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. 1 H-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 1 H-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.

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 1 H- and 13 C- 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: 1 H-MRS indicated a significant decrease in steady-state 2-HG following treatment, as expected. This was accompanied by a significant 1 H-MRS-detectable increase in glutamate. However, other metabolites previously linked to 2-HG were not altered. 13 C-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 13 C-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 1 H- and 13 C-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 1 H-MRS-detectable drop in 2-HG, a 1 H-MRS-detectable increase in glutamate, as well as a hyperpolarized 13 C-MRS-detectable change in [1- 13 C] α-KG flux, could serve as metabolic imaging biomarkers of response to 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 1 H 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 1 H MRS increase in glutamate/ glutamine occurred prior to tumor shrinkage. Cells labeled with [1-13 C]glucose and [3-13 C]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 [5-13 C]glutamate produced from [2-13 C]pyruvate and hyperpolarized [1-13 C]glutamate produced from [1-13 C]a-ketoglutarate were significantly higher in temozolomide-treated cells compared with controls. Collectively, our findings identify 1 H MRS-detectable elevation of glutamate and hyperpolarized 13 C MRS-detectable glutamate production from either pyruvate or a-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.

<div>Abstract<p>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 <i>in vivo</i>, 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.</p>Significance:<p>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.</p></div>

<div>Abstract<p>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 <i>in vivo</i>, 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.</p>Significance:<p>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.</p></div>

Mutations in isocitrate dehydrogenase 1/2 (IDHmut) are reported in 70–90% of low-grade gliomas and secondary glioblastomas. IDHmut catalyzes the reduction of a-ketoglutarate (a-KG) to 2-hydroxyglutarate (2-HG), an oncometabolite that drives tumorigenesis. Inhibition of IDHmut is therefore a rapidly emerging therapeutic approach and IDHmut inhibitors such as AG-120 and AG-881 have shown promising results in phase 1 and 2 clinical studies. The goal of this study was to identify early non-invasive metabolic biomarkers of IDHmut inhibition that can serve to moniter response to these therapies. We used 1H and 13C magnetic resonance spectroscopy (MRS) to investigate the response of two genetically-engineered IDHmut cell lines (U87-based and normal human astrocyte-based) to AG-120 and AG-881 treatment. As expected, in both cell lines, our 1H-MRS data indicated that AG-120 and AG-881 induced a significant decrease in 2-HG. Interestingly however, we also observed a significant increase in phosphocholine and glutamate, pointing to broader changes in the metabolism of treated cells and a unique MRS signature. To further investigate the increase in glutamate induced by AG-120 and AG-881 in our models, we used 13C-MRS and quantified the flux of [1-13C] glucose and [3-13C] glutamine to 13C-labeled glutamate. Our results indicate that both AG-120 and AG-881 significantly increase the flux of 13C-labeled glutamine to 13C glutamate, while the flux of 13C-labeled glucose to 13C glutamate remained unchanged. Further studies are currently underway to explore the utility of using hyperpolarized [1-13C]-glutamine and hyperpolarized [1-13C]-a-KG for monitoring flux to glutamate and 2-HG, and to validate these probes as additional biomarkers of response to IDHmut inhibition. Taken together, our studies indicate that IDHmut inhibition induces a unique MRS-detectable metabolic profile that can potentially be exploited for early non-invasive, clinically translatable detection of response to emerging IDHmut inhibitors.

BACKGROUND TERT promoter mutations that result in TERT expression are observed in over 80% of GBM and upstream inhibition of TERT expression by targeting GABPB1L is currently under investigation. In that context, non-invasive reliable biomarkers that can help detect TERT expression are needed. The aim of this research was to assess the value of magnetic resonance spectroscopy (MRS)-detectable metabolic changes as biomarkers of TERT expression in GBM. METHODS GABPB1L knock down clones (GABPB1LKD) were established by introducing Crispr Cas9 plasmid vector targeting GABPB1L into U251 cells. Two representative clones with different knock down efficiency were chosen and compared to control cells. Tumor forming capacity was evaluated by colony formation assay and magnetic resonance imaging of orthotopically implanted tumors in mice. Cells were extracted using the dual phase extraction method and 1H-MRS data of cell extracts acquired using a Bruker 500 scanner. The data was analyzed using Mnova software. Multivariate analysis was performed using the SIMCA software. RESULTS TERT expression was significantly reduced in GABPB1LKD compared to control cells depending on the GABPB1L knock down efficiency. Colony forming capacity was impaired in GABPB1LKD compared to control cells. In vivo MRI data showed significantly smaller tumor volumes in GABPB1LKD compared to control. Unbiased PCA analysis of 1H-MRS data showed separation of GABPB1LKD and control extracts and VIP scores derived from the OPLS-DA analysis, demonstrated that the common metabolites leading to separation of GABPB1LKD and control cells were aspartate, glutathione, glycerophosphocholine, glutamine, NAD(P)+, AXP. This data was confirmed by univariate analysis that revealed that aspartate, glutathione, glutamine, NAD(P)+, AXP level was significantly reduced in GABPB1LKD. CONCLUSIONS GABPB1L knock down cells that show reduced TERT expression demonstrate MRS-detectable metabolic changes. These could be translated into clinical applications, improve the monitoring of GBM patients and advance precision medicine.

<p>Supplementary Figures S1-S9 and tables S1-S6</p>