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The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is activated in more than88% of glioblastomas (GBM). New drugs targeting this pathway are currently in clinical trials. However, noninvasive assessment of treatment response remains challenging. By using magnetic resonance spectroscopy (MRS), PI3K/Akt/mTOR pathway inhibition was monitored in 3 GBM cell lines (GS-2, GBM8, and GBM6; each with a distinct pathway activating mutation) through the measurement of 2 mechanistically linked MR biomarkers: phosphocholine (PC) and hyperpolarized lactate.(31)P MRS studies showed that treatment with the PI3K inhibitor LY294002 induced significant decreases in PC to 34 %± 9% of control in GS-2 cells, 48% ± 5% in GBM8, and 45% ± 4% in GBM6. The mTOR inhibitor everolimus also induced a significant decrease in PC to 62% ± 14%, 57% ± 1%, and 58% ± 1% in GS-2, GBM8, and GBM6 cells, respectively. Using hyperpolarized (13)C MRS, we demonstrated that hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR pathway inhibition in all 3 cell lines to 51% ± 10%, 62% ± 3%, and 58% ± 2% of control with LY294002 and 72% ± 3%, 61% ± 2%, and 66% ± 3% of control with everolimus in GS-2, GBM8, and GBM6 cells, respectively. These effects were mediated by decreases in the activity and expression of choline kinase α and lactate dehydrogenase, which respectively control PC and lactate production downstream of HIF-1. Treatment with the DNA damaging agent temozolomide did not have an effect on either biomarker in any cell line. This study highlights the potential of PC and hyperpolarized lactate as noninvasive MR biomarkers of response to targeted inhibitors in GBM.

In primary central nervous system lymphoma (PCNSL), L265P mutation of the myeloid differentiation 88 (MYD88) protein is the most common, and induces a constitutive activation of IRAK4 kinase, IRAK1 phosphorylation and increased NF-kB signaling, offering a unique window for therapeutic development. We previously show that AZ1495, a potent inhibitor of IRAK4, significantly delayed progression and improved OS in L265P-mutated PCNSL models [ASH 2016], highlighting the clinical potential of IRAK inhibition. In the clinic however, early monitoring of such targeted therapies remains challenging using conventional imaging, as treatment often results in non-readily detectable anatomical changes. Here, we questioned the potential of a new neuroimaging method, hyperpolarized 13C magnetic resonance spectroscopic imaging (HP-13C MRSI), to non-invasively monitor IRAK inhibition in PCNSL for the first time. Two PCNSL patient-derived xenografts models were studied: one harboring the L265P MYD88 mutation (MYD88mut), and the other, wild type for this protein (MYD88wt). Using HP-13C MRSI, a high production of HP [1-13C] lactate was detected in both MYD88wt and MYD88mut (0.5 ± 0.04, n=2 MYD88wt; 0.61 ± 0.2, n=4 MYD88mut), indicating PCNSL presence throughout the brain. At day 2 and 4 of AZ1495 treatment, the HP [1-13C] lactate-to-pyruvate ratio remains unchanged in MYD88wt animals at all treatment time points (p=0.29 and p=0.29 at day 2 and 4 when compared to baseline, respectively) On the other hand, this ratio significantly decreased in MYD88mut animals at all time points (36.6% at day 2, p=0.07 and 56.3% at day 4, p=0.0006), in line with the previously reported effect of AZ1495 treatment on MYD88mut PCNSL only. To conlude, we showed that HP-13C MRSI can detect MYD88-mutation-specific modulations of HP lactate production following IRAK inhibition. Because HP-13C MRSI is clinically translatable and expanding rapidly, this study is of high significance for future clinical trials, to enhance monitoring of PCNSL progression and response of targeted therapies.

Today, there is no reliable noninvasive imaging method available to monitor glioblastoma (GBM) response to therapy and predict survival prior to tumor shrinkage. Dissolution Dynamic Nuclear Polarization (DNP) combined with hyperpolarized 13C Magnetic Resonance Spectroscopic Imaging (MRSI) is a novel imaging method that allows probing real-time tumor metabolism. Recent studies using 13C MRSI have shown decreased lactate production from pyruvate in GBM responsive to a dual PI3K/mTOR inhibitor and/or Temozolomide, mediated by lower expression of LDHA or PKM2 enzymes, respectively. SAHA, the histone deacetylase (HDAC) inhibitor, is a novel drug that inhibits cell proliferation by inducing cell cycle arrest followed by apoptosis. The goal of this study was to detect early HDAC inhibition using advanced 13C MRSI. Analysis of dynamic real-time cellular metabolic changes in SAHA-treated U87 live cells in bioreactors demonstrated a significant 37.7% decrease in hyperpolarized [1-13C]-lactate production. SAHA-treated cells also showed a 29.6% decrease of steady-state lactate level in cell extracts. Furthermore, we demonstrated a significant 30.3% decrease in hyperpolarized lactate-to-pyruvate ratio in SAHA-treated U87-bearing mice, which occurred prior to MRI-detectable changes in tumor size that was associated with enhanced animal survival. In order to mechanistically validate our findings, we tested the levels of expression of LDHA, MCT1 and MCT4, the main players in pyruvate-to-lactate interconversion. While expression of LDHA, the enzyme that catalyzes pyruvate-to-lactate conversion was independent of SAHA treatment, expression of both MCT1 and MCT4 transporters, that shuttle pyruvate and lactate in and out of the cell, are increased. We thus propose that increased MCT1/4 led to a decrease in lactate in response to HDAC inhibition and resulted in a reduced pyruvate-to-lactate conversion. Our findings confirm the potential translational value of the hyperpolarized lactate-to-pyruvate ratio as a biomarker for noninvasively assessing the early effects of emerging therapies for patients with GBM.

Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in 70-90% of low-grade gliomas. The mutation leads to the production of 2-hydroxyglutarate (2-HG), a novel “oncometabolite” that alters the epigenetic profile of DNA and inhibits cellular differentiation. Recent studies indicate that metabolic changes in IDH1 mutant cells extend beyond 2-HG production. We recently demonstrated that steady state levels of glutamate, lactate and phosphocholine were reduced in IDH1 mutant cells using two genetically engineered models, a U87 glioblastoma-based model and a normal human astrocyte (NHA) model. In this study, we explored the mechanism behind the reduction in glutamate levels in IDH1 mutant cells. We discovered that pyruvate dehydrogenase (PDH) activity was significantly reduced in U87 and NHA IDH1 mutant cells compared to wild-type cells. Reduced PDH activity was associated with increased inhibitory phosphorylation of PDH in both models. Examination of mRNA and protein levels for the pyruvate dehydrogenase kinases (PDKs) revealed that PDK3 expression was up-regulated in IDH1 mutant cells in both models. Levels of hypoxia inducible factor-1α (HIF-1α), an established regulator of PDK3 expression in cancer cells, was also higher in IDH1 mutant cells. Importantly, treatment of U87 and NHA IDH1 wild-type cells with 2-HG recapitulated the effects of the IDH1 mutation, with higher levels of HIF-1α and PDK3, and reduced PDH activity, in 2-HG treated cells. Importantly, pharmacological activation of PDH activity by treatment with dichloroacetate, a PDK inhibitor, completely abrogated the increase in clonogenicity conferred by the IDH1 mutation. Treatment of patient-derived mutant IDH1 neurosphere models (BT142 and BT54 cell lines) with dichloroacetate further confirmed that PDH activity is essential for cell proliferation. Thus, our results indicate that reversing the down regulation of PDH activity in IDH1 mutant glioma cells adversely affects their proliferation, a finding with therapeutic implications.

The isocitrate dehydrogenase (IDH)1 mutation is associated with accumulation of 2-hydroxyglutarate (2-HG), but the metabolic fluxes associated with 2-HG production and cellular reprogramming of mutant IDH1 cells are not fully understood. The objective of this study was to use 13C magnetic resonance spectroscopy (MRS) to probe the fate of 13C-labeled metabolites (at thermal polarization and hyperpolarized by dynamic nuclear polarization) and to monitor the glycolytic pathway, the TCA cycle, and glutamine metabolism in wild-type and mutant IDH1 glioma cells. To achieve this goal, U87 cells expressing mutant IDH1 and wild-type IDH1 were generated by transduction with a lentiviral vector coding for mutant or wild-type IDH1 respectively. MRS studies were performed using a cell perfusion (bioreactor) system. Live cells were exposed to hyperpolarized 1-13C or 2-13C-labeled pyruvic acid, 1-13C glucose or 3-13C glutamine and cell metabolism was probed using 13C MRS. We found that in mutant IDH1 cells pyruvate flux to lactate was increased relative to wild-type cells, and pyruvate flux to glutamate and 2-HG was decreased compared to pyruvate flux to glutamate in wild-type IDH1 cell. Glutamine flux to glutamate in wild-type cells, or to glutamate and 2-HG in mutant IDH1 cells was comparable. Accordingly, the total intracellular glutamate pool was reduced in mutant IDH1 cells. The drop in pyruvate flux to the TCA cycle was mediated by a significant drop in pyruvate dehydrogenase (PDH) activity, which was due to significantly increased inhibitory PDH phosphorylation. When considering 2-HG synthesis, our data also indicate that, consistent with previous work, the majority of 2-HG is produced from glutamine (82%). However in our cells a significant portion (18%) was also derived from glucose. Our findings thus point to metabolic reprogramming in IDH1 mutant cells beyond 2-HG production and highlight the value of MRS for characterizing the metabolic changes associated with the IDH1 mutation.

BACKGROUND: The IDH1 mutation is now recognized as an important driver in the etiology of low-grade brain tumors. In contrast to wild-type IDH1, which catalyzes the conversion of isocitrate to α-ketoglutarate (αKG), mutant IDH1 converts αKG into the oncometabolite 2-hydroxygluatrate (2HG). In an effort to noninvasively image the activity of mutant IDH1, we have previously shown that we can use hyperpolarized αKG together with 13C magnetic resonance spectroscopy (MRS) to noninvasively detect the production of hyperpolarized 2HG in vivo. In more recent studies we have focused on identifying additional metabolic alterations associated with the presence of the IDH1 mutation in order to develop complementary metabolic imaging approaches that might serve to inform on the presence of mutant IDH1 and its modulation with targeted therapies. METHODS: Two mutant and wild-type IDH1 cell models were generated by using either U87 glioblastoma cells or E6/E7/hTERT immortalized normal human astrocytes (NHAs) and lentivirally transducing with either wild-type IDH1 (wt cells) or wild-type and mutant IDH1 (mutant cells). Using 1H MRS we investigated cell extracts to determine steady state metabolite levels. We also determined metabolic fluxes by using 13C MRS and probing the fate of 13C-labeled precursors (at thermal equilibrium or hyperpolarized) in live cells grown in a bioreactor or in orthotopic tumors in rats. RESULTS: We found that in both U87 and NHA-based cell models not only was the IDH1 mutation associated with elevated levels of 2HG, but other metabolite levels were also altered. In particular, steady state glutamate levels were lower. This was associated with an increased flux of glutamine to 2-HG and a concomitantly reduced flux of glutamine to glutamate. The flux of glucose into the TCA cycle and towards glutamate was also reduced. At the same time the activity and expression of BCAT1 and aspartate aminotransferase dropped. Pyruvate dehydrogenase activity was also reduced, associated with an increase in its phosphorylation and the expression of pyruvate dehydrogenase kinase 3. Collectively our findings likely explain the lower glutamate levels observed in mutant IDH1 cells. Importantly, we were also able to detect a drop in the potentially translatable in vivo imaging of hyperpolarized glutamate produced from hyperpolarized αKG in mutant IDH1 orthotopic tumors. CONCLUSIONS: In addition to the production of 2HG, the presence of mutant IDH1 leads to MRS-detectable metabolic changes that could serve as complementary imaging biomarkers of mutant IDH1 activity or its inhibition with treatment. SECONDARY CATEGORY: n/a.