MOLECULAR NETWORKS OF THE EGFR-TKI RESISTANT NON-SMALL CELL LUNG CANCER: HOW TO APPLY NEW TECHNOLOGIES TO BENCH-TO-BED RESEARCH

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Abstract

Background

A secondary mutation of EGFR (T790M) accounts for 50% of all cases of acquired resistance to EGFR-TKIs in NSCLC patients with EGFR mutation. Although irreversible EGFR-TKIs are thought to be one of the strategies to overcome T790M, the efficacy of these drugs appears limited.

Methods

We generated EGFR-TKI resistant PC9GR cells with T790M by long-term gefitinib exposure of PC9 cells with exon 19 deletion. To visualize their molecular networks related to T790M, we carried out immunoaffinity purification of tyrosine-phosphorylated peptides by anti-phospho tyrosine antibodies (pTyr100) and subsequent LC-MS/MS identification in PC9 and PC9GR cells with or without erlotinib treatment.

Results

Our phosphoproteomics detected a total of 403 unique phosphosites on 266 proteins. Compared with non-treated PC9 cells, 32 pY sites (24 proteins) are decreased and 45 pY sites (33 proteins) are increased in non-treated PC9GR cells. In parental PC9 cells, 24 pY sites (19 proteins) are decreased and 12 pY sites (12 proteins) are increased by erlotinib treatment. In resistant PC9GR cells, 5 pY sites (5 proteins) are decreased and 6 pY sites (6 proteins) are increased by erlotinib treatment. The detailed analyses including different response to erlotinib between PC9 and PC9GR cells will be presented at the meeting. As a preliminary validation, we found that PC9GR cells have persistent Src kinase levels even with erlotinib treatment in our phosphoproteomics. Western blot assays also demonstrated that the irreversible EGFR-TKI BIBW-2992 (afatinib) did not alter the activation of Src family kinases in PC9GR cells. Src inhibitor dasatinib combined with afatinib abolished this Src phosphorylation along with complete suppression of downstream phospho Akt and Erk. Dasatinib enhances anti-tumor activity of afatinib in both cell proliferation assay and caspase-3 apoptosis assay in these cells. This combination showed significant in vivo tumor regression in PC9GR xenograft studies.

Conclusions

Applying global phosphoproteomics effectively visualizes the molecular networks of EGFR-TKI-resistant NSCLC related to T790M gate keeper mutation. Our preliminary validation supports further studies of irreversible EGFR inhibitors combined with dasatinib in NSCLC patients who acquired resistance to EGFR-TKI by T790M.

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