Glioblastoma (GBM) is a highly resistant cancer. Temozolomide (TMZ) is the best first-line therapy available, but TMZ response depends on the promoter methylation status of the DNA repair gene O6-methylguanine DNA methyltransferase (MGMT). MGMT methylated (MGMT-M) GBM patients have suppressed MGMT protein expression which leads to TMZ sensitization and prolonged survival. In contrast MGMT unmethylated (MGMT- UM) GBM patients are resistant to TMZ and have much shorter survivals. So far there have been no successful treatments to render MGMT-UM susceptible to TMZ and therefore there is a desperate need for novel treatment strategies for MGMT-UM. Our long-term goal is to devise strategies that can epigenetically reprogram MGMT-UM GBM patients to the TMZ susceptibility of MGMT-M GBM patients. This proposal exploits a novel epigenetic mechanism in GBM stem cells (GSCs) involving Tumor Suppressor Candidate 3 (TUSC3) that can be used to reprogram MGMT-UM to restore sensitivity to TMZ and significantly prolong survival in experimental models of GBMs. The objective in this application is to understand how TUSC3 is epigenetically regulated and how TUSC3 sensitizes GBMs to TMZ. We recently discovered using both pharmacologic epigenetic reprogramming and gain-of-function strategies that: i) TUSC3 promoter regulation in GSCs is impacted by MGMT; ii) TUSC3 significantly sensitized GSCs through suppression of DNA damage repair and cell cycle progression; and iii) TUSC3 synergized with TMZ to produce similar survival benefits in experimental models of MGMT-M and MGMT-UM GBMs. We will therefore test the hypothesis that epigenetic reactivation of TUSC3 reprograms GBMs to TMZ sensitivity and prolonged survival. In Aim 1, we will determine how TUSC3 is epigenetically regulated in GBM. In Aim 2, we will determine how TUSC3 mediates TMZ response sensitization in GBM. Mechanistic knowledge gained from these studies will enhance our understanding of novel epigenetic reprogramming mechanisms in GBM and contribute toward the optimal design of impactful trials for MGMT-UM GBMs who currently do not have good chemotherapy options.