Supplementary Figure S1. Biochemical data for UM-164 and dasatinib. Supplementary Figure S2. Western blots showing the expression of P-Src, P-EGFR and P-p38MAPK in selected TNBC cell lines. Supplementary Figure S3. UM-164 is a dual c-Src/p38 kinase inhibitor in TNBC cell lysate. Supplementary Figure S4. Signaling pathway analysis of SUM 149 cells when treated with dasatinib or UM-164 at their IC50 values (630 nM and 230 nM, respectively). Supplementary Figure S5. Effect on cell invasion of dasatinib alone and dasatinib plus a p38MAPK inhibitor, BIRB-796. Supplementary Figure S6. Effect of dasatinib alone or in combination with p38MAPK inhibitors on the level of P-EGFR/Tyr1068 and P-p38MAPK. Supplementary Figure S7. UM-164 is cytostatic and does not induce apoptosis. Supplementary Figure S8. Histopathology of major organ systems from mice treated with 140 mg/kg UM-164 for 5 days and necropsied. Supplementary Figure S9. In vivo efficacy of UM-164 compared to dasatinib in MDA-MB 231 xenografts. Supplementary Figure S10. Inhibition of p-Src in MDA-MB 468 cells by UM-164 and dasatinib. Supplementary Figure S11. Effect of UM-164, dasatinib alone and in combination with BIRB-796 in spheroid culture. Supplementary Figure S12. Wetsern blot from the UM-164 treated tumor tissues showing expression of P-38MAPK, P-EGFR and P-Src.
ARTICLE ABSTRACT
Purpose: c-Src has been shown to play a pivotal role in breast cancer progression, metastasis, and angiogenesis. In the clinic, however, the limited efficacy and high toxicity of existing c-Src inhibitors have tempered the enthusiasm for targeting c-Src. We developed a novel c-Src inhibitor (UM-164) that specifically binds the DFG-out inactive conformation of its target kinases. We hypothesized that binding the inactive kinase conformation would lead to improved pharmacologic outcomes by altering the noncatalytic functions of the targeted kinases.Experimental Design: We have analyzed the anti–triple-negative breast cancer (TNBC) activity of UM-164 in a comprehensive manner that includes in vitro cell proliferation, migration, and invasion assays (including a novel patient-derived xenograft cell line, VARI-068), along with in vivo TNBC xenografts.Results: We demonstrate that UM-164 binds the inactive kinase conformation of c-Src. Kinome-wide profiling of UM-164 identified that Src and p38 kinase families were potently inhibited by UM-164. We further demonstrate that dual c-Src/p38 inhibition is superior to mono-inhibition of c-Src or p38 alone. We demonstrate that UM-164 alters the cell localization of c-Src in TNBC cells. In xenograft models of TNBC, UM-164 resulted in a significant decrease of tumor growth compared with controls, with limited in vivo toxicity.Conclusions: In contrast with c-Src kinase inhibitors used in the clinic (1, 2), we demonstrate in vivo efficacy in xenograft models of TNBC. Our results suggest that the dual activity drug UM-164 is a promising lead compound for developing the first targeted therapeutic strategy against TNBC. Clin Cancer Res; 22(20); 5087–96. ©2016 AACR.
