ABSTRACT Telomerase reverse transcriptase (TERT) enables telomere elongation that is essential for continuous cell proliferation. TERT expression that is associated with activating mutations in the TERT promoter, is observed in virtually all glioblastoma and oligodendroglioma cases. This makes TERT the most common genetic alteration in brain tumors, and a novel therapeutic target. Noninvasive imaging of TERT expression could therefore help in distinguishing between pseudo-progression and recurrent glioma, and provide a noninvasive biomarker for assessment of treatment efficacy by TERT inhibitors. However, to date, no translational imaging approaches for TERT expression have been reported. The goal of Project 3 is to address this critical need by developing metabolic imaging biomarkers of TERT expression. Our approach is based on previous reports showing that TERT expression is associated with control of cellular redox, and our preliminary data confirming this finding and identifying additional metabolic alterations. Specifically, we have found that 1H magnetic resonance spectroscopy (MRS)-detectable levels of glutathione and the 13C MRS-detectable metabolism of hyperpolarized dehydroxyascorbate to vitamin C, are elevated in TERT-expressing cells. Additionally, hyperpolarized 13C MRS- detectable fluxes of glucose and gluconolactone via the pentose phosphate pathway to 6-phosphogluconate are elevated, as are the levels of aspartate and adenosine phosphates. We therefore hypothesize that advanced MRS metabolic imaging could be used to distinguish glioma cells expressing TERT from normal brain parenchyma and from tumor cells in which TERT expression is silenced by treatment. We will test this hypothesis as follows. In Aim 1 we will identify 1H MRS and hyperpolarized 13C MRS metabolic imaging biomarkers that are associated with TERT expression by investigating cell lines that differ only in their TERT status and determining if levels of MRS-detectable metabolic biomarkers associated with redox, and other metabolic changes can distinguish TERT-expressing from TERT non-expressing cells. In Aim 2 we will determine whether MRS- detectable biomarkers of redox can be used to monitor TERT expression in vivo by using mouse models with orthotopic TERT-expressing brain tumors, inhibiting TERT expression via genetic and/or pharmacological approaches, and determining if this inhibition can be assessed using 1H and/or hyperpolarized 13C MRS biomarkers of redox. If cell studies show that other metabolic pathways are modulated by TERT, these will also be investigated in vivo. In Aim 3 we will investigate mechanisms linking TERT expression with metabolism by assessing cellular processes known to be associated with TERT expression and determining if these processes are mechanistically linked to changes in redox-associated metabolic pathways or other MRS-detectable metabolic pathways altered by TERT. Our study is expected to lead to translatable MRS-detectable metabolic biomarkers of TERT expression that could improve glioma patient treatment and outcome.