Variants of bovine pancreatic ribonuclease (RNase A) engineered to evade the endogenous ribonuclease inhibitor protein (RI) are toxic to human cancer cells. Increasing the basicity of these variants facilitates their entry into the cytosol and thus increases their cytotoxicity. The installation of additional positive charge also has the deleterious consequence of decreasing ribonucleolytic activity or conformational stability. Here, we report that the same benefit can be availed by co-treating cells with a cationic dendrimer. We find that adding the generation 2 poly(amidoamine) dendrimer in trans increases the cytotoxicity of RI-evasive RNase A variants without decreasing their activity or stability. The increased cytotoxicity is not due to increased RI-evasion or cellular internalization, but likely results from improved translocation into the cytosol after endocytosis. These data indicate that co-treatment with highly cationic molecules could enhance the efficacy of ribonucleases as chemotherapeutic agents.

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Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that has shown efficacy in metastatic colorectal cancer, head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC). Clinical data suggests that patients whom initially respond to cetuximab eventually acquire resistance, highlighting the importance of understanding resistant mechanisms for the development of better treatment regimes for cancer patients. Recent studies have identified a role for the TAM family of receptor tyrosine kinases (Tyro, Axl, and Mer) in tumor biology, and more specifically the Axl receptor as playing a key role in promoting tumor growth and metastasis. In the current study, we utilized NSCLC and HNSCC models of acquired resistance to cetuximab and found that Axl was overexpressed, activated, and highly associated with EGFR as compared parental controls. Further, using siRNA technology and novel Axl targeting agents (antibody and tyrosine kinase inhibitor), Axl was found to regulate proliferation, EGFR activation, and MAPK signaling in cetuximab resistant clones (CtxR). Additionally, Axl mRNA expression was regulated by EGFR induced MAPK activation in resistant cells. To investigate the role of Axl in in vivo models of acquired resistance to cetuximab, de novo acquired resistance was created by prolonged treatment of cetuximab sensitive (CtxS) tumor xenografts with cetuximab. Analysis of resultant CtxR tumors demonstrated that Axl was hyperactivated and highly associated with EGFR as compared to vehicle treated tumors. In addition, CtxR tumors expressing the highest levels of phosphorylated Axl also expressed hyperactivated EGFR, supporting the role of Axl in EGFR activation. Finally, to expand these findings to other members of the TAM family, Tyro, Axl, or Mer were stably overexpressed in CtxS parental NSCLC cells and tested for cetuximab response. The results of this experimentation indicated that stable overexpression of each TAM receptor rendered CtxS cells resistant to cetuximab therapy. Collectively, the studies presented herein identify the TAM family of receptors as key players in acquired resistance to cetuximab and provide rationale for the clinical evaluation of anti-TAM receptor therapeutics in the resistant setting. Citation Format: Toni Michel Brand, Mari Iida, Kelsey L. Corrigan, Neha Luthar, Megan Hornung, Mahmoud Toulany, Parkash Gill, Ravi Salgia, Deric L. Wheeler. The TAM family of receptor tyrosine kinases play a role in acquired resistance to cetuximab. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-215. doi:10.1158/1538-7445.AM2014-LB-215