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Diffuse midline gliomas (DMGs) are lethal primary brain tumors in children. The imipridones ONC201 and ONC206 induce mitochondrial dysfunction and have emerged as promising therapies for DMG patients. However, efficacy as monotherapy is limited, identifying a need for strategies that enhance response. Another hurdle is the lack of biomarkers that report on drug-target engagement at an early timepoint after treatment onset. Here, using 1 H-magnetic resonance spectroscopy, which is a non-invasive method of quantifying metabolite pool sizes, we show that accumulation of ψ-aminobutyric acid (GABA) is an early metabolic biomarker that can be detected within a week of ONC206 treatment, when anatomical alterations are absent, in mice bearing orthotopic xenografts. Mechanistically, imipridones activate the mitochondrial protease ClpP and upregulate the stress-responsive transcription factor ATF4. ATF4, in turn, upregulates glutamate decarboxylase, which synthesizes GABA, and downregulates ABAT , which degrades GABA, leading to GABA accumulation in DMG cells and tumors. Functionally, GABA secreted by imipridone-treated cells acts in an autocrine manner via the GABAB receptor to induce expression of superoxide dismutase (SOD1), which mitigates imipridone-induced oxidative stress and, thereby, curbs apoptosis. Importantly, blocking autocrine GABA signaling using the clinical stage GABAB receptor antagonist SGS-742 exacerbates oxidative stress and synergistically induces apoptosis in combination with imipridones in DMG cells and orthotopic tumor xenografts. Collectively, we identify GABA as a unique metabolic adaptation to imipridones that can be leveraged for non-invasive assessment of drug-target engagement and therapy. Clinical translation of our studies has the potential to enable precision metabolic therapy and imaging for DMG patients. One Sentence Summary: Imipridones induce GABA accumulation in diffuse midline gliomas, an effect that can be leveraged for therapy and non-invasive imaging.
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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.
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Background: Telomere maintenance by telomerase reverse transcriptase (TERT) is essential for immortality in most cancers, including oligodendrogliomas. Agents that disrupt telomere maintenance such as the telomere uncapping agent 6-thio-2'-deoxyguanosine (6-thio-dG) are in clinical trials. We previously showed that TERT expression in oligodendrogliomas is associated with upregulation of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). We also showed that hyperpolarized δ-[1-13C]-gluconolactone metabolism to 6-phosphogluconate (6-PG) can be used to probe the PPP in glioblastomas. The goal of this study was to determine whether hyperpolarized 13C imaging using δ-[1-13C]-gluconolactone can monitor TERT expression and response to 6-thio-dG in oligodendrogliomas. Methods: We examined patient-derived oligodendroglioma cells and orthotopic tumors to assess the link between TERT and hyperpolarized δ-[1-13C]-gluconolactone metabolism. We performed in vivo imaging to assess the ability of hyperpolarized δ-[1-13C]-gluconolactone to report on TERT and response to 6-thio-dG in rats bearing orthotopic oligodendrogliomas in vivo. Results: Doxycycline-inducible TERT silencing abrogated 6-PG production from hyperpolarized δ-[1-13C]-gluconolactone in oligodendroglioma cells, consistent with the loss of G6PD activity. Rescuing TERT expression by doxycycline removal restored G6PD activity and, concomitantly, 6-PG production. 6-PG production from hyperpolarized δ-[1-13C]-gluconolactone demarcated TERT-expressing tumor from surrounding TERT-negative normal brain in vivo. Importantly, 6-thio-dG abrogated 6-PG production at an early timepoint preceding MRI-detectable alterations in rats bearing orthotopic oligodendrogliomas in vivo. Conclusions: These results indicate that hyperpolarized δ-[1-13C]-gluconolactone reports on TERT expression and early response to therapy in oligodendrogliomas. Our studies identify a novel agent for imaging tumor proliferation and treatment response in oligodendroglioma patients.
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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.
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Neoplasias Encefálicas , Glioblastoma , Telomerase , Humanos , Glioblastoma/diagnóstico por imagem , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Espectroscopia de Ressonância Magnética/métodos , Lactonas/uso terapêutico , Diagnóstico por Imagem , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Telomerase/genética , Telomerase/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/metabolismoRESUMO
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.
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Imageamento por Ressonância Magnética , Prótons , Animais , Ratos , Roedores/metabolismo , Bicarbonatos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Imagens de Fantasmas , Ácido Pirúvico/metabolismo , Lactatos , Alanina , Trifosfato de Adenosina , Desenho de EquipamentoRESUMO
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.
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Glioblastoma , Telomerase , Animais , Deutério/metabolismo , Ácido Láctico/metabolismo , Camundongos , NAD/metabolismo , Ácido Pirúvico/metabolismo , Telomerase/genética , Telomerase/metabolismoRESUMO
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.
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Glioblastoma , Telomerase , Humanos , Glioblastoma/tratamento farmacológico , Isótopos de Carbono/metabolismo , Isótopos de Carbono/uso terapêutico , Ácido Láctico/metabolismo , Biomarcadores , Telomerase/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/metabolismoRESUMO
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.
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Astrocitoma , Neoplasias Encefálicas , Glioma , Animais , Astrocitoma/diagnóstico por imagem , Astrocitoma/tratamento farmacológico , Astrocitoma/genética , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Deutério , Glioma/diagnóstico por imagem , Glioma/tratamento farmacológico , Glioma/genética , Glucose , Lactatos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Camundongos , Carga TumoralRESUMO
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.
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Neoplasias Encefálicas , Ácido Pirúvico , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Isótopos de Carbono , Cinética , Espectroscopia de Ressonância Magnética , Ácido Pirúvico/metabolismo , Ratos , Razão Sinal-RuídoRESUMO
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.
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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.
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Oligodendroglioma , Telomerase , Glucose , Humanos , Espectroscopia de Ressonância Magnética , Oligodendroglioma/diagnóstico por imagem , Oligodendroglioma/genética , Via de Pentose Fosfato , Telomerase/genética , Telomerase/metabolismoRESUMO
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.
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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.
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Neoplasias Encefálicas/genética , Homeostase do Telômero , Telômero/metabolismo , Alanina/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Isótopos de Carbono/metabolismo , Linhagem Celular Tumoral , Engenharia Genética , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Ácido Láctico/metabolismo , Masculino , Metaboloma , Modelos Biológicos , Gradação de Tumores , Proteínas de Neoplasias/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Nus , Telomerase/genética , Telomerase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although cancer has been known for decades to harbor an insatiable appetite for iron, only recently has the chemistry emerged to exploit this altered state therapeutically, by targeting the expanded cytosolic labile iron pool (LIP) of the cancer cell. The state of the art includes therapies that react with the LIP to produce cytotoxic radical species (in some cases also releasing drug payloads) and molecules that exacerbate LIP-induced oxidative stress to trigger ferroptosis. Effectively implementing LIP-targeted therapies in patients will require biomarkers to identify those tumors with the most elevated LIP and thus most likely to succumb to LIP-targeted interventions. Toward this goal, we tested whether tumor uptake of the novel LIP-sensing radiotracer 18F-TRX aligns with tumor sensitivity to LIP-targeted therapies. Methods:18F-TRX uptake was assessed in vivo among 10 subcutaneous and orthotopic human xenograft models. Glioma and renal cell carcinoma were prioritized because these tumors have the highest relative expression levels of STEAP3, the oxidoreductase that reduces ferric iron to the ferrous oxidation state, in the Broad Institute Cancer Cell Line Encyclopedia. The antitumor effects of the LIP-activated prodrug TRX-CBI, which releases the DNA alkylator CBI, were compared in mice bearing U251 or PC3 xenografts, tumors with high and intermediate levels of 18F-TRX uptake, respectively. Results:18F-TRX showed a wide range of tumor accumulation. An antitumor assessment study showed that the growth of U251 xenografts, the model with the highest 18F-TRX uptake, was potently inhibited by TRX-CBI. Moreover, the antitumor effects against U251 were significantly greater than those observed for PC3 tumors, consistent with the relative 18F-TRX-determined LIP levels in tumors before therapy. Lastly, a dosimetry study showed that the estimated effective human doses for adult male and female mice were comparable to those of other 18F-based imaging probes. Conclusion: We report the first evidence-to our knowledge-that tumor sensitivity to an LIP-targeted therapy can be predicted with a molecular imaging tool. More generally, these data bring a new dimension to the nuclear theranostic model by showing a requirement for imaging to quantify, in situ, the concentration of a metastable bioanalyte toward predicting tumor drug sensitivity.
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Compostos Radiofarmacêuticos , Animais , Linhagem Celular Tumoral , Feminino , Masculino , Camundongos , Imagem MolecularRESUMO
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.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Ácido Glutâmico/metabolismo , Isocitrato Desidrogenase/genética , Temozolomida/uso terapêutico , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Isótopos de Carbono , Feminino , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Nus , Mutação , Engenharia de Proteínas , Ácido Pirúvico/metabolismo , Distribuição Aleatória , Resultado do TratamentoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Glioma/diagnóstico por imagem , Isocitrato Desidrogenase/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Tumoral , Diaminas/farmacologia , Glioma/tratamento farmacológico , Glioma/genética , Ácido Glutâmico/metabolismo , Glutaratos/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Mutação , Espectroscopia de Prótons por Ressonância Magnética , Piridinas/farmacologiaRESUMO
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.
Assuntos
Encéfalo/diagnóstico por imagem , Glioblastoma/diagnóstico , Imageamento por Ressonância Magnética/métodos , Imagem Molecular/métodos , gama-Glutamiltransferase/metabolismo , Animais , Encéfalo/patologia , Isótopos de Carbono/administração & dosagem , Isótopos de Carbono/química , Linhagem Celular Tumoral , Dipeptídeos/administração & dosagem , Dipeptídeos/química , Estudos de Viabilidade , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Masculino , Sondas Moleculares/administração & dosagem , Sondas Moleculares/química , Ratos , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Astrócitos/metabolismo , Neoplasias Encefálicas/mortalidade , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Transformada , Glioma/mortalidade , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Estimativa de Kaplan-Meier , Camundongos , Proteínas de Neoplasias/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Processamento de Proteína Pós-Traducional , Quinoxalinas/farmacologia , Quinoxalinas/uso terapêutico , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Acetoacetatos/metabolismo , Encéfalo/metabolismo , Glioma/metabolismo , Acetoacetatos/química , Animais , Feminino , Espectroscopia de Ressonância Magnética , Camundongos , Mitocôndrias/metabolismo , Oxirredução , EspectrofotometriaRESUMO
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.