Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic mechanisms responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the erlotinib-resistant clones. RNAi-based systematic synthetic lethal screening of these candidate genes revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, immunohistochemical analysis revealed increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs resistant patients. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients.