growth and induction of apoptosis that was independent of p53 status, and was preceded by rapid inhibition of src activity. KX-01 induced apoptosis in two cell lines reported to be resistant to multi-kinase src inhibitors, MDA-MB-468 cells and BT-549. Cell cycle analysis revealed that KX-01 (50 nM, 6 hours) resulted in significant accumulation of MDA-MB-231 cells (ERα/PR/HER2/neu negative) and MCF-7 cells (ERα positive) in G2/M phase. Immunofluorescent staining for mitotic phase marker phospho-histone 3 indicated that cells had arrested in mitotic phase and many of the mitotic arrested cells were undergoing apoptosis (TUNEL), a novel cell death for a small molecule tyrosine kinase inhibitor. KX-01 induced nuclear accumulation of cyclin B1, and activation of CDK1, MPM2 and Cdc25C that is required for progression past the G2/M checkpoint. KX-01 resulted in cytochrome C release and activation of caspases 3, 6, 7, 8 and 9. A matrix design using the median-effect principle to delineate the interaction between two drugs was applied for KX-01 alone and in combination tamoxifen (TAM), paclitaxel (PAC) or doxorubicin (DOX). Combinations of KX-01 (5-75 nM) with each of these agents resulted in synergistic growth inhibition of MCF-7 cells (KX-01 + TAM) and MDA-MB-231 cells (KX-01 + DOX or PAC). In addition, synergistic induction of apoptosis was achieved by combining low doses of KX-01 with DOX, PAC or TAM. c-Src induces phosphorylation of ERα at serines 118 and 167, sites required for full receptor activity. KX-01 combined with TAM resulted in decreased phosphorylation at serine 167 that was associated with reduced transcriptional activity of ERα. In tumor xenograft models, KX-01 resulted in a dose dependent inhibition of MDA-MB-231 and MCF-7 tumor growth after 30 days (1, 2.5 or 5 mg/kg body weight, twice daily by oral gavage). In MDA-MB-231 xenografts, KX-01 reduced metastasis to bone (femur) and lung as measured by PCR for detection of human chromosome 17.These data define KX-01 as a potently active src kinase inhibitor that induces robust cell death, tumor growth inhibition and anti-metastatic effects. Combinations of KX-01 with endocrine therapy and chemotherapy present a promising new strategy for clinical management of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6104.