Outcome of glioblastoma patients after intensive care unit admission with invasive mechanical ventilation: a multicenter analysis.

PURPOSE: Patients with glioblastoma are exposed to severe symptoms and organs failures (e.g., coma or acute respiratory failure), that may require intensive care unit (ICU) admission and invasive mechanical ventilation (IMV). However, only limited data are available concerning the prognosis of patients with glioblastoma receiving IMV. We sought to describe the reasons for ICU admission, and outcomes of patients with glioblastoma requiring IMV for unplanned critical complications. METHODS: In this retrospective analysis, four certified interdisciplinary brain tumor centers performed a retrospective review of their electronic data systems. All patients with glioblastoma admitted to an in-house ICU and receiving IMV between January 2015 and December 2019 were included. Clinical and prognostic factors as well as relevant outcome parameters were evaluated by group comparisons and Kaplan Meier survival curves. RESULTS: We identified 33 glioblastoma patients with a duration of IMV of 9.2 ± 9.4 days. Main reasons for ICU admission were infection (n = 12; 34.3%) including 3 cases of Pneumocystis jirovecii pneumonia, status epilepticus (31.4%) and elevated intracranial pressure (22.9%). In-hospital mortality reached 60.6%. Younger age, low number of IMV days, better Karnofsky Performance Status Scale before admission and elevated intracranial pressure as cause of ICU admission were associated with positive prognostic outcome. CONCLUSION: We conclude that less than 50% of patients with glioblastoma have a favorable short-term outcome when unplanned ICU treatment with IMV is required. Our data mandate a careful therapy guidance and frequent reassessment of goals during ICU stay.

Molecular matched targeted therapies for primary brain tumors-a single center retrospective analysis.

PURPOSE: Molecular diagnostics including next generation gene sequencing are increasingly used to determine options for individualized therapies in brain tumor patients. We aimed to evaluate the decision-making process of molecular targeted therapies and analyze data on tolerability as well as signals for efficacy. METHODS: Via retrospective analysis, we identified primary brain tumor patients who were treated off-label with a targeted therapy at the University Hospital Frankfurt, Goethe University. We analyzed which types of molecular alterations were utilized to guide molecular off-label therapies and the diagnostic procedures for their assessment during the period from 2008 to 2021. Data on tolerability and outcomes were collected. RESULTS: 413 off-label therapies were identified with an increasing annual number for the interval after 2016. 37 interventions (9%) were targeted therapies based on molecular markers. Glioma and meningioma were the most frequent entities treated with molecular matched targeted therapies. Rare entities comprised e.g. medulloblastoma and papillary craniopharyngeoma. Molecular targeted approaches included checkpoint inhibitors, inhibitors of mTOR, FGFR, ALK, MET, ROS1, PIK3CA, CDK4/6, BRAF/MEK and PARP. Responses in the first follow-up MRI were partial response (13.5%), stable disease (29.7%) and progressive disease (46.0%). There were no new safety signals. Adverse events with fatal outcome (CTCAE grade 5) were not observed. Only, two patients discontinued treatment due to side effects. Median progression-free and overall survival were 9.1/18 months in patients with at least stable disease, and 1.8/3.6 months in those with progressive disease at the first follow-up MRI. CONCLUSION: A broad range of actionable alterations was targeted with available molecular therapeutics. However, efficacy was largely observed in entities with paradigmatic oncogenic drivers, in particular with BRAF mutations. Further research on biomarker-informed molecular matched therapies is urgently necessary.

Disruption of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α reverts key features of the neoplastic phenotype of glioma cells.

The peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a master regulator of mitochondrial biogenesis and controls metabolism by coordinating transcriptional events. Here, we interrogated whether PGC-1α is involved in tumor growth and the metabolic flexibility of glioblastoma cells. PGC-1α was expressed in a subset of established glioma cell lines and primary glioblastoma cell cultures. Furthermore, a higher PGC-1α expression was associated with an adverse outcome in the TCGA glioblastoma dataset. Suppression of PGC-1α expression by shRNA in the PGC-1α-positive U343MG glioblastoma line suppressed mitochondrial gene expression, reduced mitochondrial membrane potential, and diminished oxygen as well as glucose consumption, and lactate production. Compatible with the known PGC-1α functions in reactive oxygen species (ROS) metabolism, glioblastoma cells deficient in PGC-1α displayed ROS accumulation, had reduced RNA levels of proteins involved in ROS detoxification, and were more susceptible to death induction by H2O2 compared with control cells. PGC-1αsh cells also had impaired proliferation and migration rates in vitro and displayed less stem cell characteristics. Complementary effects were observed in PGC-1α-low LNT-229 cells engineered to overexpress PGC-1α. In an in vivo xenograft experiment, tumors formed by U343MG PGC-1αsh glioblastoma cells grew much slower than control tumors and were less invasive. Interestingly, the PGC-1α knockdown conferred protection against hypoxia-induced cell death, probably as a result of less active anabolic pathways, and this effect was associated with reduced epidermal growth factor expression and mammalian target of rapamycin signaling. In summary, PGC-1α modifies the neoplastic phenotype of glioblastoma cells toward more aggressive behavior and therefore makes PGC-1α a potential target for anti-glioblastoma therapies.

Serine-dependent redox homeostasis regulates glioblastoma cell survival.

BACKGROUND: The amino acid serine is an important substrate for biosynthesis and redox homeostasis. We investigated whether glioblastoma (GBM) cells are dependent on serine for survival under conditions of the tumour microenvironment. METHODS: Serine availability in GBM cells was modulated pharmacologically, genetically and by adjusting serine and glycine concentrations in the culture medium. Cells were investigated for regulation of serine metabolism, proliferation, sensitivity to hypoxia-induced cell death and redox homeostasis. RESULTS: Hypoxia-induced expression of phosphoglycerate dehydrogenase (PHGDH) and the mitochondrial serine hydroxymethyltransferase (SHMT2) was observed in three of five tested glioma cell lines. Nuclear factor erythroid 2-related factor (Nrf) 2 activation also induced PHGDH and SHMT2 expression in GBM cells. Low levels of endogenous PHGDH as well as PHGDH gene suppression resulted in serine dependency for cell growth. Pharmacological inhibition of PHGDH with CBR-5884 reduced proliferation and sensitised cells profoundly to hypoxia-induced cell death. This effect was accompanied by an increase in reactive oxygen species and a decrease in the NADPH/NADP+ ratio. Similarly, hypoxia-induced cell death was enhanced by PHGDH gene suppression and reduced by PHGDH overexpression. CONCLUSIONS: Serine facilitates adaptation of GBM cells to conditions of the tumour microenvironment and its metabolism could be a plausible therapeutic target.

The physiological mTOR complex 1 inhibitor DDIT4 mediates therapy resistance in glioblastoma.

BACKGROUND: Despite significant advances in the understanding of glioblastoma genetics and biology, survival is still poor. Hypoxia and nutrient depletion in the tumour microenvironment induce adaptive signalling and metabolic responses, which can influence sensitivity to therapeutic regimens. DNA damage-inducible transcript 4 (DDIT4) is a protein induced by hypoxia and in response to DNA stress. Mechanistically, DDIT4 inhibits mammalian target of rapamycin complex 1 (mTORC1) signalling by activation of the tuberous sclerosis 1/2 (TSC1/2) complex. METHODS: Using short hairpin RNA-mediated gene suppression as well as doxycycline-regulated gene induction, we developed a glioblastoma cell model to study effects of DDIT4 under conditions of the glioblastoma microenvironment and therapy. RESULTS: We found an intact DDIT4-mTORC1 signalling axis in human glioblastoma cells that was inducible by hypoxia. Temozolomide and radiotherapy also induced DDIT4 and repressed mTORC1 activity in some glioblastoma cell lines. DDIT4 gene suppression sensitised glioma cells towards hypoxia-induced cell death, while DDIT4 overexpression protected them. Additionally, in clonogenic survival analyses, DDIT4 induction conferred protection from radiotherapy and temozolomide, while DDIT4 gene suppression sensitised cells. CONCLUSIONS: We identified DDIT4 as a cell-intrinsic regulator for adaptive responses and therapy resistance in glioblastoma cells which may interfere with cell death induction by temozolomide, radiotherapy or hypoxia by inhibiting mTORC1 activity.

Second Generation mTOR Inhibitors as a Double-Edged Sword in Malignant Glioma Treatment.

Glioblastomas (GBs) frequently display activation of the epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR). mTOR exists as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). In GBs, mTORC1 inhibitors such as rapamycin have performed poorly in clinical trials, and in vitro protect GB cells from nutrient and oxygen deprivation. Next generation ATP-competitive mTOR inhibitors with affinity for both mTOR complexes have been developed, but data exploring their effects on GB metabolism are scarce. In this study, we compared the ATP-competitive mTORC1/2 inhibitors torin2, INK-128 and NVP-Bez235 to the allosteric mTORC1 inhibitor rapamycin under conditions that mimic the glioma microenvironment. In addition to inhibiting mTORC2 signaling, INK-128 and NVP-Bez235 more effectively blocked mTORC1 signaling and prompted a stronger cell growth inhibition, partly by inducing cell cycle arrest. However, under hypoxic and nutrient-poor conditions mTORC1/2 inhibitors displayed even stronger cytoprotective effects than rapamycin by reducing oxygen and glucose consumption. Thus, therapies that arrest proliferation and inhibit anabolic metabolism must be expected to improve energy homeostasis of tumor cells. These results mandate caution when treating physiologically or therapeutically induced hypoxic GBs with mTOR inhibitors.

Rescue of 2-Deoxyglucose Side Effects by Ketogenic Diet.

Cancer metabolism is characterized by extensive glucose consumption through aerobic glycolysis. No effective therapy exploiting this cancer trait has emerged so far, in part, due to the substantial side effects of the investigated drugs. In this study, we examined the side effects of a combination of isocaloric ketogenic diet (KD) with the glycolysis inhibitor 2-deoxyglucose (2-DG). Two groups of eight athymic nude mice were either fed a standard diet (SD) or a caloric unrestricted KD with a ratio of 4 g fat to 1 g protein/carbohydrate. 2-DG was investigated in commonly employed doses of 0.5 to 4 g/kg and up to 8 g/kg. Ketosis was achieved under KD (ketone bodies: SD 0.5 ± 0.14 mmol/L, KD 1.38 ± 0.28 mmol/L, p < 0.01). The intraperitoneal application of 4 g/kg of 2-DG caused a significant increase in blood glucose, which was not prevented by KD. Sedation after the 2-DG treatment was observed and a behavioral test of spontaneous motion showed that KD reduced the sedation by 2-DG (p < 0.001). A 2-DG dose escalation to 8 g/kg was lethal for 50% of the mice in the SD and for 0% of the mice in the KD group (p < 0.01). A long-term combination of KD and an oral 1 or 2 g 2-DG/kg was well-tolerated. In conclusion, KD reduces the sedative effects of 2-DG and dramatically increases the maximum tolerated dose of 2-DG. A continued combination of KD and anti-glycolytic therapy is feasible. This is, to our knowledge, the first demonstration of increased tolerance to glycolysis inhibition by KD.

Gene Suppression of Transketolase-Like Protein 1 (TKTL1) Sensitizes Glioma Cells to Hypoxia and Ionizing Radiation.

In several tumor entities, transketolase-like protein 1 (TKTL1) has been suggested to promote the nonoxidative part of the pentose phosphate pathway (PPP) and thereby to contribute to a malignant phenotype. However, its role in glioma biology has only been sparsely documented. In the present in vitro study using LNT-229 glioma cells, we analyzed the impact of TKTL1 gene suppression on basic metabolic parameters and on survival following oxygen restriction and ionizing radiation. TKTL1 was induced by hypoxia and by hypoxia-inducible factor-1α (HIF-1α). Knockdown of TKTL1 via shRNA increased the cells' demand for glucose, decreased flux through the PPP and promoted cell death under hypoxic conditions. Following irradiation, suppression of TKTL1 expression resulted in elevated levels of reactive oxygen species (ROS) and reduced clonogenic survival. In summary, our results indicate a role of TKTL1 in the adaptation of tumor cells to oxygen deprivation and in the acquisition of radioresistance. Further studies are necessary to examine whether strategies that antagonize TKTL1 function will be able to restore the sensitivity of glioma cells towards irradiation and antiangiogenic therapies in the more complex in vivo environment.

Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems.

Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01⁻1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems.

Mammalian target of rapamycin inhibition protects glioma cells from temozolomide-induced cell death.

Glioblastoma is an incurable brain tumor with a median survival below two years. Trials investigating targeted therapy with inhibitors of the kinase mTOR have produced ambiguous results. Especially combination of mTOR inhibition with standard temozolomide radiochemotherapy has resulted in reduced survival in a phase II clinical trial. To date, this phenomenon is only poorly understood. To recreate the therapeutic setting in vitro, we exposed glioblastoma cell lines to co-treatment with rapamycin and temozolomide and assessed cell viability, DNA damage and reactive oxygen species. Additionally, we employed a novel translatomic based mass spectrometry approach ("mePROD") to analyze acute changes in translated proteins. mTOR inhibition with rapamycin protected glioblastoma cells from temozolomide toxicity. Following co-treatment of temozolomide with rapamycin, an increased translation of reactive oxygen species (ROS)-detoxifying proteins was detected by mass spectrometry. This was accompanied by improved ROS-homeostasis and reduced DNA damage. Additionally, rapamycin induced the expression of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma cells with an unmethylated MGMT gene promotor. Inhibition of mTOR antagonized the cytotoxic effects of temozolomide in vitro. The induction of antioxidant defences and MGMT are two underlying candidate mechanisms. Further functional experiments in vitro and in vivo are warranted to characterize this effect that appears relevant for combinatorial therapeutic strategies.

Activating transcription factor 4 mediates adaptation of human glioblastoma cells to hypoxia and temozolomide.

The integrated stress response (ISR) is a central cellular adaptive program that is activated by diverse stressors including ER stress, hypoxia and nutrient deprivation to orchestrate responses via activating transcription factor 4 (ATF4). We hypothesized that ATF4 is essential for the adaptation of human glioblastoma (GB) cells to the conditions of the tumor microenvironment and is contributing to therapy resistance against chemotherapy. ATF4 induction in GB cells was modulated pharmacologically and genetically and investigated in the context of temozolomide treatment as well as glucose and oxygen deprivation. The relevance of the ISR was analyzed by cell death and metabolic measurements under conditions to approximate aspects of the GB microenvironment. ATF4 protein levels were induced by temozolomide treatment. In line, ATF4 gene suppressed GB cells (ATF4sh) displayed increased cell death and decreased survival after temozolomide treatment. Similar results were observed after treatment with the ISR inhibitor ISRIB. ATF4sh and ISRIB treated GB cells were sensitized to hypoxia-induced cell death. Our experimental study provides evidence for an important role of ATF4 for the adaptation of human GB cells to conditions of the tumor microenvironment characterized by low oxygen and nutrient availability and for the development of temozolomide resistance. Inhibiting the ISR in GB cells could therefore be a promising therapeutic approach.

Cetuximab-Mediated Protection from Hypoxia- Induced Cell Death: Implications for Therapy Sequence in Colorectal Cancer.

Monoclonal antibodies like cetuximab, targeting the epidermal growth factor receptor (EGFR), and bevacizumab, targeting the vascular endothelial growth factor (VEGF), are an integral part of treatment regimens for metastasized colorectal cancer. However, inhibition of the EGFR has been shown to protect human glioma cells from cell death under hypoxic conditions. In colon carcinoma cells, the consequences of EGFR blockade in hypoxia (e.g., induced by bevacizumab) have not been evaluated yet. LIM1215 and SW948 colon carcinoma and LNT-229 glioblastoma cells were treated with cetuximab, PD153035, and erlotinib and analyzed for cell density and viability. The sequential administration of either cetuximab followed by bevacizumab (CET->BEV) or bevacizumab followed by cetuximab (BEV->CET) was investigated in a LIM1215 (KRAS wildtype) and SW948 (KRAS mutant) xenograft mouse model. In vitro, cetuximab protected from hypoxia. In the LIM1215 model, a survival benefit with cetuximab and bevacizumab monotherapy was observed, but only the sequence CET->BEV showed an additional benefit. This effect was confirmed in the SW948 model. Our observations support the hypothesis that bevacizumab modulates the tumor microenvironment (e.g., by inducing hypoxia) where cetuximab could trigger protective effects when administered later on. The sequence CET->BEV therefore seems to be superior as possible mutual adverse effects are bypassed.

Activation of Epidermal Growth Factor Receptor Sensitizes Glioblastoma Cells to Hypoxia-Induced Cell Death.

BACKGROUND: The epidermal growth factor receptor (EGFR) signaling pathway is genetically activated in approximately 50% of glioblastomas (GBs). Its inhibition has been explored clinically but produced disappointing results, potentially due to metabolic effects that protect GB cells against nutrient deprivation and hypoxia. Here, we hypothesized that EGFR activation could disable metabolic adaptation and define a GB cell population sensitive to starvation. METHODS: Using genetically engineered GB cells to model different types of EGFR activation, we analyzed changes in metabolism and cell survival under conditions of the tumor microenvironment. RESULTS: We found that expression of mutant EGFRvIII as well as EGF stimulation of EGFR-overexpressing cells impaired physiological adaptation to starvation and rendered cells sensitive to hypoxia-induced cell death. This was preceded by adenosine triphosphate (ATP) depletion and an increase in glycolysis. Furthermore, EGFRvIII mutant cells had higher levels of mitochondrial superoxides potentially due to decreased metabolic flux into the serine synthesis pathway which was associated with a decrease in the NADPH/NADP+ ratio. CONCLUSIONS: The finding that EGFR activation renders GB cells susceptible to starvation could help to identify a subgroup of patients more likely to benefit from starvation-inducing therapies.

AMPK activation protects astrocytes from hypoxia­induced cell death.

Adenosine monophosphate (AMP)­activated protein kinase (AMPK) is a major cellular energy sensor that is activated by an increase in the AMP/adenosine triphosphate (ATP) ratio. This causes the initiation of adaptive cellular programs, leading to the inhibition of anabolic pathways and increasing ATP synthesis. AMPK indirectly inhibits mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a serine/threonine kinase and central regulator of cell growth and metabolism, which integrates various growth inhibitory signals, such as the depletion of glucose, amino acids, ATP and oxygen. While neuroprotective approaches by definition focus on neurons, that are more sensitive under cell stress conditions, astrocytes play an important role in the cerebral energy homeostasis during ischemia. Therefore, the protection of astrocytic cells or other glial cells may contribute to the preservation of neuronal integrity and function. In the present study, it was thus hypothesized that a preventive induction of energy deprivation­activated signaling pathways via AMPK may protect astrocytes from hypoxia and glucose deprivation. Hypoxia­induced cell death was measured in a paradigm of hypoxia and partial glucose deprivation in vitro in the immortalized human astrocytic cell line SVG. Both the glycolysis inhibitor 2­deoxy­d­glucose (2DG) and the AMPK activator A­769662 induced the phosphorylation of AMPK, resulting in mTORC1 inhibition, as evidenced by a decrease in the phosphorylation of the target ribosomal protein S6 (RPS6). Treatment with both 2DG and A­769662 also decreased glucose consumption and lactate production. Furthermore, A­769662, but not 2DG induced an increase in oxygen consumption, possibly indicating a more efficient glucose utilization through oxidative phosphorylation. Hypoxia­induced cell death was profoundly reduced by treatment with 2DG or A­769662. On the whole, the findings of the present study demonstrate, that AMPK activation via 2DG or A­769662 protects astrocytes under hypoxic and glucose­depleted conditions.

Delayed Occurrence of Hypertrophic Olivary Degeneration after Therapy of Posterior Fossa Tumors: A Single Institution Retrospective Analysis.

(1) Background: A lesion within the dentato-rubro-olivary pathway (DROP) in the posterior fossa can cause secondary neurodegeneration of the inferior olivary nucleus: so-called hypertrophic olivary degeneration (HOD). The clinical syndrome of HOD occurs slowly over months and may be overlooked in progressive neuro-oncological diseases. Posterior fossa tumors are often located near these strategic structures. The goal of this study was to analyze the systematics of HOD occurrence in neuro-oncological patients. (2) Methods: The neuroradiological database of the university healthcare center was scanned for HOD-related terms from 2010 to 2019. After excluding patients with other causes of HOD, 12 datasets from neuro-oncological patients were analyzed under predetermined criteria. (3) Results: Patients received multimodal tumor treatments including neurosurgery, radiotherapy, and chemotherapy. HOD occurred both unilaterally (left n = 4; right n = 5) and bilaterally (n = 3). Though the mass effect of posterior fossa tumors had already affected strategic structures of the DROP, none of the patients showed signs of HOD on MRI until therapeutic measures including neurosurgery affecting the DROP were applied. HOD was visible on MRI within a median of 6 months after the neurosurgical intervention. In 67%, the presumed underlying surgical lesion in the DROP lay in the contralateral dentate nucleus. (4) Conclusion: In a selected cohort of neuro-oncological patients, therapeutic lesions within the DROP were associated with HOD occurrence.

Subependymomas - Characteristics of a "Leave me Alone" Lesion. / Subependymome ­ Charakteristika einer "leave me alone"-Läsion.

PURPOSE: Intracranial subependymomas are rare, mostly asymptomatic tumours, which are often found incidentally and therefore did not receive much attention in previous literature. By being classified as benign grade I in the WHO classification of tumours of the central nervous system, they are given a special status compared to the other ependymal tumours. Tumor recurrences are a rarity, spinal "drop metastases" do not occur. While etiological, pathological and therapeutic characteristics have been subject of several publications over the last few decades and have meanwhile been well studied, the imaging characteristics are much less well received. MATERIAL AND METHOD: Retrospective analysis of our relatively large group of 33 patients with subependymoma, including 4 patients with a mixture of subependymomas with ependymal cell fractions in terms of imaging and clinical aspects and with reference to a current literature review. RESULTS: Subependymomas have typical image morphologic characteristics that differentiate them from tumors of other entities, however, the rare subgroup of histopathological mixtures of subependymomas with ependymal cell fractions has no distinctly different imaging properties. CONCLUSIONS: Knowing the imaging characteristics of subpendymoma and their differential diagnoses is of particular importance in order to be able to decide between the necessity of follow-up controls, an early invasive diagnosis or, depending on the entity, tumor resection. KEY POINTS: · Subependymomas have typical imaging characteristics that are clearly distinguishable from other entities.. · Increased incidence in middle/ older aged men, most frequent localization: 4th ventricle.. · Symptomatic subependymomas, often located in lateral ventricles, are usually characterized by hydrocephalus.. · Radiological identification of mixed subependymoma with ependymal cell fractions is not possible.. · Image based differentiation from other entities is important for the procedure.. CITATION FORMAT: · Kammerer S, Mueller-Eschner M, Lauer A et al. Subependymomas - Characteristics of a "Leave me Alone" Lesion. Fortschr Röntgenstr 2018; 190: 955 - 966.