Abstract
Aurora kinase A (AURKA) has emerged as a drug target for glioblastoma (GBM). However, resistance to therapy remains a critical issue. By integration of transcriptome, chromatin ...immunoprecipitation sequencing (CHIP-seq), Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), proteomic and metabolite screening followed by carbon tracing and extracellular flux analyses we show that genetic and pharmacological AURKA inhibition elicits metabolic reprogramming mediated by inhibition of MYC targets and concomitant activation of Peroxisome Proliferator Activated Receptor Alpha (PPARA) signaling. While glycolysis is suppressed by AURKA inhibition, we note an increase in the oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO), which was accompanied by an increase of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Combining AURKA inhibitors with inhibitors of FAO extends overall survival in orthotopic GBM PDX models. Taken together, these data suggest that simultaneous targeting of oxidative metabolism and AURKAi might be a potential novel therapy against recalcitrant malignancies.
Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via ...the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.
INTRODUCTION Pineal tumors are rare entities, with pathology ranging from benign to highly malignant. The most common tumors in this region are germinomas, which are highly radiosensitive. However, ...other malignant tumors of this region, such glioblastoma, pineoblastomas and sarcomas are resistant to current therapies with a poor prognosis. Therapeutic advancements have been limited by the absence of tumor models and lack of readily available tissue specimen. METHODS C57BL/6 mice with Trp53f/f; PTEN f/f floxed sites, and a stop-floxed YFP were used to harvest pineal glands by microdissection that were then dissociated and plated in SATO media. A PDGF-IRES-CRE virus was added to cells for Cre mediated deletion of p53 and PTEN, with expression of PDGF-B and YFP. Immunofluorescence and Western blotting were used to determine lineage expression patterns and to confirm expression of YFP, indicating viral infection with transformation. PCR was also used to confirm transgenic changes. Cell viability assays were performed using Cell Titer Glo bioluminescence. Orthotopic stereotactic injections were used for in vivo assays with serial MRIs to track tumor growth. RESULTS Immunofluorescence of non-infected gland cells demonstrated a mixed population of cells including synaptophysin positive, indicating pineal parenchymal cells. 3 independent highly proliferative cell lines were generated from separate harvests, shown to be YFP positive and PCR demonstrated p53/PTEN loss confirming viral transformation. Western blots were negative for synaptophysin. Treatment with the pan-HDAC inhibitor, panobinostat, resulted in decreased cell viability indicating sensitivity to this chemotherapeutic agent. Orthotopic injections formed tumors in the pineal region that demonstrated heterogeneous staining with areas of GFAP, Nestin positivity and retention of YFP expression. CONCLUSION Ex vivo transformation of transgenic mouse pineal glands is a robust method for the generation of malignant pineal tumors, with an in vivo tumor formation pattern similar to that of human tumors, and responsiveness to HDAC inhibitors. Further studies are warranted to characterize these tumors.
Topotecan is cytotoxic to glioma cells but is clinically ineffective because of drug delivery limitations. Systemic delivery is limited by toxicity and insufficient brain penetrance, and, to date, ...convection-enhanced delivery (CED) has been restricted to a single treatment of restricted duration. To address this problem, we engineered a subcutaneously implanted catheter-pump system capable of repeated, chronic (prolonged, pulsatile) CED of topotecan into the brain and tested its safety and biological effects in patients with recurrent glioblastoma.
We did a single-centre, open-label, single-arm, phase 1b clinical trial at Columbia University Irving Medical Center (New York, NY, USA). Eligible patients were at least 18 years of age with solitary, histologically confirmed recurrent glioblastoma showing radiographic progression after surgery, radiotherapy, and chemotherapy, and a Karnofsky Performance Status of at least 70. Five patients had catheters stereotactically implanted into the glioma-infiltrated peritumoural brain and connected to subcutaneously implanted pumps that infused 146 μM topotecan 200 μL/h for 48 h, followed by a 5–7-day washout period before the next infusion, with four total infusions. After the fourth infusion, the pump was removed and the tumour was resected. The primary endpoint of the study was safety of the treatment regimen as defined by presence of serious adverse events. Analyses were done in all treated patients. The trial is closed, and is registered with ClinicalTrials.gov, NCT03154996.
Between Jan 22, 2018, and July 8, 2019, chronic CED of topotecan was successfully completed safely in all five patients, and was well tolerated without substantial complications. The only grade 3 adverse event related to treatment was intraoperative supplemental motor area syndrome (one 20% of five patients in the treatment group), and there were no grade 4 adverse events. Other serious adverse events were related to surgical resection and not the study treatment. Median follow-up was 12 months (IQR 10–17) from pump explant. Post-treatment tissue analysis showed that topotecan significantly reduced proliferating tumour cells in all five patients.
In this small patient cohort, we showed that chronic CED of topotecan is a potentially safe and active therapy for recurrent glioblastoma. Our analysis provided a unique tissue-based assessment of treatment response without the need for large patient numbers. This novel delivery of topotecan overcomes limitations in delivery and treatment response assessment for patients with glioblastoma and could be applicable for other anti-glioma drugs or other CNS diseases. Further studies are warranted to determine the effect of this drug delivery approach on clinical outcomes.
US National Institutes of Health, The William Rhodes and Louise Tilzer Rhodes Center for Glioblastoma, the Michael Weiner Glioblastoma Research Into Treatment Fund, the Gary and Yael Fegel Foundation, and The Khatib Foundation.
Glioblastoma (GBM) diffusely infiltrates the brain and intermingles with non-neoplastic brain cells, including astrocytes, neurons and microglia/myeloid cells. This complex mixture of cell types ...forms the biological context for therapeutic response and tumor recurrence. We used single-nucleus RNA sequencing and spatial transcriptomics to determine the cellular composition and transcriptional states in primary and recurrent glioma and identified three compositional 'tissue-states' defined by cohabitation patterns between specific subpopulations of neoplastic and non-neoplastic brain cells. These tissue-states correlated with radiographic, histopathologic, and prognostic features and were enriched in distinct metabolic pathways. Fatty acid biosynthesis was enriched in the tissue-state defined by the cohabitation of astrocyte-like/mesenchymal glioma cells, reactive astrocytes, and macrophages, and was associated with recurrent GBM and shorter survival. Treating acute slices of GBM with a fatty acid synthesis inhibitor depleted the transcriptional signature of this pernicious tissue-state. These findings point to therapies that target interdependencies in the GBM microenvironment.
INTRODUCTION Ferroptosis is an iron dependent form of regulated cell death mediated by lipid peroxides, which has proven to be a promising avenue for cancer treatment. METHODS In vitro assays were ...performed using glioma cell lines previously established by our laboratory. Cell viability was assessed using bioluminescence. Flow cytometry was used to determine changes in lipid peroxidation (BODIPY-C11) and reactive oxygen species (ROS) (H2DCFDA) in normal and CMD conditions. In vivo studies were performed using stereotactic orthotopic injections in syngeneic mice, fed control or CMD diet. Subsets were also treated with radiation to assess synergy by measuring tumor burden via bioluminescence and survival. RESULTS Both murine and human glioma cell lines are sensitive to ferroptosis induction. CMD sensitized tumor cell lines to ferroptosis by RSL-3, and IKE, and reduced glutathione levels. Short-term CMD dramatically increased lipid peroxidation and ROS in response to ferroptosis inducers. CMD treatment also increased stereotypic transcriptional markers of ferroptosis in glioma cell lines. Ex-vivo organotypic slices taken from primary human glioma surgical specimen showed similar findings. Co-treatment with CMD and radiation showed synergistic cell death in glioma cell lines. This was accompanied by a synergistic increase in lipid peroxidation in the co-treatment group. In vivo, the CMD diet was well tolerated. Co-treatment with radiation significantly reduced tumor size and prolonged survival (P = .0093). CONCLUSION These data show that CMD synergizes with radiation treatment, and may be a promising therapy to improve the efficacy of radiation treatment in glioma.
The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is uncertain but mitochondrial dysfunction may play an important role. Ketones promote mitochondrial energy production and membrane ...stabilization.
SOD1-G93A transgenic ALS mice were fed a ketogenic diet (KD) based on known formulations for humans. Motor performance, longevity, and motor neuron counts were measured in treated and disease controls. Because mitochondrial dysfunction plays a central role in neuronal cell death in ALS, we also studied the effect that the principal ketone body, D-beta-3 hydroxybutyrate (DBH), has on mitochondrial ATP generation and neuroprotection. Blood ketones were > 3.5 times higher in KD fed animals compared to controls. KD fed mice lost 50% of baseline motor performance 25 days later than disease controls. KD animals weighed 4.6 g more than disease control animals at study endpoint; the interaction between diet and change in weight was significant (p = 0.047). In spinal cord sections obtained at the study endpoint, there were more motor neurons in KD fed animals (p = 0.030). DBH prevented rotenone mediated inhibition of mitochondrial complex I but not malonate inhibition of complex II. Rotenone neurotoxicity in SMI-32 immunopositive motor neurons was also inhibited by DBH.
This is the first study showing that diet, specifically a KD, alters the progression of the clinical and biological manifestations of the G93A SOD1 transgenic mouse model of ALS. These effects may be due to the ability of ketone bodies to promote ATP synthesis and bypass inhibition of complex I in the mitochondrial respiratory chain.
Isocitrate Dehydrogenase-1 (IDH1) is commonly mutated in lower-grade diffuse gliomas. The IDH1R132H mutation is an important diagnostic tool for tumor diagnosis and prognosis; however, its role in ...glioma development, and its impact on response to therapy, is not fully understood. We developed a murine model of proneural IDH1R132H-mutated glioma that shows elevated production of 2-hydroxyglutarate (2-HG) and increased trimethylation of lysine residue K27 on histone H3 (H3K27me3) compared to IDH1 wild-type tumors. We found that using Tazemetostat to inhibit the methyltransferase for H3K27, Enhancer of Zeste 2 (EZH2), reduced H3K27me3 levels and increased acetylation on H3K27. We also found that, although the histone deacetylase inhibitor (HDACi) Panobinostat was less cytotoxic in IDH1R132H-mutated cells (either isolated from murine glioma or oligodendrocyte progenitor cells infected in vitro with a retrovirus expressing IDH1R132H) compared to IDH1-wild-type cells, combination treatment with Tazemetostat is synergistic in both mutant and wild-type models. These findings indicate a novel therapeutic strategy for IDH1-mutated gliomas that targets the specific epigenetic alteration in these tumors.
The Warburg effect is a tumor-related phenomenon that could potentially be targeted therapeutically. Here, we showed that glioblastoma (GBM) cultures and patients' tumors harbored super-enhancers in ...several genes related to the Warburg effect. By conducting a transcriptome analysis followed by ChIP-Seq coupled with a comprehensive metabolite analysis in GBM models, we found that FDA-approved global (panobinostat, vorinostat) and selective (romidepsin) histone deacetylase (HDAC) inhibitors elicited metabolic reprogramming in concert with disruption of several Warburg effect-related super-enhancers. Extracellular flux and carbon-tracing analyses revealed that HDAC inhibitors blunted glycolysis in a c-Myc-dependent manner and lowered ATP levels. This resulted in the engagement of oxidative phosphorylation (OXPHOS) driven by elevated fatty acid oxidation (FAO), rendering GBM cells dependent on these pathways. Mechanistically, interference with HDAC1/-2 elicited a suppression of c-Myc protein levels and a concomitant increase in 2 transcriptional drivers of oxidative metabolism, PGC1α and PPARD, suggesting an inverse relationship. Rescue and ChIP experiments indicated that c-Myc bound to the promoter regions of PGC1α and PPARD to counteract their upregulation driven by HDAC1/-2 inhibition. Finally, we demonstrated that combination treatment with HDAC and FAO inhibitors extended animal survival in patient-derived xenograft model systems in vivo more potently than single treatments in the absence of toxicity.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive impairments in memory and cognition. Extracellular accumulation of soluble high-molecular-weight (HMW) A beta ...oligomers has been proposed to be largely responsible for AD dementia and memory deficits in the Tg2576 mice, a model of AD. In this study, we found that a naturally derived grape seed polyphenolic extract can significantly inhibit amyloid beta -protein aggregation into high-molecular-weight oligomers in vitro. When orally administered to Tg2576 mice, this polyphenolic preparation significantly attenuates AD-type cognitive deterioration coincidentally with reduced HMW soluble oligomeric A beta in the brain. Our study suggests that grape seed-derived polyphenolics may be useful agents to prevent or treat AD.
PDF
