Suresh Udutha

Background: The 1p/19q codeletion is a hallmark of oligodendrogliomas. The goal of this study was to exploit the metabolic vulnerabilities induced by the 1p/19q codeletion for the treatment and imaging of oligodendrogliomas. Methods: We used stable isotope tracing, mass spectrometry, and genetic and pharmacological approaches to interrogate [U-13C]-glucose metabolism in patient-derived oligodendroglioma models (SF10417, BT88, BT54, TS603, NCH612). We examined whether tracing [6,6′-2H]-glucose metabolism using deuterium metabolic imaging (DMI) provided an early readout of treatment response. Results: The glycolytic enzyme enolase 1 (ENO1; chromosome 1p36.23) was downregulated in patient-derived oligodendroglioma cells and patient tissue due to the 1p/19q codeletion and histone hypermethylation. Conversely, inactivation of the CIC transcriptional repressor, driven by activated mitogen-activated protein kinase (MAPK) signaling, upregulated the ENO2 isoform specifically in oligodendrogliomas. Genetic ablation of ENO2 or pharmacological inhibition using POMHEX inhibited proliferation with nanomolar potency but was not cytotoxic to oligodendroglioma cells. Mechanistically, ENO2 loss abrogated [U-13C]-glucose metabolism to lactate but shunted glucose toward biosynthesis of serine and purine nucleotides, an effect that was driven by the rate-limiting enzyme for serine synthesis, phosphoglycerate dehydrogenase (PHGDH). Importantly, combining the PHGDH inhibitor D8 with POMHEX resulted in synthetic lethality in vitro and induced tumor regression in vivo. Furthermore, DMI of lactate production from [6,6′-2H]-glucose provided an early readout of response to combination therapy that preceded MRI-detectable alterations and reflected extended survival. Conclusions: We have identified ENO2 and PHGDH as metabolic vulnerabilities induced by the 1p/19q codeletion in oligodendrogliomas and [6,6′-2H]-glucose as a noninvasive tracer of early response to therapy.

Hyperactivation of glucose metabolism to lactate is a metabolic hallmark of cancer. However, the functional role of lactate in pediatric diffuse midline glioma (DMG) cells is unclear. Here, using stable isotope tracing and loss-of-function studies in clinically relevant patient-derived DMG models, we show that the oncogenic histone H3K27M mutation epigenetically up-regulates the rate-limiting glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and drives lactate production from [U- ¹³ C]-glucose in DMGs. Mechanistically, lactate posttranslationally activates the nucleoside diphosphate kinase NME1 through lactylation and facilitates the synthesis of nucleoside triphosphates that are essential for DNA replication and tumor proliferation. This mechanistic link between glycolysis and nucleotide biosynthesis provides the opportunity for deuterium metabolic imaging of tumor growth and response to therapy. Spatially mapping ² H-lactate production from [6,6- ² H]-glucose allows visualization of the metabolically active tumor lesion and provides an early readout of response to standard of care and targeted therapy that precedes extended survival and reflects pharmacodynamic alterations in tumor tissues in preclinical DMG models in vivo at clinical field strength (3 T). Overall, we have identified an H3K27M-lactate-NME1 axis that drives DMG proliferation and facilitates noninvasive in vivo metabolic imaging of DMGs.

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.