Abstract BACKGROUND Diffuse Midline Glioma (DMG) is a highly aggressive (CNS WHO Grade IV) pediatric brain tumor characterized by the histone mutation H3K27M, resulting in global hypomethylation of ...histones. H3K27M mutant cells are highly dependent on methionine and methionine Adenosyltransferase 2A (MAT2A), a central regulator of the methionine cycle, representing a key vulnerability in H3K27M gliomas. MAT2A is upregulated by methyltransferase-like protein 16 (METTL16), which deposits N6-methyladenosine (m6A) on a subset of RNA residues to regulate gene expression. MAT2A is a sensor of methyl group donor S-adenosylmethionine (SAM) that, when placed, increases MAT2A intron splicing to compensate for a decrease in MAT2A function. METHODS To investigate our hypothesis, H3K27M histone mutation knockout cell lines were established using CRISPR Cas9 (Jabado lab) in DIPG4 cells. shRNA-mediated knockdown was used to attenuate METTL16 expression in DIPG4 H3K27M cells, and growth was assessed via sulforhodamine B (SRB) assay. RESULTS In silico analysis of normal brain tissue compared to low-grade glioma and high-grade glioma demonstrated increased METTL16 at both the RNA and protein levels (p=<0.0001). Therefore, we postulated that targeting METTL16 would affect H3K27M growth. Results showed increased cell death compared to control cells by day 4 (p=0.0034) and day 8 (p=<0.0001). Furthermore, knockdown of METTL16 in DMG cells with H3K27M decreased m6A levels on poly (A) RNA (p=0.049). CONCLUSIONS Our study highlights the vulnerability of H3K27M mutant cells to METTL16 modulation, elucidating underlying molecular mechanisms and paving the way for innovative therapeutic strategies targeting this axis.
A key aberrant biological difference between tumor cells and normal differentiated cells is altered metabolism, whereby cancer cells acquire a number of stable genetic and epigenetic alterations to ...retain proliferation, survive under unfavorable microenvironments and invade into surrounding tissues. A classic biochemical adaptation is the metabolic shift to aerobic glycolysis rather than mitochondrial oxidative phosphorylation, regardless of oxygen availability, a phenomenon termed the "Warburg Effect". Aerobic glycolysis, characterized by high glucose uptake, low oxygen consumption and elevated production of lactate, is associated with a survival advantage as well as the generation of substrates such as fatty acids, amino acids and nucleotides necessary in rapidly proliferating cells. This review discusses the role of key metabolic enzymes and their association with aerobic glycolysis in Glioblastoma Multiforme (GBM), an aggressive, highly glycolytic and deadly brain tumor. Targeting key metabolic enzymes involved in modulating the "Warburg Effect" may provide a novel therapeutic approach either singularly or in combination with existing therapies in GBMs.
Glioma stem-like cells (GSC) in glioblastoma (GBM) structure tumor cells into a hierarchical organization and are postulated to be recalcitrant to conventional treatments, resulting in fatal relapse ...of the disease. A better understanding of these cells would be essential for meaningful and lasting treatments. In this issue of
, Virolle and colleagues report a fascinating phenotype whereby the extracellular signal-regulated kinase (ERK) pathway regulates a mechanism of dedifferentiation of GBM cells into a stem-like state expressing markers of pluripotency through an EGFR-ERK-EGR1-dependent axis. This aptly termed "toggle switch" may contribute to maintenance of GSCs, promote intratumor heterogeneity, and potentially provide innovative treatment options.
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Abstract
Pediatric-type diffuse high grade gliomas are aggressive brain cancers in children which to date lack effective treatment options and remain largely understudied. Even though genomic markers ...including histone H3 K27M and G34R mutations have been identified, their functional implications on signaling networks remain to be described. Phosphoproteomics might be able to uncover activated signaling pathways which could open up new paths for treatment. Here we analyzed formalin-fixed paraffin-embedded (FFPE) sections from K27M mutated, G34R mutated and histone H3 wild type patient tumors (n = 14) using phosphoproteomics. Our workflow not only enables the comprehensive characterization of phosphoserines and threonines but also of phosphotyrosines which, despite being rare modifications (0.1-1% of all phosphorylations), are known to play an important role in cancer. We found that signaling networks were distinct between the different tumor subtypes. For phosphotyrosines, tumor subtype accounted for the largest source of variation in the data (44% of variance) as determined by principle component analysis. The separation by subtype could also be seen from serine and threonine phosphoproteomics and proteomics, but was not as striking as for the phosphotyrosine data. Differential abundance analysis, partial least squares discriminant analysis and self-organizing maps revealed deregulated signaling networks between subtypes. The most downregulated pathway in K27M compared to wild type tumors was enriched for proteins involved in the epigenetic regulation of gene expression (FDR<5%), which is in accordance with the remodeling of the epigenome taking place in K27M mutated tumors. In addition, we could identify signaling networks (e.g. EGFR signaling) which were activated in a few individual patients independent of their subtype and might be targetable. Our study provides insights into the signaling landscape of pediatric-type diffuse high grade gliomas. Recognizing deregulated signaling networks across subtypes and in individual patients could offer new avenues for personalized therapy.
Abstract
Diffuse midline gliomas (DMGs) and other high-grade gliomas (HGG) are the most commonly lethal intrinsic brain tumors of childhood. Accordingly, novel treatment approaches are needed that ...consider the heterogeneity of these tumors. Using an extensive pharmacological screen, we identified several two-drug combinations that were particularly effective against a range of DMG and HGG cell lines, the most active being the combination of the histone deacetylase inhibitor, panobinostat, and a proteasome inhibitor, marizomib, both in clinical trials, which synergistically induced tumor-specific apoptosis. However, at clinically achievable concentrations, a small population of resistant tumor cells survived and dominated, mimicking the clinical scenario, which prompted our development of drug resistance models as mechanistic tools. Resistance mechanisms were examined using RNA sequencing of drug-resistant versus -naïve cells. NMNAT2, a critical mediator of NAD synthesis, was overexpressed in DIPGs in the context of high baseline QPRT expression, suggesting a critical role of NAD synthesis and utilization pathways in resistance. Gene-set enrichment analysis suggested a key role for glycolytic pathways in the resistance phenotype. In support of this hypothesis, we observed increased levels of mitochondrial mass and NAD and ATP in resistant cells compared to drug-naïve cells, as well as upregulation of glycolytic intermediates by LC/MS. Conversely, inhibition of OXPHOS and glycolysis pathways resensitized resistant glioma cells to apoptosis in vitro. To test this hypothesis in vivo, mice were injected with drug-naïve or resistant DIPG-013 glioma cells and were exposed to an OXPHOS and glycolysis inhibitor, lonidamine. Lonidamine alone extended survival of naïve (18.6 + 1.4 d vs. 25 + 1.6 d, p=0.0003) and resistant (18.7 + 1 d vs. 62 + 1 d, p=0.0002) DIPG13p models (Log-Rank). This supports the application of metabolic therapy as a novel strategy for treating resistant MGs that has activity in a clinically relevant DIPG model. Supported by NIHS10OD023402, the DIPG/DMG Collaborative, the CNI, and Connor’s Cure.
Abstract
Medulloblastoma (MB), the most common type of embryonal tumor in children, is a fast-growing and heterogeneous brain tumor arising in the cerebellum. The molecular characterization of MB ...reveals four major subgroups (WNT, SHH, Group 3, and Group 4) and Group 3 is the most aggressive and malignant, characterized by frequent metastasis at diagnosis and the worst prognosis. We have recently found that BAF60C/SMARCD3 is highly expressed in Group 3 MB and associated with poor prognosis. Mechanistically, BAF60C/SMARCD3 hijacks a neurodevelopmental epigenetic program mediated by Disabled1 (DAB1)-Reelin signaling to promote metastatic dissemination in MB. Using single-cell RNA sequencing data of developing mouse and human cerebellum, we found that BAF60C/SMARCD3 expression in Purkinje cells (PCs) is upregulated during embryonic and early postnatal stages, but downregulated during the late postnatal stage of cerebellar development. To examine the effect of BAF60C/SMARCD3 depletion on PC migration and positioning during embryonic and postnatal stages of cerebellar development, we generated targeted knockout mice by crossing the PC-specific Cre mouse (L7Cre-2 and FoxP2-Cre) with the Smarcd3flox/flox mouse. We found that BAF60C/SMARCD3 depletion significantly influences embryo development in FoxP2-Cre; Smarcd3flox/flox but not in L7Cre-2; Smarcd3flox/flox mice, further supporting the expression of BAF60C/SMARCD3 in early stage (Foxp2 +) but not in the late stage (L7/Pcp2+) during cerebellum development. To determine whether BAF60C/SMARCD3 contributes to MB development, we injected lentivirus-carrying constitutively active MYCS62D with/without BAF60C/SMARCD3 in C57BL/6J mice. Interestingly, we found that MYCS62D with BAF60C/SMARCD3 can induce a spontaneous Group3-like MB with strong metastatic dissemination compared to MYCS62D without BAF60C/SMARCD3. These results demonstrate that BAF60C/SMARCD3 plays a pivotal role in PC biology and cerebellar development, and tumor cells hijack BAF60C/SMARCD3-Reelin signaling to promote tumor metastasis in MB.
PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely unknown. Here we show that PARK2 is altered ...in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitric oxide synthase (eNOS), enhanced levels of reactive oxygen species, and a concomitant increase in oxidized nitric oxide levels, thereby promoting the inhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylation in supporting cell survival and proliferation under conditions of energy deprivation.
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•PARK2 negatively regulates the PI3K/Akt pathway•PARK2 depletion promotes PTEN inactivation by S-nitrosylation and ubiquitination•AMPK activation triggers PTEN S-nitrosylation in the absence of PARK2 depletion•PARK2 loss and PTEN loss display striking cooperativity to promote tumorigenesis in vivo
Gupta et al. reveal an important missing piece in the dynamic signaling and metabolic network governing PI3K/Akt activation. PARK2 inactivation connects energy and oxidative stress to Akt activation via redox-mediated inactivation of PTEN by S-nitrosylation to support cell survival under conditions of energy deprivation.
Dysregulated growth factor receptor pathways, RNA modifications, and metabolism each promote tumor heterogeneity. Here, we demonstrate that platelet-derived growth factor (PDGF) signaling induces N
...-methyladenosine (m
A) accumulation in glioblastoma (GBM) stem cells (GSCs) to regulate mitophagy. PDGF ligands stimulate early growth response 1 (EGR1) transcription to induce methyltransferase-like 3 (METTL3) to promote GSC proliferation and self-renewal. Targeting the PDGF-METTL3 axis inhibits mitophagy by regulating m
A modification of optineurin (OPTN). Forced OPTN expression phenocopies PDGF inhibition, and OPTN levels portend longer survival of GBM patients; these results suggest a tumor-suppressive role for OPTN. Pharmacologic targeting of METTL3 augments anti-tumor efficacy of PDGF receptor (PDGFR) and mitophagy inhibitors in vitro and in vivo. Collectively, we define PDGF signaling as an upstream regulator of oncogenic m
A regulation, driving tumor metabolism to promote cancer stem cell maintenance, highlighting PDGF-METTL3-OPTN signaling as a GBM therapeutic target.
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