Monitoring response to a clinically relevant IDH inhibitor in glioma-Hyperpolarized 13C magnetic resonance spectroscopy approaches.

Background: Mutant isocitrate dehydrogenase (IDHmut) catalyzes 2-hydroxyglutarate (2HG) production and is considered a therapeutic target for IDHmut tumors. However, response is mostly associated with inhibition of tumor growth. Response assessment via anatomic imaging is therefore challenging. Our goal was to directly detect IDHmut inhibition using a new hyperpolarized (HP) 13C magnetic resonance spectroscopy-based approach to noninvasively assess α-ketoglutarate (αKG) metabolism to 2HG and glutamate. Methods: We studied IDHmut-expressing normal human astrocyte (NHAIDH1mut) cells and rats with BT257 tumors, and assessed response to the IDHmut inhibitor BAY-1436032 (n ≥ 4). We developed a new 13C Echo Planar Spectroscopic Imaging sequence with an optimized RF pulse to monitor the fate of HP [1-13C]αKG and [5-12C,1-13C]αKG with a 2.5 × 2.5 × 8 mm3 spatial resolution. Results: Cell studies confirmed that BAY-1436032-treatment leads to a drop in HP 2HG and an increase in HP glutamate detectable with both HP substrates. Data using HP [5-12C,1-13C]αKG also demonstrated that its conversion to 2HG is detectable without the proximal 1.1% natural abundance [5-13C]αKG signal. In vivo studies showed that glutamate is produced in normal brains but no 2HG is detectable. In tumor-bearing rats, we detected the production of both 2HG and glutamate, and BAY-1436032-treatment led to a drop in 2HG and an increase in glutamate. Using HP [5-12C,1-13C]αKG we detected metabolism with an signal-to-noise ratio of 23 for 2HG and 17 for glutamate. Conclusions: Our findings point to the clinical potential of HP αKG, which recently received FDA investigational new drug approval for research, for noninvasive localized imaging of IDHmut status.

Telomerase reverse transcriptase induces targetable alterations in glutathione and nucleotide biosynthesis in glioblastomas.

Telomerase reverse transcriptase (TERT) is essential for glioblastoma (GBM) proliferation. Delineating metabolic vulnerabilities induced by TERT can lead to novel GBM therapies. We previously showed that TERT upregulates glutathione (GSH) pool size in GBMs. Here, we show that TERT acts via the FOXO1 transcription factor to upregulate expression of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme of de novo GSH synthesis. Inhibiting GCLC using siRNA or buthionine sulfoximine (BSO) reduces synthesis of 13 C-GSH from [U- 13 C]-glutamine and inhibits clonogenicity. However, GCLC inhibition does not induce cell death, an effect that is associated with elevated [U- 13 C]-glutamine metabolism to glutamate and pyrimidine nucleotide biosynthesis. Mechanistically, GCLC inhibition activates MYC and leads to compensatory upregulation of two key glutamine-utilizing enzymes i.e., glutaminase (GLS), which generates glutamate from glutamine, and CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotatase), the enzyme that converts glutamine to the pyrimidine nucleotide precursor dihydroorotate. We then examined the therapeutic potential of inhibiting GLS and CAD in combination with GCLC. 6-diazo-5-oxy-L-norleucin (DON) is a potent inhibitor of glutamine-utilizing enzymes including GLS and CAD. The combination of BSO and DON suppresses GSH and pyrimidine nucleotide biosynthesis and is synergistically lethal in GBM cells. Importantly, in vivo stable isotope tracing indicates that combined treatment with JHU-083 (a brain-penetrant prodrug of DON) and BSO abrogates synthesis of GSH and pyrimidine nucleotides from [U- 13 C]-glutamine and induces tumor shrinkage in mice bearing intracranial GBM xenografts. Collectively, our studies exploit a mechanistic understanding of TERT biology to identify synthetically lethal metabolic vulnerabilities in GBMs. SIGNIFICANCE: Using in vivo stable isotope tracing, metabolomics, and loss-of-function studies, we demonstrate that TERT expression is associated with metabolic alterations that can be synergistically targeted for therapy in glioblastomas.

Hyperpolarized δ-[1- 13C]gluconolactone imaging visualizes response to TERT or GABPB1 targeting therapy for glioblastoma.

TERT promoter mutations are a hallmark of glioblastoma (GBM). Accordingly, TERT and GABPB1, a subunit of the upstream mutant TERT promoter transcription factor GABP, are being considered as promising therapeutic targets in GBM. We recently reported that the expression of TERT or GABP1 modulates flux via the pentose phosphate pathway (PPP). Here, we investigated whether 13C magnetic resonance spectroscopy (MRS) of hyperpolarized (HP) δ- [1-13C]gluconolactone can serve to image the reduction in PPP flux following TERT or GABPB1 silencing. We investigated two different human GBM cell lines stably expressing shRNAs targeting TERT or GABPB1, as well as doxycycline-inducible shTERT or shGABPB1cells. MRS studies were performed on live cells and in vivo tumors, and dynamic sets of 13C MR spectra were acquired following injection of HP δ-[1-13C]gluconolactone. HP 6-phosphogluconolactone (6PG), the product of δ-[1-13C]gluconolactone via the PPP, was significantly reduced in TERT or GABPB1-silenced cells or tumors compared to controls in all our models. Furthermore, a positive correlation between TERT expression and 6PG levels was observed. Our data indicate that HP δ-[1-13C]gluconolactone, an imaging tool with translational potential, could serve to monitor TERT expression and its silencing with therapies that target either TERT or GABPB1 in mutant TERT promoter GBM patients.

A 13C/31P surface coil to visualize metabolism and energetics in the rodent brain at 3 Tesla.

PURPOSE: We constructed a 13C/31P surface coil at 3 T for studying cancer metabolism and bioenergetics. In a single scan session, hyperpolarized 13C-pyruvate MRS and 31P MRS was carried out for a healthy rat brain. METHODS: All experiments were carried out at 3 Tesla. The multinuclear surface coil was designed as two coplanar loops each tuned to either the 13C or 31P operating frequency with an LCC trap on the 13C loop. A commercial volume proton coil was used for anatomical localization and B0 shimming. Single tuned coils operating at either the 13C or 31P frequency were built to evaluate the relative performance of the multinuclear coil. Coil performance metrics consisted of measuring Q factor ratio, calculating system input power using a single-pulse acquisition, and acquiring SNR and flip angle maps using 2D CSI sequences. To observe in vivo spectra, a bolus of hyperpolarized [1-13C] pyruvate was administered via tail vein. In vivo13C and endogenous 31P spectra were obtained in a single scan session using 1D slice selective acquisitions. RESULTS: When 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 of 13C labeled lactate, alanine, and bicarbonate in addition to ATP from 31P MRS. CONCLUSIONS: The 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.

Deuterium Metabolic Imaging Reports on TERT Expression and Early Response to Therapy in Cancer.

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.

Imaging biomarkers of TERT or GABPB1 silencing in TERT-positive glioblastoma.

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.

Deuterium magnetic resonance spectroscopy enables noninvasive metabolic imaging of tumor burden and response to therapy in low-grade gliomas.

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.

Acquisition and quantification pipeline for in vivo hyperpolarized 13 C MR spectroscopy.

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.

Imaging 6-Phosphogluconolactonase Activity in Brain Tumors In Vivo Using Hyperpolarized δ-[1-13C]gluconolactone.

INTRODUCTION: The pentose phosphate pathway (PPP) is essential for NADPH generation and redox homeostasis in cancer, including glioblastomas. However, the precise contribution to redox and tumor proliferation of the second PPP enzyme 6-phosphogluconolactonase (PGLS), which converts 6-phospho-δ-gluconolactone to 6-phosphogluconate (6PG), remains unclear. Furthermore, non-invasive methods of assessing PGLS activity are lacking. The goal of this study was to examine the role of PGLS in glioblastomas and assess the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive imaging. METHODS: To interrogate the function of PGLS in redox, PGLS expression was silenced in U87, U251 and GS2 glioblastoma cells by RNA interference and levels of NADPH and reduced glutathione (GSH) measured. Clonogenicity assays were used to assess the effect of PGLS silencing on glioblastoma proliferation. Hyperpolarized δ-[1-13C]gluconolactone metabolism to 6PG was assessed in live cells treated with the chemotherapeutic agent temozolomide (TMZ) or with vehicle control. 13C 2D echo-planar spectroscopic imaging (EPSI) studies of hyperpolarized δ-[1-13C]gluconolactone metabolism were performed on rats bearing orthotopic glioblastoma tumors or tumor-free controls on a 3T spectrometer. Longitudinal 2D EPSI studies of hyperpolarized δ-[1-13C]gluconolactone metabolism and T2-weighted magnetic resonance imaging (MRI) were performed in rats bearing orthotopic U251 tumors following treatment with TMZ to examine the ability of hyperpolarized δ-[1-13C]gluconolactone to report on treatment response. RESULTS: PGLS knockdown downregulated NADPH and GSH, elevated oxidative stress and inhibited clonogenicity in all models. Conversely, PGLS expression and activity and steady-state NADPH and GSH were higher in tumor tissues from rats bearing orthotopic glioblastoma xenografts relative to contralateral brain and tumor-free brain. Importantly, [1-13C]6PG production from hyperpolarized δ-[1-13C]gluconolactone was observed in live glioblastoma cells and was significantly reduced by treatment with TMZ. Furthermore, hyperpolarized δ-[1-13C]gluconolactone metabolism to [1-13C]6PG could differentiate tumor from contralateral normal brain in vivo. Notably, TMZ significantly reduced 6PG production from hyperpolarized δ-[1-13C]gluconolactone at an early timepoint prior to volumetric alterations as assessed by anatomical imaging. CONCLUSIONS: Collectively, we have, for the first time, identified a role for PGLS activity in glioblastoma proliferation and validated the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive in vivo imaging of glioblastomas and their response to therapy.

Metabolic imaging detects elevated glucose flux through the pentose phosphate pathway associated with TERT expression in low-grade gliomas.

BACKGROUND: Telomerase reverse transcriptase (TERT) is essential for tumor proliferation, including in low-grade oligodendrogliomas (LGOGs). Since TERT is silenced in normal cells, it is also a therapeutic target. Therefore, noninvasive methods of imaging TERT are needed. Here, we examined the link between TERT expression and metabolism in LGOGs, with the goal of leveraging this information for noninvasive magnetic resonance spectroscopy (MRS)-based metabolic imaging of LGOGs. METHODS: Immortalized normal human astrocytes with doxycycline-inducible TERT silencing, patient-derived LGOG cells, orthotopic tumors, and LGOG patient biopsies were studied to determine the mechanistic link between TERT expression and glucose metabolism. The ability of hyperpolarized [U-13C, U-2H]-glucose to noninvasively assess TERT expression was tested in live cells and orthotopic tumors. RESULTS: TERT expression was associated with elevated glucose flux through the pentose phosphate pathway (PPP), elevated NADPH, which is a major product of the PPP, and elevated glutathione, which is maintained in a reduced state by NADPH. Importantly, hyperpolarized [U-13C, U-2H]-glucose metabolism via the PPP noninvasively reported on TERT expression and response to TERT inhibition in patient-derived LGOG cells and orthotopic tumors. Mechanistically, TERT acted via the sirtuin SIRT2 to upregulate the glucose transporter GLUT1 and the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase. CONCLUSIONS: We have, for the first time, leveraged a mechanistic understanding of TERT-associated metabolic reprogramming for noninvasive imaging of LGOGs using hyperpolarized [U-13C, U-2H]-glucose. Our findings provide a novel way of imaging a hallmark of tumor immortality and have the potential to improve diagnosis and treatment response assessment for LGOG patients.

Early Noninvasive Metabolic Biomarkers of Mutant IDH Inhibition in Glioma.

Approximately 80% of low-grade glioma (LGGs) harbor mutant isocitrate dehydrogenase 1/2 (IDH1/2) driver mutations leading to accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Thus, inhibition of mutant IDH is considered a potential therapeutic target. Several mutant IDH inhibitors are currently in clinical trials, including AG-881 and BAY-1436032. However, to date, early detection of response remains a challenge. In this study we used high resolution 1H magnetic resonance spectroscopy (1H-MRS) to identify early noninvasive MR (Magnetic Resonance)-detectable metabolic biomarkers of response to mutant IDH inhibition. In vivo 1H-MRS was performed on mice orthotopically-implanted with either genetically engineered (U87IDHmut) or patient-derived (BT257 and SF10417) mutant IDH1 cells. Treatment with either AG-881 or BAY-1436032 induced a significant reduction in 2-HG. Moreover, both inhibitors led to a significant early and sustained increase in glutamate and the sum of glutamate and glutamine (GLX) in all three models. A transient early increase in N-acetylaspartate (NAA) was also observed. Importantly, all models demonstrated enhanced animal survival following both treatments and the metabolic alterations were observed prior to any detectable differences in tumor volume between control and treated tumors. Our study therefore identifies potential translatable early metabolic biomarkers of drug delivery, mutant IDH inhibition and glioma response to treatment with emerging clinically relevant therapies.

Non-invasive assessment of telomere maintenance mechanisms in brain tumors.

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.

Glutamate Is a Noninvasive Metabolic Biomarker of IDH1-Mutant Glioma Response to Temozolomide Treatment.

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.

Patient-derived cells from recurrent tumors that model the evolution of IDH-mutant glioma.

BACKGROUND: IDH-mutant lower-grade gliomas (LGGs) evolve under the selective pressure of therapy, but well-characterized patient-derived cells (PDCs) modeling evolutionary stages are lacking. IDH-mutant LGGs may develop therapeutic resistance associated with chemotherapy-driven hypermutation and malignant progression. The aim of this study was to establish and characterize PDCs, single-cell-derived PDCs (scPDCs), and xenografts (PDX) of IDH1-mutant recurrences representing distinct stages of tumor evolution. METHODS: We derived and validated cell cultures from IDH1-mutant recurrences of astrocytoma and oligodendroglioma. We used exome sequencing and phylogenetic reconstruction to examine the evolutionary stage represented by PDCs, scPDCs, and PDX relative to corresponding spatiotemporal tumor tissue and germline DNA. PDCs were also characterized for growth and tumor immortality phenotypes, and PDX were examined histologically. RESULTS: The integrated astrocytoma phylogeny revealed 2 independent founder clonal expansions of hypermutated (HM) cells in tumor tissue that are faithfully represented by independent PDCs. The oligodendroglioma phylogeny showed more than 4000 temozolomide-associated mutations shared among tumor samples, PDCs, scPDCs, and PDX, suggesting a shared monoclonal origin. The PDCs from both subtypes exhibited hallmarks of tumorigenesis, retention of subtype-defining genomic features, production of 2-hydroxyglutarate, and subtype-specific telomere maintenance mechanisms that confer tumor cell immortality. The oligodendroglioma PDCs formed infiltrative intracranial tumors with characteristic histology. CONCLUSIONS: These PDCs, scPDCs, and PDX are unique and versatile community resources that model the heterogeneous clonal origins and functions of recurrent IDH1-mutant LGGs. The integrated phylogenies advance our knowledge of the complex evolution and immense mutational load of IDH1-mutant HM glioma.

MR-detectable metabolic biomarkers of response to mutant IDH inhibition in low-grade glioma.

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.

In vivo detection of γ-glutamyl-transferase up-regulation in glioma using hyperpolarized γ-glutamyl-[1-13C]glycine.

Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. γ-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP γ-glutamyl-[1-13C]glycine and acquiring dynamic 13C data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of γ-glutamyl-[1-13C]glycine and its product [1-13C]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in γ-glutamyl-[1-13C]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1-13C]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1-13C]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP γ-glutamyl-[1-13C]glycine to monitor GGT expression in the brain and thus to detect glioblastoma.

First hyperpolarized [2-13C]pyruvate MR studies of human brain metabolism.

We developed methods for the preparation of hyperpolarized (HP) sterile [2-13C]pyruvate to test its feasibility in first-ever human NMR studies following FDA-IND & IRB approval. Spectral results using this MR stable-isotope imaging approach demonstrated the feasibility of investigating human cerebral energy metabolism by measuring the dynamic conversion of HP [2-13C]pyruvate to [2-13C]lactate and [5-13C]glutamate in the brain of four healthy volunteers. Metabolite kinetics, signal-to-noise (SNR) and area-under-curve (AUC) ratios, and calculated [2-13C]pyruvate to [2-13C]lactate conversion rates (kPL) were measured and showed similar but not identical inter-subject values. The kPL measurements were equivalent with prior human HP [1-13C]pyruvate measurements.

PI3K/mTOR inhibition of IDH1 mutant glioma leads to reduced 2HG production that is associated with increased survival.

70-90% of low-grade gliomas and secondary glioblastomas are characterized by mutations in isocitrate dehydrogenase 1 (IDHmut). IDHmut produces the oncometabolite 2-hydroxyglutarate (2HG), which drives tumorigenesis in these tumors. The phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway represents an attractive therapeutic target for IDHmut gliomas, but noninvasive indicators of drug target modulation are lacking. The goal of this study was therefore to identify magnetic resonance spectroscopy (MRS)-detectable metabolic biomarkers associated with IDHmut glioma response to the dual PI3K/(mTOR) inhibitor XL765. 1H-MRS of two cell lines genetically modified to express IDHmut showed that XL765 induced a significant reduction in several intracellular metabolites including 2HG. Importantly, examination of an orthotopic IDHmut tumor model showed that enhanced animal survival following XL765 treatment was associated with a significant in vivo 1H-MRS detectable reduction in 2HG but not with significant inhibition in tumor growth. Further validation is required, but our results indicate that 2HG could serve as a potential noninvasive MRS-detectable metabolic biomarker of IDHmut glioma response to PI3K/mTOR inhibition.

In vivo investigation of hyperpolarized [1,3-13C2]acetoacetate as a metabolic probe in normal brain and in glioma.

Dysregulation in NAD+/NADH levels is associated with increased cell division and elevated levels of reactive oxygen species in rapidly proliferating cancer cells. Conversion of the ketone body acetoacetate (AcAc) to ß-hydroxybutyrate (ß-HB) by the mitochondrial enzyme ß-hydroxybutyrate dehydrogenase (BDH) depends upon NADH availability. The ß-HB-to-AcAc ratio is therefore expected to reflect mitochondrial redox. Previous studies reported the potential of hyperpolarized 13C-AcAc to monitor mitochondrial redox in cells, perfused organs and in vivo. However, the ability of hyperpolarized 13C-AcAc to cross the blood brain barrier (BBB) and its potential to monitor brain metabolism remained unknown. Our goal was to assess the value of hyperpolarized [1,3-13C2]AcAc in healthy and tumor-bearing mice in vivo. Following hyperpolarized [1,3-13C2]AcAc injection, production of [1,3-13C2]ß-HB was detected in normal and tumor-bearing mice. Significantly higher levels of [1-13C]AcAc and lower [1-13C]ß-HB-to-[1-13C]AcAc ratios were observed in tumor-bearing mice. These results were consistent with decreased BDH activity in tumors and associated with increased total cellular NAD+/NADH. Our study confirmed that AcAc crosses the BBB and can be used for monitoring metabolism in the brain. It highlights the potential of AcAc for future clinical translation and its potential utility for monitoring metabolic changes associated with glioma, and other neurological disorders.

HDAC inhibition in glioblastoma monitored by hyperpolarized 13 C MRSI.

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized 13 C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real-time dynamic metabolic changes using a bioreactor system with live vorinostat-treated or control cells. We found a significant 40% decrease in the 13 C MRS-detectable ratio of hyperpolarized [1-13 C]lactate to hyperpolarized [1-13 C]pyruvate, [1-13 C]Lac/Pyr, and a 37% decrease in the pseudo-rate constant, kPL , for hyperpolarized [1-13 C]lactate production, in vorinostat-treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat-treated cells. In vivo 13 C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1-13 C]Lac/Pyr when comparing vorinostat-treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC-inhibited tumors. Thus, the 13 C MRSI-detectable decrease in hyperpolarized [1-13 C]lactate production could serve as a biomarker of response to HDAC inhibitors.