Purpose:Telomere maintenance is a hallmark of cancer. Most tumors maintain telomere length via reactivation of telomerase reverse transcriptase (TERT) expression. Identifying clinically translatable imaging biomarkers of TERT can enable non-invasive assessment of tumor proliferation and response to therapy. Methods: We used RNA interference, doxycycline-inducible expression systems and pharmacological inhibitors to mechanistically delineate the association between TERT and metabolism in preclinical patient-derived tumor models. Deuterium magnetic resonance spectroscopy (2H-MRS), which is a novel, translational metabolic imaging modality, was used for imaging TERT in cells and tumor-bearing mice in vivo. Results: Our results indicate that TERT expression is associated with elevated NADH in multiple cancers, including glioblastoma, oligodendroglioma, melanoma, neuroblastoma, and hepatocellular carcinoma. Mechanistically, TERT acts via the metabolic regulator FOXO1 to upregulate nicotinamide phosphoribosyl transferase, which is the key enzyme for NAD+ biosynthesis, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts NAD+ to NADH. Since NADH is essential for pyruvate flux to lactate, we show that 2H-MRS-based assessment of lactate production from [U-2H]-pyruvate reports on TERT expression in preclinical tumor models in vivo, including at clinical field strength (3T). Importantly, [U-2H]-pyruvate reports on early response to therapy in mice bearing orthotopic patient-derived gliomas at early timepoints before radiographic alterations can be visualized by magnetic resonance imaging. Conclusions: Elevated NADH is a metabolic consequence of TERT expression in cancer. Importantly, [U-2H]-pyruvate reports on early response to therapy, prior to anatomical alterations, thereby providing clinicians with a novel tool for assessment of tumor burden and treatment response in cancer.

Background The alternative lengthening of telomeres (ALT) pathway is essential for tumor proliferation in astrocytomas. The goal of this study was to identify metabolic alterations linked to the ALT pathway that can be exploited for non-invasive magnetic resonance spectroscopy (MRS)-based imaging of astrocytomas in vivo. Methods Genetic and pharmacological methods were used to dissect the association between the ALT pathway and glucose metabolism in genetically-engineered and patient-derived astrocytoma models. 2H-MRS was used for non-invasive imaging of ALT-linked modulation of glycolytic flux in mice bearing orthotopic astrocytomas in vivo. Results The ALT pathway was associated with higher activity of the rate-limiting glycolytic enzyme phosphofructokinase-1 and concomitantly elevated flux of glucose to lactate in astrocytoma cells. Silencing the ALT pathway or treating with the poly (ADP-ribose) polymerase inhibitor niraparib that induces telomeric-fusion in ALT-dependent astrocytoma cells abrogated glycolytic flux. Importantly, this metabolic reprogramming could be non-invasively visualized by 2H-MRS. Lactate production from [6,6’- 2H]-glucose was higher in ALT-dependent astrocytoma tumors relative to normal brain in vivo. Furthermore, treatment of orthotopic astrocytoma-bearing mice with niraparib reduced lactate production from [6,6’- 2H]-glucose at early timepoints when alterations in tumor volume could not be detected by anatomical imaging, pointing to the ability of [6,6’- 2H]-glucose to report on pseudoprogression in vivo. Conclusions We have mechanistically linked the ALT pathway to elevated glycolytic flux and demonstrated the ability of [6,6’- 2H]-glucose to non-invasively assess tumor burden and response to therapy in astrocytomas. Our findings point to a novel, clinically translatable method for metabolic imaging of astrocytoma patients.

<div>AbstractPurpose:<p>Telomere maintenance is a hallmark of cancer. Most tumors maintain telomere length via reactivation of telomerase reverse transcriptase (TERT) expression. Identifying clinically translatable imaging biomarkers of TERT can enable noninvasive assessment of tumor proliferation and response to therapy.</p>Experimental Design:<p>We used RNAi, doxycycline-inducible expression systems, and pharmacologic inhibitors to mechanistically delineate the association between TERT and metabolism in preclinical patient-derived tumor models. Deuterium magnetic resonance spectroscopy (²H-MRS), which is a novel, translational metabolic imaging modality, was used for imaging TERT in cells and tumor-bearing mice <i>in vivo</i>.</p>Results:<p>Our results indicate that TERT expression is associated with elevated NADH in multiple cancers, including glioblastoma, oligodendroglioma, melanoma, neuroblastoma, and hepatocellular carcinoma. Mechanistically, TERT acts via the metabolic regulator FOXO1 to upregulate nicotinamide phosphoribosyl transferase, which is the key enzyme for NAD+ biosynthesis, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts NAD+ to NADH. Because NADH is essential for pyruvate flux to lactate, we show that ²H-MRS–based assessment of lactate production from [U-²H]-pyruvate reports on TERT expression in preclinical tumor models <i>in vivo</i>, including at clinical field strength (3T). Importantly, [U-²H]-pyruvate reports on early response to therapy in mice bearing orthotopic patient-derived gliomas at early timepoints before radiographic alterations can be visualized by MRI.</p>Conclusions:<p>Elevated NADH is a metabolic consequence of TERT expression in cancer. Importantly, [U-²H]-pyruvate reports on early response to therapy, prior to anatomic alterations, thereby providing clinicians with a novel tool for assessment of tumor burden and treatment response in cancer.</p></div>

<div>AbstractPurpose:<p>Telomere maintenance is a hallmark of cancer. Most tumors maintain telomere length via reactivation of telomerase reverse transcriptase (TERT) expression. Identifying clinically translatable imaging biomarkers of TERT can enable noninvasive assessment of tumor proliferation and response to therapy.</p>Experimental Design:<p>We used RNAi, doxycycline-inducible expression systems, and pharmacologic inhibitors to mechanistically delineate the association between TERT and metabolism in preclinical patient-derived tumor models. Deuterium magnetic resonance spectroscopy (²H-MRS), which is a novel, translational metabolic imaging modality, was used for imaging TERT in cells and tumor-bearing mice <i>in vivo</i>.</p>Results:<p>Our results indicate that TERT expression is associated with elevated NADH in multiple cancers, including glioblastoma, oligodendroglioma, melanoma, neuroblastoma, and hepatocellular carcinoma. Mechanistically, TERT acts via the metabolic regulator FOXO1 to upregulate nicotinamide phosphoribosyl transferase, which is the key enzyme for NAD+ biosynthesis, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts NAD+ to NADH. Because NADH is essential for pyruvate flux to lactate, we show that ²H-MRS–based assessment of lactate production from [U-²H]-pyruvate reports on TERT expression in preclinical tumor models <i>in vivo</i>, including at clinical field strength (3T). Importantly, [U-²H]-pyruvate reports on early response to therapy in mice bearing orthotopic patient-derived gliomas at early timepoints before radiographic alterations can be visualized by MRI.</p>Conclusions:<p>Elevated NADH is a metabolic consequence of TERT expression in cancer. Importantly, [U-²H]-pyruvate reports on early response to therapy, prior to anatomic alterations, thereby providing clinicians with a novel tool for assessment of tumor burden and treatment response in cancer.</p></div>

The Warburg effect, characterized by elevated glucose uptake and flux to lactate, is a metabolic hallmark of cancer. Recent studies have identified deuterium 2H-magnetic resonance spectroscopy (MRS) using 6,6’-2H-glucose as a novel method of imaging the Warburg effect in high-grade primary glioblastomas (GBMs). However, its utility for imaging low-grade gliomas has not been tested. The goal of this study was to determine whether 6,6’-2H-glucose can be used for imaging tumor burden and treatment response in mutant isocitrate dehydrogenase (IDHmut) low-grade gliomas in vivo. We examined mice bearing orthotopic tumors of the patient-derived BT257 astrocytoma model. 1H-MRS, providing a readout of steady-state metabolite levels, confirmed the presence of 2-hydroxyglutarate, the product of IDHmut, in BT257 tumor tissue but not normal brain. Previous studies comparing IDHmut gliomas with GBMs suggest that IDHmut gliomas undergo lactate dehydrogenase silencing, potentially leading to a non-glycolytic phenotype. Nevertheless, our results indicated that, compared to normal brain, glucose uptake and concomitant flux to lactate were significantly higher in BT257 tumor tissue. Importantly, 6,6’-2H-glucose metabolism to lactate was observed in BT257 tumor-bearing mice, but not tumor-free mice. Furthermore, imaging studies confirmed spatial localization of lactate production to the tumor vs. contralateral normal brain. We then examined the ability of 6,6’-2H-glucose to assess treatment response. Poly-(adenosine 5′-diphosphate-ribose) polymerase inhibitors (PARPi) inhibit IDHmut glioma growth and are in clinical trials for IDHmut glioma patients. Treatment with the PARPi niraparib reduced 6,6’-2H-glucose flux to lactate in BT257 tumor-bearing mice. Importantly, this reduction was observed at early time-points when no difference in tumor volume could be detected using anatomical imaging, pointing to the ability of 6,6’-2H-glucose to assess pseudoprogression. Collectively, our results suggest that IDHmut gliomas display a glycolytic phenotype amenable to non-invasive 2H-MRS-based imaging of tumor burden and treatment response.