Preclinical Development Rb–Raf-1 Interaction Disruptor RRD-251 Induces Apoptosis in Metastatic Melanoma Cells and Synergizes with Dacarbazine

Metastatic melanoma is an aggressive cancer with very low response rate against conventional chemotherapeutic agents such as dacarbazine (DTIC). Inhibitor of Rb–Raf-1 interaction RRD-251 was tested against the melanoma cell lines SK-MEL-28, SK-MEL-5, and SK-MEL-2. RRD-251 was found to be a potent inhibitor of melanoma cell proliferation, irrespective of V600E B-Raf mutation status of the cell lines. In a SK-MEL-28 xenograft experiment, RRD-251 exerted a significant suppression of tumor growth compared with vehicle (P 1⁄4 0.003). Similar to in vitro effects, tumors from RRD-251–treated animals showed decreased Rb–Raf-1 interaction in vivo. Growth suppressive effects of RRD-251were associatedwith induction of apoptosis as well as a G1 arrest, with an accompanying decrease in S-phase cells. RRD-251 inhibited Rb phosphorylation and downregulated E2F1 protein levels in these cells. Real-time PCR analysis showed that RRD-251 caused downregulation of cell-cycle regulatory genes thymidylate synthase (TS) and cdc6 as well as the antiapoptotic gene Mcl-1. Combinatorial treatment of RRD-251 and DTIC resulted in a significantly higher apoptosis in DTIC resistant cell lines SK-MEL-28 and SK-MEL-5, as revealed by increased caspase-3 activity and PARP cleavage. Because aberrant Rb/E2F pathway is associated with melanoma progression and resistance to apoptosis, these results suggest that the Rb–Raf-1 inhibitor could be an effective agent for melanoma treatment, either alone or in combination with DTIC. Mol Cancer Ther; 9(12); 3330–41. 2010 AACR.