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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 noninvasive assessment of tumor proliferation and response to therapy. Experimental Design: 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 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. 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 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 MRI. Conclusions: 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.

Table S1. GS-2 orthotopic tumor model: in vivo single voxel 1H MRS; Table S2. GS-2 orthotopic tumor model: ex vivo 1H HR-MAS MR Spectroscopy; Table S3. U87-MG orthotopic tumor model: ex vivo 1H HR-MAS MR Spectroscopy.

Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1mut) genes as they progressed through their lifespan. IDH1mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1WT or IDH1mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1mut cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo. Clonal populations of postcrisis IDH1mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680–9. ©2016 AACR.

Purpose: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts.Experimental Design: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s).Results: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met–activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts.Conclusion: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance. Clin Cancer Res; 19(7); 1773–83. ©2012 AACR.

Supplementary Figures S1-S9 and tables S1-S6

PDF file - 1353K, Supplementary Table S1. Patient and tumor characteristics from the 22 bevacizumab-resistant versus 22 bevacizumab-na�ve glioblastomas from whom paired pre-treatment tissue was available. Supplementary Table S2. Altered transcripts in 15 bevacizumab-resistant glioblastomas compared to before treatment. Supplementary Table S3. Altered transcripts in 15 bevacizumab-resistant glioblastomas compared to before treatment. Supplementary Table S4. Altered transcripts in bevacizumab-naive recurrent glioblastomas compared to their paired glioblastomas at diagnosis. Supplemetary Table S5. FISH Results. Supplementary Table S6. Patient and tumor characteristics from 22 bevacizumab-na�ve recurrent glioblastomas (control group) from whom paired pre-treatment tissue was available.

CQ and BAF restore phospholipid levels and inhibit clonogenicity of IDHmut glioma cells.

CQ and BAF do not affect ER area or phospholipid levels in IDHwt glioma cells.

2-HG induces a reduction in ER area in IDHmut glioma cells.

PDF file - 102K

Supplementary Data from Deuterium Metabolic Imaging Reports on TERT Expression and Early Response to Therapy in Cancer

Tumor metabolism is reprogrammed to meet the demands of proliferating cancer cells. In particular, cancer cells upregulate synthesis of the membrane phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) in order to allow for rapid membrane turnover. Nonetheless, we show here that, in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas, which produce the oncometabolite 2-hydroxyglutarate (2-HG), PtdCho and PtdE biosynthesis is downregulated and results in lower levels of both phospholipids when compared with wild-type IDH1 cells. 2-HG inhibited collagen-4-prolyl hydroxylase activity, leading to accumulation of misfolded procollagen-IV in the endoplasmic reticulum (ER) of both genetically engineered and patient-derived IDHmut glioma models. The resulting ER stress triggered increased expression of FAM134b, which mediated autophagic degradation of the ER (ER-phagy) and a reduction in the ER area. Because the ER is the site of phospholipid synthesis, ER-phagy led to reduced PtdCho and PtdE biosynthesis. Inhibition of ER-phagy via pharmacological or molecular approaches restored phospholipid biosynthesis in IDHmut glioma cells, triggered apoptotic cell death, inhibited tumor growth, and prolonged the survival of orthotopic IDHmut glioma-bearing mice, pointing to a potential therapeutic opportunity. Glioma patient biopsies also exhibited increased ER-phagy and downregulation of PtdCho and PtdE levels in IDHmut samples compared with wild-type, clinically validating our observations. Collectively, this study provides detailed and clinically relevant insights into the functional link between oncometabolite-driven ER-phagy and phospholipid biosynthesis in IDHmut gliomas.Significance: Downregulation of phospholipid biosynthesis via ER-phagy is essential for proliferation and clonogenicity of mutant IDH1 gliomas, a finding with immediate therapeutic implications. Cancer Res; 78(9); 2290–304. ©2018 AACR.

2-HG inhibits PtdCho and PtdE biosynthesis.

SNVs identified by exome-sequencing.

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PDF file - 3201K, Supplementary Figure S1. Real time RT PCR analysis of BRGs compared to paired pre-treatment GBMs; Supplementary Figure S2. FISH reveals no change in c-Met amplification after bevacizumab resistance as compared to before; Supplementary Figure S3. Changes in Pearson correlation coefficients to 3 previously described molecular GBM subtypes in BRGs and bevacizumab-na�ve GBMs after recurrence; Supplementary Figure S4. C-Met phosphorylation increases after bevacizumab resistance; Supplementary Figure S5. Increased expression of phosphorylated FAK, a downstream effector of c-Met, in bevacizumab-resistant glioblastomas (BRGs) compared to before treatment; Supplementary Figure S6. Increased expression of phosphorylated STAT3, a downstream effector of c-Met, in bevacizumab-resistant glioblastomas (BRGs) compared to before treatment; Supplementary Figure S7. Hypoxia after bevacizumab resistance versus bevacizumab-na�ve recurrence; Supplementary Figure S8. Endothelial cells do not contribute to increased c-Met expression after bevacizumab resistance; Supplementary Figure S9. Hypoxia increases HGF expression in U87-IgG and U87-BevR cells; Supplementary Figure S10. Xenografts established from BRGs analyzed by immunohistochemistry; Supplementary Figure S11. Immunofluorescent staining of xenografts established from bevacizumab-na�ve GBMs; Supplementary Figure S12. Hematoxylin and Eosin staining of xenografts established from bevacizumab-na�ve GBMs; Supplementary Figure S13. Comparison of xenografts from BRGs versus xenografts from bevacizumab-na�ve GBMs; Supplementary Figure S14. Murine MRI of intracranial BRG-derived xenografts reveals loss of the ability of VEGF blockade to reduce vascular permeability and fractional blood volume; Supplementary Figure S15. Reduced c-Met expression after transducing BRG-derived SF7796 xenograft cells with c-Met shRNA; Supplementary Figure S16. Effects of c-Met knockdown on brain slice invasion in U87-BevR cells; Supplementary Figure S17. Altered morphology in BRG cells after transduction with shRNA targeting c-Met; Supplementary Figure S18. C-Met knockdown alters brain slice invasion in BRG-derived SF7796 cells; Supplementary Figure S19. Hematoxylin and eosin stainings of intracranial SF7796/shControl and SF7796/shCmet1 xenografts; Supplementary Figure S20. Transduction of U87 with retrovirus derived from pBABE-Puro-Tpr-met leads to increased Tpr-met phosphorylation.

Phospholipid biosynthesis is down-regulated in IDHmut glioma cells.

Silencing FAM134b, Atg5 or Atg7 restores phospholipid biosynthesis in IDHmut glioma cells.

2-HG induces ER-phagy in IDHmut glioma cells.

Although lower grade gliomas are driven by mutations in the isocitrate dehydrogenase 1 (IDH1) gene and are less aggressive than primary glioblastoma, they nonetheless generally recur. IDH1-mutant patients are increasingly being treated with temozolomide, but early detection of response remains a challenge and there is a need for complementary imaging methods to assess response to therapy prior to tumor shrinkage. The goal of this study was to determine the value of magnetic resonance spectroscopy (MRS)–based metabolic changes for detection of response to temozolomide in both genetically engineered and patient-derived mutant IDH1 models. Using 1H MRS in combination with chemometrics identified several metabolic alterations in temozolomide-treated cells, including a significant increase in steady-state glutamate levels. This was confirmed in vivo, where the observed 1H MRS increase in glutamate/glutamine occurred prior to tumor shrinkage. Cells labeled with [1–13C]glucose and [3–13C]glutamine, the principal sources of cellular glutamate, showed that flux to glutamate both from glucose via the tricarboxylic acid cycle and from glutamine were increased following temozolomide treatment. In line with these results, hyperpolarized [5–13C]glutamate produced from [2–13C]pyruvate and hyperpolarized [1–13C]glutamate produced from [1–13C]α-ketoglutarate were significantly higher in temozolomide-treated cells compared with controls. Collectively, our findings identify 1H MRS-detectable elevation of glutamate and hyperpolarized 13C MRS-detectable glutamate production from either pyruvate or α-ketoglutarate as potential translatable metabolic biomarkers of response to temozolomide treatment in mutant IDH1 glioma.Significance:These findings show that glutamate can be used as a noninvasive, imageable metabolic marker for early assessment of tumor response to temozolomide, with the potential to improve treatment strategies for mutant IDH1 patients.

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 noninvasive assessment of tumor proliferation and response to therapy.Experimental Design: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 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. 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 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 MRI.Conclusions: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.

ER-phagy is linked to phospholipid biosynthesis in BT54 neurospheres (A-C), orthotopic IDHmut tumor xenografts (D-J) and IDHmut patient biopsies (K-Q).

Effect of 2-HG on cell proliferation of IDH1 wild-type cells for the U87 (A) and NHA (B) models.

Supplementary Figure 4 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

Supplementary Figure 3 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

The recent development of hyperpolarized 13C magnetic resonance spectroscopic imaging provides a novel method for in vivo metabolic imaging with potential applications for detection of cancer and response to treatment. Chemotherapy-induced apoptosis was shown to decrease the flux of hyperpolarized 13C label from pyruvate to lactate due to depletion of NADH, the coenzyme of lactate dehydrogenase. In contrast, we show here that in PC-3MM2 tumors, inhibition of platelet-derived growth factor receptor with imatinib reduces the conversion of hyperpolarized pyruvate to lactate by lowering the expression of lactate dehydrogenase itself. This was accompanied by reduced expression of vascular endothelial growth factor and glutaminase, and is likely mediated by reduced expression of their transcriptional factors hypoxia-inducible factor-1 and c-Myc. Our results indicate that hyperpolarized 13C MRSI could potentially detect the molecular effect of various cell signaling inhibitors, thus providing a radiation-free method to predict tumor response. Cancer Res; 70(19); 7400–10. ©2010 AACR.

SuppFigure3. Stable suppression of PKM2 expression in G55 cells significantly alters intra-cellular concentrations of pyruvate and lactate. (*, p

Comparison of PDH activity in the various models used in this study.

Supplementary Figure 1 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

Supplementary Figure 2 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

Supplementary Methods, Figure Legends 1-4 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

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 noninvasive 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 noninvasive 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 the 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.

Purpose The goal of this study was to combine a specialized acquisition method with a new quantification pipeline to accurately and efficiently probe the metabolism of hyperpolarized 13 C-labeled compounds in vivo. In this study, we tested our approach on [2-13 C]pyruvate and [1-13 C]α-ketoglutarate data in rat orthotopic brain tumor models at 3T. Methods We used a multiband metabolite-specific radiofrequency (RF) excitation in combination with a variable flip angle scheme to minimize substrate polarization loss and measure fast metabolic processes. We then applied spectral-temporal denoising using singular value decomposition to enhance spectral quality. This was combined with LCModel-based automatic 13 C spectral fitting and flip angle correction to separate overlapping signals and rapidly quantify the different metabolites. Results Denoising improved the metabolite signal-to-noise ratio (SNR) by approximately 5. It also improved the accuracy of metabolite quantification as evidenced by a significant reduction of the Cramer Rao lower bounds. Furthermore, the use of the automated and user-independent LCModel-based quantification approach could be performed rapidly, with the kinetic quantification of eight metabolite peaks in a 12-spectrum array achieved in less than 1 minute. Conclusion The specialized acquisition method combined with denoising and a new quantification pipeline using LCModel for the first time for hyperpolarized 13 C data enhanced our ability to monitor the metabolism of [2-13 C]pyruvate and [1-13 C]α-ketoglutarate in rat orthotopic brain tumor models in vivo. This approach could be broadly applicable to other hyperpolarized agents both preclinically and in the clinical setting.

Background TERT promoter mutations are observed in 80% of wild-type IDH glioblastoma (GBM). Moreover, the upstream TERT transcription factor GABPB1 was recently identified as a cancer-specific therapeutic target for tumors harboring a TERT promoter mutation. In that context, noninvasive imaging biomarkers are needed for the detection of TERT modulation. Methods Multiple GBM models were investigated as cells and in vivo tumors and the impact of TERT silencing, either directly or by targeting GABPB1, was determined using 1H and hyperpolarized 13C magnetic resonance spectroscopy (MRS). Changes in associated metabolic enzymes were also investigated. Results 1H-MRS revealed that lactate and glutathione (GSH) were the most significantly altered metabolites when either TERT or GABPB1 was silenced, and lactate and GSH levels were correlated with cellular TERT expression. Consistent with the drop in lactate, 13C-MRS showed that hyperpolarized [1-13C]lactate production from [1-13C]pyruvate was also reduced when TERT was silenced. Mechanistically, the reduction in GSH was associated with a reduction in pentose phosphate pathway flux, reduced activity of glucose-6-phosphate dehydrogenase, and reduced NADPH. The drop in lactate and hyperpolarized lactate were associated with reductions in glycolytic flux, NADH, and expression/activity of GLUT1, monocarboxylate transporters, and lactate dehydrogenase A. Conclusions Our study indicates that MRS-detectable GSH, lactate, and lactate production could serve as metabolic biomarkers of response to emerging TERT-targeted therapies for GBM with activating TERT promoter mutations. Importantly these biomarkers are readily translatable to the clinic, and thus could ultimately improve GBM patient management.

We constructed aAll experiments were carried out at 3 Tesla. The multinuclear surface coil was designed as two coplanar loops each tuned to either theWhen compared with single tuned surface coils, the multinuclear coil performance showed a decrease in Q factor ratio, SNR, and transmit efficiency. Flip angle maps showed adequate flip angles within the phantom when the transmit voltage was set using an external phantom. Results show good detection ofThe coil enables obtaining complementary information within a scan session, thus reducing the number of trials and minimizing biological variability for studies of metabolism and bioenergetics.

Telomere maintenance is a universal hallmark of cancer. Most tumors including low-grade oligodendrogliomas use telomerase reverse transcriptase (TERT) expression for telomere maintenance while astrocytomas use the alternative lengthening of telomeres (ALT) pathway. Although TERT and ALT are hallmarks of tumor proliferation and attractive therapeutic targets, translational methods of imaging TERT and ALT are lacking. Here we show that TERT and ALT are associated with unique 1H-magnetic resonance spectroscopy (MRS)-detectable metabolic signatures in genetically-engineered and patient-derived glioma models and patient biopsies. Importantly, we have leveraged this information to mechanistically validate hyperpolarized [1-13C]-alanine flux to pyruvate as an imaging biomarker of ALT status and hyperpolarized [1-13C]-alanine flux to lactate as an imaging biomarker of TERT status in low-grade gliomas. Collectively, we have identified metabolic biomarkers of TERT and ALT status that provide a way of integrating critical oncogenic information into non-invasive imaging modalities that can improve tumor diagnosis and treatment response monitoring., Telomerase expression and the alternative lengthening of telomeres pathway are hallmarks of cancer. Here, the authors show that, in primary brain tumors, these features are correlated with metabolic signatures detectable by metabolic imaging, suggesting that they can be used to non-invasively monitor telomere maintenance in brain tumours.

Author(s): Molloy, Abigail R; Najac, Chloe; Viswanath, Pavithra; Lakhani, Aliya; Subramani, Elavarasan; Batsios, Georgios; Radoul, Marina; Gillespie, Anne Marie; Pieper, Russell O; Ronen, Sabrina M | Abstract: Mutations in isocitrate dehydrogenase 1 (IDH1mut) are reported in 70-90% of low-grade gliomas and secondary glioblastomas. IDH1mut catalyzes the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), an oncometabolite which drives tumorigenesis. Inhibition of IDH1mut is therefore an emerging therapeutic approach, and inhibitors such as AG-120 and AG-881 have shown promising results in phase 1 and 2 clinical studies. However, detection of response to these therapies prior to changes in tumor growth can be challenging. The goal of this study was to identify non-invasive clinically translatable metabolic imaging biomarkers of IDH1mut inhibition that can serve to assess response. Methods: IDH1mut inhibition was confirmed using an enzyme assay and 1H- and 13C- magnetic resonance spectroscopy (MRS) were used to investigate the metabolic effects of AG-120 and AG-881 on two genetically engineered IDH1mut-expressing cell lines, NHAIDH1mut and U87IDH1mut. Results: 1H-MRS indicated a significant decrease in steady-state 2-HG following treatment, as expected. This was accompanied by a significant 1H-MRS-detectable increase in glutamate. However, other metabolites previously linked to 2-HG were not altered. 13C-MRS also showed that the steady-state changes in glutamate were associated with a modulation in the flux of glutamine to both glutamate and 2-HG. Finally, hyperpolarized 13C-MRS was used to show that the flux of α-KG to both glutamate and 2-HG was modulated by treatment. Conclusion: In this study, we identified potential 1H- and 13C-MRS-detectable biomarkers of response to IDH1mut inhibition in gliomas. Although further studies are needed to evaluate the utility of these biomarkers in vivo, we expect that in addition to a 1H-MRS-detectable drop in 2-HG, a 1H-MRS-detectable increase in glutamate, as well as a hyperpolarized 13C-MRS-detectable change in [1-13C] α-KG flux, could serve as metabolic imaging biomarkers of response to treatment.

Metabolic reprogramming is a fundamental hallmark of cancer, which can be exploited for non-invasive tumor imaging. Deuterium magnetic resonance spectroscopy (2H-MRS) recently emerged as a novel, translational method of interrogating flux from 2H-labeled substrates to metabolic products. However, to date, preclinical studies have been performed in vivo, an endeavor which suffers from low-throughput and potential wastage of animal life, especially when considering studies of treatment response. Developing in vitro assays for monitoring metabolism of 2H-labeled substrates will enhance throughput, lead to the rapid evaluation of new 2H-based probes and enable identification of treatment response biomarkers, thereby allowing the best 2H-based probes to be translated for further in vivo assessment. The goal of this study was to develop a preclinical cell-based platform for quantifying metabolism of 2H-labeled probes in brain tumor models. Since the Warburg effect, which is characterized by elevated glycolytic production of lactate, is a metabolic phenotype of cancer, including brain tumors, we examined metabolism of 2H-glucose or 2H-pyruvate in patient-derived glioblastoma (GBM6) and oligodendroglioma (BT88) cells and compared to normal human astrocytes (NHACONTROL). Following incubation in media containing [6,6’-2H]glucose or [U-2H]pyruvate, 2H-MR spectra obtained from live cell suspensions showed elevated 2H-lactate production in GBM6 and BT88 cells relative to NHACONTROL. Importantly, 2H-lactate production from [6,6’-2H]glucose or from [U-2H]pyruvate was reduced in GBM6 or BT88 cells subjected to irradiation and temozolomide, which is standard of care for glioma patients, pointing to the utility of this method for detecting response to therapy. Collectively, we have, for the first time, demonstrated the ability to quantify metabolism of 2H-MRS probes in live cell suspensions and validated the utility of our assay for differentiating tumor from normal cells and assessing response to therapy. Our studies will expedite the identification of novel 2H-MRS probes for imaging brain tumors and potentially other types of cancer.

Glioma patient management relies heavily on magnetic resonance imaging (MRI). However, MRI is often inadequate for assessment of tumor burden and pseudoprogression. Non-invasive methods that report on molecular pathways such as telomere maintenance that drive tumor proliferation are needed. Among brain tumors, low-grade astrocytomas (LGAs) use the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. The goal of this study was to identify ALT-linked metabolic alterations that can be exploited for non-invasive magnetic resonance spectroscopy (MRS)-based imaging of LGAs. We examined the patient-derived BT257 model and compared neurospheres that are ALT-dependent (BT257 ALT+) with those in which the ALT pathway has been silenced (BT257 ALT-). Our studies suggest that expression and activity of the rate-limiting glycolytic enzyme phosphofructokinase-1 are significantly higher in BT257 ALT+ neurospheres relative to ALT-, an effect that is associated with elevated glucose flux to lactate. Studies indicate that poly(ADP-ribose) polymerase inhibitors such as niraparib selectively induce telomeric fusion and cell death in ALT-dependent cells. We find that the telomeric fusion-mediated cytotoxicity of niraparib is associated with significantly reduced glycolytic flux in BT257 ALT+ neurospheres. We then examined whether 2H-MRS using [6,6’-2H]-glucose, which is a clinically translatable method of imaging glycolytic flux, can be used to monitor the ALT pathway in vivo. [6,6’-2H]-glucose flux to lactate is elevated in tumor relative to normal brain in mice bearing orthotopic BT257 tumors. Importantly, following treatment of BT257 tumor-bearing mice with niraparib, lactate production from [6,6’-2H]-glucose is significantly reduced at early timepoints when alterations in tumor volume cannot be observed by MRI, pointing to the ability of [6,6’-2H]-glucose to report on pseudoprogression in vivo. Collectively, our studies mechanistically link the ALT pathway with elevated glycolytic flux via phosphofructokinase-1 and identify deuterium metabolic imaging as a novel, non-invasive method of imaging tumor burden and treatment response in LGAs in vivo.

SUMMARYTelomerase reverse transcriptase (TERT) expression is indispensable for tumor immortality. Non-invasive methods of imaging TERT can, therefore, report on tumor proliferation and response to therapy. Here, we show that TERT expression is associated with elevated levels of the redox metabolite 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 NADH biosynthesis. Importantly, deuterium magnetic resonance spectroscopy (2H-MRS), which is a novel, clinically translatable metabolic imaging modality, can be leveraged for imaging TERT-linked NADH in preclinical tumor models in vivo. Since NADH is essential for pyruvate flux to lactate, 2H-MRS following administration of 2H-labeled pyruvate non-invasively visualizes TERT expression and reports on early response to therapy. Collectively, our study provides insights into the mechanisms of TERT-linked metabolic reprogramming and, importantly, establishes 2H-MRS as a pan-cancer strategy for imaging tumor immortality.