1 Cancer Therapy : Preclinical 2 Peptides Mimicking the Unique ARTS-XIAP Binding Site 3 Promote Apoptotic Cell Death in Cultured Cancer Cells 4 5

7 Abstract 8 Purpose: XIAP [X-linked inhibitor of apoptosis (IAP) protein] is the best characterized mammalian 9 caspase inhibitor. XIAP is frequently overexpressed in a variety of human tumors, and genetic inactivation of 10 XIAP in mice protects against lymphoma. Therefore, XIAP is an attractive target for anticancer therapy. IAP 11 antagonists based on a conserved IAP-binding motif (IBM), often referred to as "Smac-mimetics," are 12 currently being evaluated for cancer therapy in the clinic. ARTS (Sept4_i2) is a mitochondrial proapoptotic 13 protein which promotes apoptosis by directly binding and inhibiting XIAP via a mechanism that is distinct 14 from all other known IAP antagonists. Here, we investigated the ability of peptides derived from ARTS to 15 antagonize XIAP and promote apoptosis in cancer cell lines. 16 Experimental Design: The ability of synthetic peptides, derived from the C-terminus of ARTS, to bind to 17 XIAP, stimulate XIAPdegradation and induce apoptosiswas examined.We compared the response of several 18 cancer cell lines to different ARTS-derived peptides. Pull-down assayswere used to examine binding to XIAP, 19 and apoptosis was evaluated using terminal deoxynucleotidyl transferase–mediated dUTP nick end 20 labeling, caspase activation, and Western blot analyses of caspase substrates. 21 Results: The C-terminus of ARTS contains a unique sequence, termed ARTS-IBM (AIBM), which is 22 important for binding to XIAP and cell killing. AIBMpeptides can bind to XIAP-BIR3, penetrate cancer cells, 23 reduce XIAP levels, and promote apoptosis. 24 Conclusions: Short synthetic peptides derived from the C-terminus of ARTS are sufficient for binding to 25 XIAP and can induce apoptosis in cancer cells. These results provide proof-of-concept for the feasibility of 26 developing ARTS-based anticancer therapeutics. Clin Cancer Res; 18(00); 1–10. !2012 AACR. 27 28 29