Synergistic effects of a small molecule EGFR/HER2 inhibitor in combination with a triple angiokinase inhibitor in colorectal cancer xenografts refractory to combinations of cetuximab and bevacizumab

Backgound: Combining different classes of targeted agents represents a major challenge in modern cancer therapy. Agents targeting EGFR‐ or VEGF‐signaling are approved for treatment of metastatic colorectal cancer paving the way for novel therapeutic strategies combining the two classes of agents. Unexpectedly, large clinical studies with combinations of EGFR‐targeted antibodies and bevacizumab showed no added benefit compared to bevacizumab alone. It is currently unclear whether these findings are linked to incompatibility between the antibodies or rather to incompatibility between the two classes of targeted agents. Experimental: Combinations of two small orally active molecules, the irreversible EGFR and HER2 inhibitor BIBW2992 (B2) and the triple angiokinase inhibitor BIBF1120 (B1) were characterized in human colorectal cancer cell lines in vitro and in mice using the HT‐29 colorectal xenograft model that is BRAF V600E mutated. Growth inhibitory and cytotoxic effects were characterized by flow cytometry complemented by immunological analyses for p27Kip1, phospho‐Erk and phospho‐Akt in cellular models and by quantitative immunohistochemistry analyses of EdU incorporation and TUNEL staining in tumor xenografts. Results: Exposure of colorectal cancer cells to B1 or to B2 resulted in growth inhibition linked to prolonged p27Kip1‐mediated G1 arrest, which in most cell lines was accompanied by delayed apoptosis. Combinations of B1 and B2 resulted in increased cell death in all cellular models studied. In xenografts, four weeks treatment with combinations of B1 and B2 were associated with a clear synergistic effect on tumor growth inhibition compared to either agent alone. In contrast, no additional activity was observed for combinations of cetuximab and bevacizumab in the same xenograft model. Quantitative immunohistochemistry analyses revealed that all 4 agents inhibited in vivo DNA synthesis, as shown by EdU incorporation, and that none of the combinations tested showed superiority. TUNEL staining was not increased following single agent treatment with B1, cetuximab and bevacizumab while B2 resulted in increased TUNEL staining. Combinations of B1 and B2 were characterized by a pronounced increase in TUNEL staining, while no increase was observed for combinations of cetuximab and bevacizumab. Conclusions: We here show that combinations of small molecules that simultaneously target the HER and the angiogenic pathways show synergistic cytotoxic and tumor‐growth inhibitory activities in the HT‐29 colorectal xenograft model which is refractory to the combinations of cetuximab and bevacizumab. Therefore, the lack of superior clinical activity for combinations of cetuximab/panitumumab and bevacizumab is not due to an inherent incompatibility between the 2 classes of targeted agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A240.