Vamsidhara Vemireddy

Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse, human glioblastomas in the mouse brain in vivo

Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.

In vivo chemical exchange saturation transfer imaging allows early detection of a therapeutic response in glioblastoma

Significance The prognosis and management of patients with glioma is vastly different depending on whether one detects tumor progression or treatment effects. Although the gold standard in the evaluation of treatment efficacy involves MRI, the currently available imaging methods often do not suffice to make the final decision. Our study demonstrated that amide proton transfer (APT) imaging, one subset of chemical exchange saturation transfer imaging, can detect molecular signals in glioma induced by short-term chemotherapy with temozolomide. These molecular events precede morphologic changes. The APT signal did not decrease in tumors resistant to chemotherapy. APT imaging may provide a useful prognostic biomarker of treatment response or tumor progression in glioma. Glioblastoma multiforme (GBM), which account for more than 50% of all gliomas, is among the deadliest of all human cancers. Given the dismal prognosis of GBM, it would be advantageous to identify early biomarkers of a response to therapy to avoid continuing ineffective treatments and to initiate other therapeutic strategies. The present in vivo longitudinal study in an orthotopic mouse model demonstrates quantitative assessment of early treatment response during short-term chemotherapy with temozolomide (TMZ) by amide proton transfer (APT) imaging. In a GBM line, only one course of TMZ (3 d exposure and 4 d rest) at a dose of 80 mg/kg resulted in substantial reduction in APT signal compared with untreated control animals, in which the APT signal continued to increase. Although there were no detectable differences in tumor volume, cell density, or apoptosis rate between groups, levels of Ki67 (index of cell proliferation) were substantially reduced in treated tumors. In another TMZ-resistant GBM line, the APT signal and levels of Ki67 increased despite the same course of TMZ treatment. As metabolite changes are known to occur early in the time course of chemotherapy and precede morphologic changes, these results suggest that the APT signal in glioma may be a useful functional biomarker of treatment response or degree of tumor progression. Thus, APT imaging may serve as a sensitive biomarker of early treatment response and could potentially replace invasive biopsies to provide a definitive diagnosis. This would have a major impact on the clinical management of patients with glioma.

Prospective Longitudinal Analysis of 2-Hydroxyglutarate Magnetic Resonance Spectroscopy Identifies Broad Clinical Utility for the Management of Patients With IDH-Mutant Glioma

Purpose Proton magnetic resonance spectroscopy (MRS) of the brain can detect 2-hydroxyglutarate (2HG), the oncometabolite produced in neoplasms harboring a mutation in the gene coding for isocitrate dehydrogenase ( IDH). We conducted a prospective longitudinal imaging study to determine whether quantitative assessment of 2HG by MRS could serve as a noninvasive clinical imaging biomarker for IDH-mutated gliomas. Patients and Methods 2HG MRS was performed in 136 patients using point-resolved spectroscopy at 3 T in parallel with standard clinical magnetic resonance imaging and assessment. Data were analyzed in patient cohorts representing the major phases of the glioma clinical course and were further subgrouped by histology and treatment type to evaluate 2HG. Histologic correlations were performed. Results Quantitative 2HG MRS was technically and biologically reproducible. 2HG concentration > 1 mM could be reliably detected with high confidence. During the period of indolent disease, 2HG concentration varied by less than ± 1 mM, and it increased sharply with tumor progression. 2HG concentration was positively correlated with tumor cellularity and significantly differed between high- and lower-grade gliomas. In response to cytotoxic therapy, 2HG concentration decreased rapidly in 1p/19q codeleted oligodendrogliomas and with a slower time course in astrocytomas and mixed gliomas. The magnitude and time course of the decrease in 2HG concentration and magnitude of the decrease in tumor volume did not differ between oligodendrogliomas treated with temozolomide or carmustine. Criteria for 2HG MRS were established to make a presumptive molecular diagnosis of an IDH mutation in gliomas technically unable to undergo a surgical procedure. Conclusion 2HG concentration as measured by MRS was reproducible and reliably reflected the disease state. These data provide a basis for incorporating 2HG MRS into clinical management of IDH-mutated gliomas.

Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors.

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2‐13C2]glucose. The [3‐13C]lactate/[2,3‐13C2]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, 13C–13C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, 13C multiplets of γ‐aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patients primary renal mass and brain metastasis were all strongly immunopositive for the 67‐kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that 13C‐labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy. Copyright

EGFR wild type antagonizes EGFRvIII-mediated activation of Met in glioblastoma

Epidermal growth factor receptor (EGFR)vIII is the most common EGFR mutant found in glioblastoma (GBM). EGFRvIII does not bind ligand, is highly oncogenic and is usually coexpressed with EGFR wild type (EGFRwt). EGFRvIII activates Met, and Met contributes to EGFRvIII-mediated oncogenicity and resistance to treatment. Here, we report that addition of EGF results in a rapid loss of EGFRvIII-driven Met phosphorylation in glioma cells. Met is associated with EGFRvIII in a physical complex. Addition of EGF results in a dissociation of the EGFRvIII–Met complex with a concomitant loss of Met phosphorylation. Consistent with the abrogation of Met activation, addition of EGF results in the inhibition of EGFRvIII-mediated resistance to chemotherapy. Thus, our study suggests that ligand in the milieu of EGFRvIII-expressing GBM cells is likely to influence the EGFRvIII–Met interaction and resistance to treatment, and highlights a novel antagonistic interaction between EGFRwt and EGFRvIII in glioma cells.

DNA double-strand breaks cooperate with loss of Ink4 and Arf tumor suppressors to generate glioblastomas with frequent Met amplification

Glioblastomas (GBM) are highly radioresistant and lethal brain tumors. Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are a risk factor for the development of GBM. In this study, we systematically examined the contribution of IR-induced DSBs to GBM development using transgenic mouse models harboring brain-targeted deletions of key tumor suppressors frequently lost in GBM, namely Ink4a, Ink4b, Arf and/or PTEN. Using low linear energy transfer (LET) X-rays to generate simple breaks or high LET HZE particles (Fe ions) to generate complex breaks, we found that DSBs induce high-grade gliomas in these mice which, otherwise, do not develop gliomas spontaneously. Loss of Ink4a and Arf was sufficient to trigger IR-induced glioma development but additional loss of Ink4b significantly increased tumor incidence. We analyzed IR-induced tumors for copy number alterations to identify oncogenic changes that were generated and selected for as a consequence of stochastic DSB events. We found Met amplification to be the most significant oncogenic event in these radiation-induced gliomas. Importantly, Met activation resulted in the expression of Sox2, a GBM cancer stem cell marker, and was obligatory for tumor formation. In sum, these results indicate that radiation-induced DSBs cooperate with loss of Ink4 and Arf tumor suppressors to generate high-grade gliomas that are commonly driven by Met amplification and activation.

Hepatic gluconeogenesis influences 13C enrichment in lactate in human brain tumors during metabolism of [1,2-13C]acetate

(13)C-enriched compounds are readily metabolized in human malignancies. Fragments of the tumor, acquired by biopsy or surgical resection, may be acid-extracted and (13)C NMR spectroscopy of metabolites such as glutamate, glutamine, 2-hydroxyglutarate, lactate and others provide a rich source of information about tumor metabolism in situ. Recently we observed (13)C-(13)C spin-spin coupling in (13)C NMR spectra of lactate in brain tumors removed from patients who were infused with [1,2-(13)C]acetate prior to the surgery. We found, in four patients, that infusion of (13)C-enriched acetate was associated with synthesis of (13)C-enriched glucose, detectable in plasma. (13)C labeled glucose derived from [1,2-(13)C]acetate metabolism in the liver and the brain pyruvate recycling in the tumor together lead to the production of the (13)C labeled lactate pool in the brain tumor. Their combined contribution to acetate metabolism in the brain tumors was less than 4.0%, significantly lower than the direct oxidation of acetate in the citric acid cycle in tumors.

1H MRS characterization of neurochemical profiles in orthotopic mouse models of human brain tumors

Glioblastoma (GBM), the most common primary brain tumor, is resistant to currently available treatments. The development of mouse models of human GBM has provided a tool for studying mechanisms involved in tumor initiation and growth as well as a platform for preclinical investigation of new drugs. In this study we used 1H MR spectroscopy to study the neurochemical profile of a human orthotopic tumor (HOT) mouse model of human GBM. The goal of this study was to evaluate differences in metabolite concentrations in the GBM HOT mice when compared with normal mouse brain in order to determine if MRS could reliably differentiate tumor from normal brain. A TE =19 ms PRESS sequence at 9.4 T was used for measuring metabolite levels in 12 GBM mice and 8 healthy mice. Levels for 12 metabolites and for lipids/macromolecules at 0.9 ppm and at 1.3 ppm were reliably detected in all mouse spectra. The tumors had significantly lower concentrations of total creatine, GABA, glutamate, total N‐acetylaspartate, aspartate, lipids/macromolecules at 0.9 ppm, and lipids/macromolecules at 1.3 ppm than did the brains of normal mice. The concentrations of glycine and lactate, however, were significantly higher in tumors than in normal brain. Copyright

Measurement of 13C turnover into glutamate and glutamine pools in brain tumor patients

Malignant brain tumors are known to utilize acetate as an alternate carbon source in the citric acid cycle for their bioenergetics. 13C NMR‐based isotopomer analysis has been used to measure turnover of 13C‐acetate carbons into glutamate and glutamine pools in tumors. Plasma from the patients infused with [1,2‐13C]acetate further revealed the presence of 13C isotopomers of glutamine, glucose, and lactate in the circulation that were generated due to metabolism of [1,2‐13C]acetate by peripheral organs. In the tumor cells, [4‐13C] and [3,4‐13C]glutamate and glutamine isotopomers were generated from blood‐borne 13C‐labeled glucose and lactate which were formed due to [1,2‐13C[acetate metabolism of peripheral tissues. [4,5‐13C] and [3,4,5‐13C]glutamate and glutamine isotopomers were produced from [1,2‐13C]acetyl‐CoA that was derived from direct oxidation of [1,2‐13C] acetate in the tumor. Major portion of C4 13C fractional enrichment of glutamate (93.3 ± 0.02%) and glutamine (90.9 ± 0.03%) were derived from [1,2‐13C]acetate‐derived acetyl‐CoA.

Abstract 1111: Preclinical evaluation of CP868,596, a novel PDGFRα Inhibitor for treatment of glioblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Recent large scale genomic studies have shown that ∼13% of glioblastoma (GBM) have a copy number gain of the gene encoding for platelet-derived growth factor receptor alpha (PDGFRα). Animal studies have shown that PDGFRα signaling can drive tumor growth. Clinical studies, however, have failed to show efficacy when patients with recurrent GBM were treated with non-specific PDGFR inhibitors (Imatinib, Sunitinib). Drawing firm conclusions from such studies is limited by lack of patient stratification by PDGFRα expression and lack of biological data demonstrating effective target inhibition. CP-868,596 is a benzimidazole which is orally bioavailable, and has IC50s of 0.9 nM and 1.8 nM for PDGFRα and PDGFRb, respectively. Phase I trials of CP-868,596 have shown a favorable safety profile and serum concentrations up to 2000 nM. We are pursuing a systematic in vitro and in vivo preclinical evaluation to assess potential clinical efficacy of CP-868,596 for the treatment of glioma. Methods: To evaluate IC50 for CP868,596, we have genetically engineered conditional Ink4a/Arf-/- mouse astrocytes to stably co-express both the human PDGFRα and its cognate ligand, PDGF-AA. Expression levels of PDGFRα and PDGF-AA levels were adjusted so they are comparable to those we have found in GBM tumors. Results and Discussion: Immunoblot analysis shows that PDGFRα phosphorylation and its downstream signaling pathway (pAKT, phospho-S6 ribosomal protein, and p-4EBP1) activated either by autocrine and/or exogenous PDGF-AA ligand (1um) is completely blocked by CP868,596 (concentrations 0.1-1um). INK4a/Arf-/-, PDGFRα/PDGF-AA astrocytes, when implanted into the NOD-SCID mouse brain or subcutaneous space (flank), form orthotopic /heterotopic tumors, respectively. Using these glioma models, intratumoral CP868,596 levels and the ability of the drug to block PDGFRα phosphorylation is being evaluated (dose range 0.1-5 ug/kg/hr IV). While multiple signal transduction pathways are implicated in pathogenesis of GBM, CP868,596 may only be effective in those GBM where PDGFR is the dominant ‘driver’ of tumorigenesis. To test this hypothesis we have established a panel of primary GBM orthotopic mouse tumor lines which overexpress PDGFRα/PDGF-AA with and without EGFR and/or cMet gene overexpression and will evaluate response to treatment with CP868,596. In addition, a Phase II treatment trial in adult glioma patients is being initiated. Data from the in vitro and in vivo studies will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1111. doi:10.1158/1538-7445.AM2011-1111