23. Uncovering Mechanisms of Resistance to Cetuximab by Insertional Mutagenesis in Heterotopically-Engrafted Human Colorectal Cancers

Abstract

The development of high-throughput technologies has made possible the identification of cancer-driver mutations, which account for the growth and spreading of cancer cells. The discovery of these specific cancer biomarkers led to the development of anti-cancer targeted therapies, which hit in a specific manner cell pathways directly involved in tumor progression. This new class of therapeutic agents, which comprehend small molecules and antibodies, has been shown to be more effective and less toxic than conventional chemotherapy in advanced forms of cancer. However, the inevitable development of acquired resistance, due to the acquisition of multiple mutations or activation of compensatory pathways, has limited their success. A paradigm for this concept are the anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibodies, cetuximab and panitumumab, which are Federal Drug Administration (FDA)-approved agent for the treatment of EGFR-expressing metastatic colorectal cancer (mCRC). Unfortunately, despite the massive initial reduction, response is transient and tumors become refractory within 12-18 months.We take advantage of a lentiviral vector (LV)- based insertional mutagenesis platform to induce cetuximab-resistance in Patient-Derived Xenografts (PDXs) of mCRC, with the final goal of uncovering the molecular mechanism of the resistance. Insertional mutagenesis has been successfully used in our lab to explore novel genes involved in the resistance to lapatinib in two breast cancer cell lines and to erlotinib in a pancreatic cancer cell line. Given these encouraging results we now aim to apply this technology to human cancer specimens to better reflect the real clinical response. Established PDXs from cetuximab-sensitive liver metastatic lesions of CRC have been excised, chemically digested to single cell suspension and consequently transduced with a genotoxic LV, harboring the hyperactive enhancer/promoter of Spleen Focus Forming Virus in the Long Terminal Repeats (LV-SF-LTR) sequences or a non-genotoxic LV, containing self inactivating LTR sequences. Transduced tumor cells have been re-implanted in NSG mice to reconstitute the tumors. Once engrafted, mice have been divided in two groups receiving either cetuximab or the drug-vehicle. LV-SF-LTR transduction of a large amount of cells with a high vector copy number will allow us to randomly hit genes responsible for the resistance to the tumor-specific targeted therapy and deregulate their expression. Therefore, exposure to the treatment will not rest tumor growth in these tumors. LAM-PCR and deep sequencing analysis performed on drug-sensitive and drug-resistant tumors will be used to map the integration sites of the LVs and consequently to identify LV-induced gene deregulations responsible for the pharmacological resistance.The identification of the biomarkers accounting for the drug-resistance is a real challenge that will allow screening for rational drug combinations to reverse resistance, improving the life expectancy of CRC patients.