Kumar Pichumani

Background: Metabolic alterations during transformation of low-grade gliomas (LGGs) into high-grade glioblastomas (GBMs) remain incompletely understood. Particularly, IDH wildtype (IDHwt) diffuse astrocytomas harboring TERT promoter (TERTp) mutations, classified as molecular GBM under the 2021 WHO classification, may exhibit distinct metabolic and epigenetic features compared to histological WHO Grade 4 GBMs. Here, we conducted a detailed metabolomic comparison of tumor specimens from a patient initially diagnosed with WHO Grade 2 IDHwt diffuse astrocytoma carrying TERTp mutation, who subsequently progressed to a histologically confirmed WHO Grade 4 GBM upon recurrence.Case presentation: A 66-year-old female patient underwent surgical resection of a WHO Grade 2 diffuse astrocytoma in April 2018. Molecular testing revealed IDH1-wildtype status and TERTp mutation, classifying the tumor as a molecular GBM. Following ~3.5 years of clinical stability, magnetic resonance imaging detected tumor recurrence. The patient underwent a second craniotomy in February 2022, with histopathology confirming progression to WHO Grade 4 GBM. Using untargeted proton nuclear magnetic resonance (¹H NMR) spectroscopy, we analyzed aqueous-methanol and chloroform phases from methanol-chloroform-water extraction of tumor tissue from both time points. Compared to the primary tumor, the aqueous-methanol phase of the recurrent WHO Grade 4 GBM specimen showed decreased levels of neuronal and glial markers including N-acetylaspartate, myo-inositol, and scyllo-inositol. Elevated metabolites included phosphocholine, phosphoethanolamine, glycine, taurine, hypotaurine, branched-chain amino acids (leucine, isoleucine, valine), and notably alanine, which increased approximately 6-fold. Alanine likely serves as an alternative carbon source supporting tumor proliferation and aggressiveness. The chloroform phase showed the presence of cholesterol in both tumors; however, cholesteryl ester (CE) was detected only in the recurrent tumor. The CE-to-cholesterol ratio of 0.44 in the recurrent tumor suggests significant cholesterol esterification during malignant progression.Conclusion: Our findings identify alanine accumulation and increased cholesterol esterification as key metabolic features accompanying malignant transformation from molecular GBM to histological WHO grade-4 GBM. These metabolic changes may serve as biomarkers of tumor progression and recurrence. Importantly, alanine detection via magnetic resonance spectroscopy offers promising potential for non-invasive glioma diagnostics. Furthermore, targeting cholesterol esterification pathways using Acyl-CoA:cholesterol acyltransferase inhibitors could provide a novel therapeutic approach, especially for low-grade astrocytomas with high risk of malignant progression and recurrence.

We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesis and unraveling their metabolic phenotypes provide new therapeutic insights. Glutamine catabolism is key to the growth and proliferation of tumors. Here, we investigated the metabolomics of freshly resected meningiomas and glutamine metabolism in patient-derived meningioma cells. ¹H NMR spectroscopy of tumor tissues from meningioma patients was used to differentiate the metabolite profiles of grade-I and grade-II meningiomas. Glutamine metabolism was examined using ¹³C/¹⁵N glutamine tracer, in 5 patient-derived meningioma cells. Alanine, lactate, glutamate, glutamine, and glycine were predominantly elevated only in grade-II meningiomas by 74%, 76%, 35%, 75%, and 33%, respectively, with alanine and glutamine levels being statistically significant (P ≤ .02). ¹³C/¹⁵N glutamine tracer experiments revealed that both grade-I and -II meningiomas actively metabolize glutamine to generate various key carbon intermediates including alanine and proline that are necessary for the tumor growth. Also, it is shown that glutaminase (GLS1) inhibitor, CB-839 is highly effective in downregulating glutamine metabolism and decreasing proliferation in meningioma cells. Alanine and glutamine/glutamate are mainly elevated in grade-II meningiomas. Grade-I meningiomas possess relatively higher glutamine metabolism providing carbon/nitrogen for the biosynthesis of key nonessential amino acids. GLS1 inhibitor (CB-839) is very effective in downregulating glutamine metabolic pathways in grade-I meningiomas leading to decreased cellular proliferation.

Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of GBM to use galactose (Gal) as a fuel via the Leloir pathway is investigated. Methods: Gene transcript data were accessed to determine the association between expression of genes of the Leloir pathway and patient outcomes. Growth studies were performed on five primary patient-derived GBM cultures using Glc-free media supplemented with Gal. The role of Glut3/Glut14 in sugar import was investigated using antibody inhibition of hexose transport. A specific inhibitor of GALK1 (Cpd36) was used to inhibit Gal catabolism. Gal metabolism was examined using proton, carbon and phosphorous NMR spectroscopy, with ¹³C-labeled Glc and Gal as tracers. Results: Data analysis from published databases revealed that elevated levels of mRNA transcripts of SLC2A3 (Glut3), SLC2A14 (Glut14) and key Leloir pathway enzymes correlate with poor patient outcomes. GBM cultures proliferated when grown solely on Gal in Glc-free media and switching Glc-grown GBM cells into Gal-enriched/Glc-free media produced elevated levels of Glut3 and/or Glut14 enzymes. The ¹³C NMR-based metabolic flux analysis demonstrated a fully functional Leloir pathway and elevated pentose phosphate pathway activity for efficient Gal metabolism in GBM cells. Conclusion: Expression of Glut3 and/or Glut14 together with the enzymes of the Leloir pathway allows GBM to transport and metabolize Gal at physiological glucose concentrations, providing GBM cells with an alternate energy source. The presence of this pathway in GBM and its selective targeting may provide new treatment strategies.

Background: Rathke’s Cleft Cysts (RCCs) are rare epithelial cysts arising from remnants of the Rathke pouch in the pituitary gland. A subset of these lesions enlarge and produce a mass effect with consequent hypopituitarism, and may result in visual loss. Moreover, some RCCs with a high intra-cystic protein content may mimic cystic pituitary adenoma, which makes their differential diagnosis ambiguous. Currently, medical professionals have no definitive way to distinguish RCCs from pituitary adenomas. Therefore, preoperative confirmation of RCCs would be of help to medical professionals for the management and proper surgical decision making. The goal of this study is to identify molecular markers in RCCs. Methods: We characterized aqueous and chloroform extracts of surgically resected RCCs and pituitary adenomas using ex vivo ¹H NMR spectroscopy. Results: All RCCs exclusively showed the presence of mucopolysaccharides which are glycosaminoglycans (GAGs) made up of disaccharides of aminosugars and uronic sugars. Conclusion: GAGs can be used as metabolite marker for the detection of RCCs and this knowledge will lay the groundwork for the development of a non-invasive, in vivo magnetic resonance spectroscopy methodology for the differential diagnosis of RCCs and pituitary adenomas using clinical MRI scanners.

The diagnosis of various histological subtypes of pituitary tumors is made using serum based hormone panel test. However, certain subtypes secrete more than one hormone, making the diagnosis ambiguous. Here, we performed ¹H-NMR based metabolomic analysis of serum and whole-blood from luteinizing/follicle-stimulating (LH/FSH)-secreting (n = 24), prolactinomas (n = 14), and non-functional (NF) (n = 9) tumors. We found elevated levels of betahydroxybutyrate (BHB) in serum and whole-blood (WB) of prolactinomas (0.481 ± 0.211/0.329 ± 0.228 mM in serum/WB), but it was statistically significant (p ≤ 0.0033, Bonferroni correction) only in serum when compared with LH/FSH-secreting tumor patients (0.269 ± 0.139/0.167 ± 0.113 mM in serum/WB). Phenylalanine in NF tumors was found to be elevated in both serum and WB when compared with prolactinomas but it met the statistical significance criteria (p ≤ 0.0028) only in the serum. Alanine (p ≤ 0.011), tyrosine (p ≤ 0.014) and formate (p ≤ 0.011) were also elevated in NF tumors but none showed statistically significance when compared with prolactinomas. Quantification of BHB and the above amino acids in the circulation may aid in the development of blood-based in vitro diagnostic methods which can supplement the currently used serum hormone panel in the diagnosis of various subtypes of pituitary tumors.

Altered metabolism is considered one of the hallmarks of cancer. The findings that malignant brain tumors and brain metastases utilize acetate as an alternative nutrient are relatively recent and offer new avenues for investigation of altered metabolism in human cancers. Here, we describe comprehensively the details of the ¹³C NMR-based isotopomer methodology to measure in vivo acetate utilization in brain tumor patients, including the contribution from acetate metabolism of peripheral tissues. Methods described in this chapter can be readily extended to other cancer types.

Pituitary adenomas (PAs) are benign growths arising from epithelial cells in the adenohypophysis of the pituitary gland. To date, there has been no detailed metabolic characterization of PAs of various subtypes. In this study, we report nuclear magnetic resonance (NMR) based metabolomic analysis of surgically resected tumors from forty five pituitary tumor patients [gonadotropic (LH/FSH-secreting) = 17; prolactinomas (PRL-secreting) = 11, Cushing’s disease (ACTH-secreting) = 4, non-functional = 5, and mixed = 8] who underwent transsphenoidal selective adenomectomy. Compared to LH/FSH-secreting tumors, PRL-secreting tumors showed statistically significant decrease in the levels of N-acetylaspartate (NAA), myo-inositol (mI), scyllo-inositol (sI), glycine, taurine, phosphoethanolamine (PE) and increase in the levels of glutamine. When compared with LH/FSH-secreting tumors, ACTH-secreting tumors showed statistically significant decrease in the levels of sI, glycine, PE and increase in the levels of aspartate. Although lipid extracts of PAs showed the presence of many common lipid molecules, only glycerophosphoethanolamine (GPE) showed statistically significant decrease in PRL, ACTH and non-functional subtypes when compared to LH/FSH-secreting tumors. Changes observed in these metabolite concentrations among various subtypes of PAs reflect metabolic heterogeneity in these tumors and may pave the way towards the development of metabolic markers to distinguish various immunohistochemical subtypes of PAs.

Meningiomas are tumors arising from meninges, the membranes surrounding the brain and the spinal cord. Majority of meningiomas (especially the grade-1) are benign and grow slowly. However, atypical meningiomas (grade-2) exhibit increased cellular proliferation, and grow at much faster rate than benign meningiomas. Moreover, atypical meningiomas have higher probability to recur compared to benign meningiomas. The goal of this study is to identify potential metabolic markers that can be used to distinguish meningioma tumors of grade-1 and atypical grade-2. We have collected tumor tissues from fifteen meningioma patients (grade-1 = 10 and grade-2 = 5) during surgical removal of the tumor mass at the Houston Methodist Hospital. We have employed ex vivo 1H magnetic resonance spectroscopy of tumor tissue extracts to determine whether there are any metabolic differences between grade-1 and grade-2 meningiomas. We have detected the following aqueous metabolites in the tumors that we have studied: leucine, isoleucine, valine, lactate, alanine, acetate, glutamate, succinate, glutamine, aspartate, creatine. phosphocreatine, phosphocholine/glycerophosphocholine, glycine, phosphoethanolamine, myoinositol, and glucose. Quantitative analysis of these metabolites revealed for the first time that alanine and glutamine were highly elevated in grade-2 meningiomas compared to grade-1 tumors. The levels of alanine and glutamine in grade-1 are 2.93 ± 1.20 and 4.81 ± 2.37 µmol/g, while in grade-2 tumors, the levels are 4.95 ± 2.47 and 7.13 ± 4.23 µmol/g respectively. Elevated levels of alanine has been detected previously in grade -1 meningioma patients. However, there has been no study to date that compares various metabolite levels between grade-1 and grade-2 (atypical) meningiomas. Glutamine-derived glutamate is known as the key precursor in alanine biosynthesis. We examine using a larger patient cohort whether there is any correlation between the levels of alanine and glutamine in meningiomas of these two grades.

Recently, scalp application of alternating electric fields has shown therapeutic benefit in patients with glioblastoma multiforme (GBM). The U.S. Food and Drug Administration has approved this new “Tumor Treating Fields (TTF)” therapy provided by Novocure (trade name Optune) as monotherapy for recurrent GBM and in combination with temozolomide for newly diagnosed GBM. Two multicenter Phase III clinical trials demonstrate that in the former condition TTF treatment after standard of care chemotherapy only increases median survival from 6 to 6.6 months, and in the latter condition combination chemo-TTF therapy, compared to chemotherapy alone, prolongs progression free survival from 4 to 7.1 months. Although this treatment is highly promising, its underlying biophysical and molecular mechanisms are unclear. “Transcranial Rotating Permanent Magnet Stimulation” (TRPMS) is a similar approach in which non-invasive multifocal magnetic stimulation produces the required alternating electric fields by electromagnetic induction. Ongoing pilot TRPMS trials in chronic ischemic stroke and other neurological patients reveal a complete absence of significant TRPMS device-related adverse effects. Here, we report pre-clinical tests of the hypothesis that TRPMS-based alternating electric fields cause depolarization of the mitochondrial membrane potential (MMP) and alter mitochondrial oxidation of nutrients, leading to reactive oxygen species (ROS) and cytochrome C-mediated apoptosis in patient-derived GBM cells. Using 13C NMR spectroscopybased isotopomer analysis in GBM cells we observe that after 3 hours of treatment with TRPMS-induced electric field stimulation there is an increased metabolic flux of glycolysis and decreased mitochondrial glucose oxidation in the TCA cycle. Fluorescence microscopy with MitoTracker demonstrates changes in staining intensity that are consistent with TRPMS-induced disruption of mitochondrial networks due to depolarization of MMP, processes that culminate in apoptosis triggered by release of ROS and cytochrome C. These findings, if supported by in-vivo data in mouse models, would suggest an important role of TRPMS in GBM treatment.