Within the context of the “Combi-Targeting” concept, we designed RB24 to inhibit EGFR or Her2 phosphorylation and to further degrade upon hydrolysis to RB10 (another EGFR/Her2 inhibitor) + a DNA methylating species. In vitro analyses using fluorescence microscopy, comet and Annexin-V binding assays showed that RB24 released RB10 in the perinuclear region, damaged DNA and induced apoptosis in the human MDA-MB-435 breast cancer cells. Transfection of the latter cells with ErbB1 or ErbB2 correlated with 2-3 fold higher levels of DNA damage, enhanced cell death by apoptosis and increased intensity of fluorescence associated with RB10 in the perinuclear region. The selective enhancement of DNA lesions in the transfected cells was abolished by co-incubation with exogenous RB10, suggesting that intact RB24 bound to its cognate perinuclear sites prior to releasing the alkylating species. Analysis of key signaling proteins in the cells demonstrated a DNA-damage dependent activation of JNK and down-regulation of Bad through inhibition of EGFR or Her2 phosphorylation by RB24. This translated in to a 10-20-fold stronger cytocidal activity by RB24 when compared with its clinical counterpart Temodal®.The results suggest a targeting model based on a by-stander effect that increases the levels of DNA damage in the transfectants and a cooperative enhancement of apoptosis through JNK activation and down regulation of Bad. This convergent mechanism may account for the remarkable potency of RB24 against the oncogenic expressing transfectants. The results in toto indicate that the Combi-Targeting concept may well represent a novel strategy to enhance the potency of alkylators in EGFR- and Her2-expressing cells.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA