The heparan sulfate 6-O-endosulfatase (SULF2) promotes growth and metastasis of solid tumors. We recently identified that cytosine methylation of the SULF2 promoter is associated with better survival of resected lung adenocarcinoma patients, and now also demonstrates a marginal improvement in survival of advanced non-small cell lung cancer (NSCLC) patients receiving standard chemotherapy (hazard ratio0.63, P0.07). Subsequent studies focused on investigating the effect of methylation on SULF2 expression and its genome-wide impact. The genes and pathways modulated by epigenetic inactivation of SULF2 and the effects on sensitivity to chemotherapy were characterized in vitro and in vivo. Silencing SULF2 through small interfering RNA or methylation primarily increased expression of interferon-inducible genes including ISG15, a marker for increased sensitivity to topoisomerase-1 inhibitors such as camptothecin (CPT). NSCLC cell lines with methylated SULF2 (SULF2M) express 60-fold higher ISG15 compared with SULF2 unmethylated (SULF2U) NSCLC cell lines and normal human bronchial epithelial cells. In vitro, SULF2M and high ISG15 (ISG15H)-expressing NSCLC cell lines were 134-fold more sensitive to CPT than SULF2U and low ISG15 (ISG15L)-expressing cell lines. Topotecan, a soluble analog of CPT and FDA-approved anticancer drug, dramatically arrested the growth of SULF2M-ISG15H, but not SULF2U-ISG15L lung tumors in nude mice (P0.002). Similarly, high ISG15 expression that is comparable to the topotecan (TPT)-sensitive NSCLC cell lines was found in tumors from 25% of NSCLC patients compared with normal lung, indicating a potential to identify and target the most sensitive NSCLC subpopulation for personalized TPT therapy.