Abstract Dexamethasone (DEX) is routinely prescribed in brain tumor patients to limit vasogenic edema but may also exacerbate immunosuppression and adversely affect survival. The wide spectrum of dosing and individual variation in glucocorticoid (GC) response makes it difficult to assess the impact of DEX exposures. A potential marker of steroid pathway activation and GC load affecting the immune system are induced changes in chromatin structure marked by DNA methylation. We identified DEX-responsive DNA methylation sites in blood leukocytes from glioma patients treated with the drug at various doses and times during the course of their disease. Using weighted co-methylation network analysis, we show that DEX-induced hypomethylation includes well-known regulators of GC receptor (GR) sensitivity (e.g., FK506 binding protein 51: FKBP5) and inflammation (e.g., myeloperoxidase: MPO) and is enriched at genomic locations containing glucocorticoid receptor (GR) binding sites. Elastic net regression modeling was used to train a multilocus GC methylation index (GCMI) that discriminates current DEX users and non-users. GCMI scores showed wide interindividual variation among cases and DEX naïve control subjects. Using independent samples of DEX naïve and exposed glioma patients we show that the GCMI is a sensitive and specific indicator of DEX exposure. GCMI measured in non-glioma controls indicated sensitivity to non-DEX steroid treatments (e.g. prednisolone, fluticasone). Subjects with elevated neutrophil and decreased lymphocyte counts demonstrated high GCMI scores, reflecting the clinically relevant in vivo impact of this marker. Among 195 IDH wildtype and hTERT non-mutant glioma subjects, the GCMI was associated with a HR of 1.11 (95% CI 1.06–1.17) p< 0.0001 in Cox survival models that included age and tumor grade. We conclude that epigenetic remodeling in the peripheral immune compartment in response to DEX exposures is a rich source of potentially powerful markers of individual response to GC pathway activation and associated alterations in the immune response.

Abstract Lineage-specific DNA methylation marks differentiate leukocyte cell types while individual biological aging mechanisms impact other methylation alterations. Human glioma incidence and survival times have been shown to be associated with aberrant immune profiles and have a strong dependency on age. Here we developed a single epigenetic analysis framework to evaluate both immune cell fractions and epigenetic age in peripheral blood. We examined these measures in archived blood from 197 triple-negative glioma patients (TNG; IDH wildtype, 1p19q intact and TERT wildtype) and 312 frequency-matched controls from the SF Bay Area Adult Glioma Study (AGS). Significant differences were observed with TNG cases having lower CD4 and CD8 T cell, natural killer, and B cell fractions, and higher neutrophil fractions than controls. TNG cases were significantly older than controls in two of three epigenetic age estimates; however, there was no difference in epigenetic age acceleration once immune cell proportions were considered. For the TNG cases, we augmented results from several machine learning methods to delineate risk groups of TNG patients with significantly different overall survival. We compared survival models built by recursive partitioning, random forest, and elastic net methods. The final model was chosen by repeated bootstrap sampling via the Brier score loss function and validated in an independent set of 72 IDH-mutant only or TERT-mutant only glioma patients also from the AGS. The final model indicated important interactions between immune cell fractions (including CD4 and CD8 T cells and neutrophils) and treatment, age, and dexamethasone status when adjusted for the main effects of epigenetic age, glioblastoma status, and the neutrophil-to-lymphocyte ratio. The capacity of immunomethylomics to capture diverse, clinically relevant information and the simplicity of its implementation make this a powerful tool for personalized patient evaluation in the neuro-oncology clinic.

The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4, which is homozygously deleted in nearly one-third of cases. As loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 (ME2) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3. The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis. Here we show that ME3 depletion selectively kills ME2-null PDAC cells in a manner consistent with an essential function for ME3 in ME2-null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime 'collateral lethality' therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease.