Abstract C4: TLN-4601 is a novel tumor selective pro-apoptotic agent acting independently of Bax, Bak or Bcl-2 expression

Abstract

Abstract Background: TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through Thallion's proprietary DECIPHER® technology platform. The compound has demonstrated broad in vitro cytotoxicity and in vivo tumor growth inhibition. Phase I clinical testing has shown the compound to be safe and well tolerated, with early signs of anti-tumor activity. Although considerable research efforts have focused on the ability of TLN-4601 to inhibit the Ras/MAPK signalling pathway, the compound has also been shown to induce apoptosis through caspase activation. The objectives of the present work were to further elucidate the molecular events, pathways and temporal patterns associated with TLN-4601-induced apoptosis. Material and Methods: Protein extracts from Jurkat cells (a human T cell lymphoblast-like cell line), Jurkat cells with caspase 8 knocked-down, Jurkat cells over-expressing Bcl-2, mouse embryonic fibroblasts isolated from Bax/Bak double knock-out mice, and both quiescent and PHA-L stimulated human peripheral blood lymphocytes (PBLs) treated with TLN-4601 over different exposure times were analyzed by Western blot to evaluate the induction of apoptotic markers, including cytochrome c release, caspase activation and PARP cleavage. Apoptosis was assessed by DNA laddering and sub-G1 DNA content. The effect of TLN-4601 on cell viability were measured using commercial Caspase-3/7 activity and ATP assays. Results: TLN-4601 caused rapid apoptotic cell death in Jurkat cells as characterized by mitochondrial cytochrome c release, caspase activation, loss of cellular ATP and DNA laddering. Equal sensitivity of the wild-type and caspase 8 knocked-down Jurkat cells to TLN-4601 suggests that the extrinsic apoptotic pathway was not involved in mediating this activity. Interestingly, TLN-4601 exposure resulted in cytochrome c release independently of the apoptosis-inducing proteins Bax and Bak. Furthermore, the over-expression of the anti-apoptotic protein Bcl-2 did not affect the ability of TLN-4601 to induce cell death. Finally, a short exposure of TLN-4601 exhibited a preferential selectivity for triggering apoptosis in Jurkat cells as compared to either quiescent and PHA-L stimulated PBLs. Conclusions: TLN-4601 mediates rapid cell death via the intrinsic apoptotic pathway independent of Bax, Bak or Bcl-2 expression. Moreover, the differential sensitivity to TLN-4601 observed between Jurkat cells and PBLs highlights the potential for a usable therapeutic margin between cancer and normal cells. Taken together, these properties represent a very promising profile for an anti-cancer agent and suggest that further investigation of TLN-4601 in hematological malignancies is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C4.