Abstract Glioblastoma (GBM) is the most malignant brain cancer, with extremely poor prognosis in patients. GBM tumors are characterized by distinct molecular subtypes, known as proneural (PN), classical, and mesenchymal (MES). Inherited heterogeneity and aberrant aggressiveness contribute to therapy resistance and frequent recurrence of malignant GBM tumors. In addition, GBM tumors also contain a small subpopulation of tumor-initiating or stem-like cancer cells (GSCs). Gene expression profiling studies from our laboratory showed that patient-derived GSCs can also be classified into two subtypes, PN and MES, that are phenotypically similar to clinical GBM. Among three thousand genes that are differentially expressed between PN and MES-like GSCs, Lymphocyte Antigen 6 Complex, Locus K (LY6K) was identified as one of the top differentially expressed genes. LY6K is a GPI-anchored protein from the LY6 family. Several members of the LY6 family have been implicated in human cancers, including breast, esophageal, and lung cancers. Moreover, the function of LY6K in GBM has not been reported. Here, we examined the roles of LY6K upregulation in GBM tumorigenesis and investigated the underlying mechanism of LY6K action. We hypothesized that high levels of LY6K in GSCs promotes GBM tumorigenesis. Based on our other preliminary data, we further hypothesized that LY6K functions by enhancing ERK activation, thus promoting radioresistance. To test our hypotheses, we first examined the role of LY6K in advancing GBM tumorigenic behaviors of GSCs. We performed in vitro and in vivo tumorigenicity assays and showed that the presence of LY6K significantly increases GSC cell growth and glioma sphere forming ability in vitro and promotes tumor formation in orthotopic brain xenograft mouse models. The underlying mechanism governing LY6K expression was found to be DNA promoter methylation. Moreover, irradiation strongly induces LY6K expression via demethylation of LY6K promoter in GSCs with otherwise undetectable levels of LY6K. Furthermore, we investigated the mechanism by which LY6K contributes to GSC tumorigenicity. We observed that there is a strong relationship between LY6K and ERK signaling in GSCs and U87 glioma cells. The presence of LY6K stimulates ERK activation and subsequently augments cell proliferation of GSCs. Lastly, we identified the GPI-anchor domain of LY6K as a key region in enhancing ERK activation and are currently examining the mechanistic properties of this domain. The mechanistic insights gained from our studies will advance current knowledge of aberrantly upregulated LY6K and ERK signaling enhancement in promoting GBM tumorigenicity. Citation Format: Namratha G. Sastry, Tianzhi Huang, Angel Alvarez, Rajendra Pangeni, Xiao Song, Xuechao Wan, John Kessler, Ichiro Nakano, Bo Hu, Shi-Yuan Cheng. Novel roles of LY6K in glioblastoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3432.