Identification of dexamethasone-induced metabolic vulnerabilities in glioblastoma

Glioblastoma represents the most aggressive and common high-grade (IV) malignant brain tumour in adults. Despite many efforts have been done in order to ameliorate the clinical outcome of this disease, the standard of care has not substantially changed in the last 20 years, and is based on surgical resection followed by radiotherapy and chemotherapy with the alkylating agent temozolomide. Nonetheless, the aggressiveness and recurrence of this tumour make the prognosis still very poor and the median survival for glioblastoma patients is 12-18 months from the time of diagnosis. Since 1960’s, patients presenting with clinical symptoms associated with glioblastoma are invariably treated with dexamethasone, a potent anti-inflammatory drug that reduces the peritumoral oedema, and alleviates the neurological symptoms caused by the increased intracranial pressure. Given the clinical relevance of dexamethasone for the management of glioblastoma patients, several studies have been performed to understand the effects of dexamethasone on patients’ survival. A retrospective clinical analysis of three independent glioma patient cohorts found that steroids treatment associates with shorter survival. As indicated by the name, glucocorticoids regulate glucose metabolism. Dexamethasone increases glycaemia, and this has been linked to a shorter survival of glioblastoma patients. Nevertheless, because of its effectiveness, affordability and accessibility, dexamethasone will remain a mainstay of glioblastoma patients’ therapy. On these bases, we employed naïve patient-derived cell lines that have been demonstrated to retain the pathophysiological features of the tumour in the patients to identify the metabolic effects of dexamethasone on glioblastoma cells and characterize specific drug-induced vulnerabilities. In particular, we studied the metabolic reactions altered by dexamethasone treatment with the aim to exploit them as therapeutic targets. Therefore, we cultured the cells in serumfree physiological media (Plasmax) to retain the stem cells subpopulation and expose them to the same metabolites concentrations found in human plasma. We found that the glucocorticoid receptor, the dexamethasone main target, was expressed in all cell lines, and translocated to the nucleus upon dexamethasone treatment. While the effects of dexamethasone on proliferation were cell linedependent, we profiled a dexamethasone-dependent transcriptional signature common to all cell lines. Orthogonal transcriptomic and metabolomics analyses identified nicotinamide N-methyltransferase, NNMT, to be transcriptionally and functionally upregulated by dexamethasone. NNMT transfers methyl groups from S-adenosyl-methionine (SAM) to nicotinamide, producing N1-methylnicotinamide. Dexamethasone-mediated NNMT over-activation caused a shortage in SAM and an increase in N1-methylnicotinamide levels in all cell lines. Unexpectedly, dexamethasone treatment did not sensitize glioblastoma cells to sub-physiological and growth-limiting concentrations of methionine. We validated these findings in 10 naïve human glioblastoma cell lines and in glioblastoma tumours orthotopically implanted in immunocompromised mice. In vivo, dexamethasone decreased the methionine levels in tumour and contralateral brain tissue without altering its circulating levels. Dexamethasone administration significantly decreased tumour volume assessed by MRI and proliferation index. Notably, the levels of NNMT and N1-methylnicotinamide were markedly higher in tumour tissue compared to contralateral normal brain, and these differences were amplified by dexamethasone treatment. Moreover, we demonstrated that the activity of NNMT is increased in tumour tissues derived from glioblastoma patients, compared to adjacent oedematous brain tissue. These results suggest that NNMT activity could be targeted for the development of a novel PET tracer for the visualization of invasive tumours, aiding the diagnosis and the response to therapy of glioblastoma patients. Moreover, these tumour-specific dexamethasone-induced metabolic alterations may lead to a rationally designed therapeutic plan for glioblastomas with heterogeneous mutational status