In vivo efficacy of AT-101, an orally active pan Bcl-2 family protein inhibitor in combination with docetaxel or erlotinib for non small cell lung cancer therapy

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Certain members of the Bcl-2 family of proteins (Bcl-2, Bcl-xL and Mcl-1) are over-expressed in lung cancers and are generally recognized to play an important role in the resistance to current anti-cancer therapies. A549 and H460 Non Small Cell Lung Cancer (NSCLC) cell lines are typically resistant to cytotoxic chemotherapy. AT-101 is an orally active, pan-Bcl-2 family inhibitor (Bcl-2, Bcl-xL, Mcl-1, Bcl-w) currently in clinical development in several phase II trials. Here we evaluated the effects of AT-101 on NSCLC cell line survival both as a single agent and in combination with traditional or targeted anti-cancer agents.
 Methods: Cell growth inhibition was measured using WST-based assays in A549 and H460 NSCLC cells. In vivo, the ability of AT-101 to potentiate the anti-cancer effect of the chemotherapeutic agent docetaxel or epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (erlotinib) was evaluated in an A549 xenograft model. Pharmacodynamic studies were performed after combined therapy of AT-101 and erlotinib. Apoptosis induced in vivo by AT-101 alone or combined with erlotinib was confirmed by using TUNEL assay and angiogenesis was analyzed with CD31 immunostaining.
 Results: AT-101 induces a dose-dependent cell killing effect in NSCLC cell lines with a mean IC₅₀ of 2.27uM (A549) and 3.12uM (H460), compared to previously published data with the small molecule Bcl-2/Bcl-xL inhibitor ABT-737 (IC₅₀=11.009 uM in A549 cell line and >100uM in H460). In vitro, after 48 hours of exposure to AT-101 at 10 uM, 40% of A549 cells undergo apoptosis under normal serum conditions.We examined different in vivo pulse dosing schedules (once a week, or 3 days a week) for AT-101 therapy both as a single agent and also in combination with docetaxel. In vivo, combined treatment with AT-101 (15mg/kg, p.o., daily) and docetaxel (8 mg/kg, i.v., once weekly) synergistically suppress subcutaneous A549 cells tumor growth compared with treatment with either agent alone. The combination shows anti-tumor efficacy with a T/C value of 0.441 whereas the single agent treatments are less effective (T/C value =0.668 for AT-101 alone and 0.682 for docetaxel) (P<0.001). A pulse dosing schedule of AT-101 (60mg/kg) for 3 days a week combined with docetaxel (30mg/kg, q3w), shows tumor inhibition with a T/C value = 0.341 whereas the single agent treatments have a T/C value =0.650 for AT-101 alone and 0.656 for docetaxel, (P<0.001). In the A549 xenograft model, combined therapy of AT-101 (25 mg/kg, p.o., daily) with erlotinib (80 mg/kg,) is more effective than either agent alone (T/C value of 0.281, versus T/C=0.654 for AT-101 alone and 0.603 for erlotinib alone, respectively) (P<0.001). No significant toxicities were observed in combination as compared with monotherapy. Pharmacodynamic studies show that erlotinib alone or combined with AT-101 can decrease EGFR phosphorylation in mice bearing A549 tumor. TUNEL assay experiments have shown that apoptosis induced by the combination therapy is more robust than with either single agent.Expression in situ of CD31 demonstrates that AT-101 alone or in combination with erlotinib can lower the number and distribution of endothelial cells. Taken together, these data suggest that molecular targeted therapy with the pan Bcl-2 inhibitor AT-101 may improve the outcome of current therapy for NSCLC.