ARTICLE ABSTRACTCancer stem cells (CSC) have garnered significant attention as a therapeutic focus, based on evidence that they may represent an etiologic root of treatment-resistant cells. Indeed, expression of the multidrug resistance protein ATP-binding cassette subfamily G member 2 (ABCG2) confers chemoresistance to CSCs, where it serves as a potential biomarker and therapeutic target. Here, we show that afatinib, a small-molecule inhibitor of the tyrosine kinases EGFR, HER2, and HER4, preferentially eliminated side population cells with CSC character, in both cell lines and patient-derived leukemia cells, by decreasing ABCG2 expression. In these cells, afatinib also acted in parallel to suppress self-renewal capacity and tumorigenicity. Combining afatinib with the DNA-damaging drug topotecan enhanced the antitumor effect of topotecan in vitro and in vivo. Mechanistic investigations suggested that ABCG2 suppression by afatinib did not proceed by proteolysis through the ubiquitin-dependent proteosome, lysosome, or calpain. Instead, we found that afatinib increased DNA methyltransferase activity, thereby leading to methylation of the ABCG2 promoter and to a decrease in ABCG2 message level. Taken together, our results advocate the use of afatinib in combination with conventional chemotherapeutic drugs to improve efficacy by improving CSC eradication. Cancer Res; 74(16); 4431–45. ©2014 AACR.