A chemical screen for medulloblastoma identifies quercetin as a putative radiosensitizer

Treatment of medulloblastoma in children fails in approximately 30% of patients, and is often accompanied by severe late sequelae. Therefore, more effective drugs are needed that spare normal tissue and diminish long-term side effects. Since radiotherapy plays a pivotal role in the treatment of medulloblastoma, we set out to identify novel drugs that could potentiate the effect of ionizing radiation. Thereto, a small molecule library, consisting of 960 chemical compounds, was screened for its ability to sensitize towards irradiation. This small molecule screen identified the flavonoid quercetin as a novel radiosensitizer for the medulloblastoma cell lines DAOY, D283-med, and, to a lesser extent, D458-med at low micromolar concentrations and irradiation doses used in fractionated radiation schemes. Quercetin did not affect the proliferation of neural precursor cells or normal human fibroblasts. Importantly, in vivo experiments confirmed the radiosensitizing properties of quercetin. Administration of this flavonoid at the time of irradiation significantly prolonged survival in orthotopically xenografted mice. Together, these findings indicate that quercetin is a potent radiosensitizer for medulloblastoma cells that may be a promising lead for the treatment of medulloblastoma in patients

Identification of a Druggable Pathway Controlling Glioblastoma Invasiveness

Diffuse and uncontrollable brain invasion is a hallmark of glioblastoma (GBM), but its mechanism is understood poorly. We developed a 3D ex vivo organotypic model to study GBM invasion. We demonstrate that invading GBM cells upregulate a network of extracellular matrix (ECM) components, including multiple collagens, whose expression correlates strongly with grade and clinical outcome. We identify interferon regulatory factor 3 (IRF3) as a transcriptional repressor of ECM factors and show that IRF3 acts as a suppressor of GBM invasion. Therapeutic activation of IRF3 by inhibiting casein kinase 2 (CK2)—a negative regulator of IRF3—downregulated the expression of ECM factors and suppressed GBM invasion in ex vivo and in vivo models across a panel of patient-derived GBM cell lines representative of the main molecular GBM subtypes. Our data provide mechanistic insight into the invasive capacity of GBM tumors and identify a potential therapy to inhibit GBM invasion

Glycan modification of glioblastoma-derived extracellular vesicles enhances receptor-mediated targeting of dendritic cells

Glioblastoma is the most prevalent and aggressive primary brain tumour for which total tumour lysate-pulsed dendritic cell vaccination is currently under clinical evaluation. Glioblastoma extracellular vesicles (EVs) may represent an enriched cell-free source of tumour-associated (neo-) antigens to pulse dendritic cells (DCs) for the initiation of an anti-tumour immune response. Capture and uptake of EVs by DCs could occur in a receptor-mediated and presumably glycan-dependent way, yet the glycan composition of glioblastoma EVs is unknown. Here, we set out to characterize the glycocalyx composition of glioblastoma EVs by lectin-binding ELISA and comprehensive immunogold transmission electron microscopy (immuno-TEM). The surface glycan profile of human glioblastoma cell line-derived EVs (50–200 nm) was dominated by α-2,3- and α-2,6 linked sialic acid-capped complex N-glycans and bi-antennary N-glycans. Since sialic acids can trigger immune inhibitory sialic acid–binding Ig-like lectin (Siglec) receptors, we screened for Siglec ligands on the EVs. Glioblastoma EVs showed significant binding to Siglec-9, which is highly expressed on DCs. Surprisingly, however, glioblastoma EVs lack glycans that could bind Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), a receptor that mediates uptake and induction of CD4+ and CD8+ T cell activation. Therefore, we explored whether modification of the EV glycan surface could reduce immune inhibitory Siglec binding, while enhancing EV internalization by DCs in a DC-SIGN dependent manner. Desialylation with a pan-sialic acid hydrolase led to reduction of sialic acid expression on EVs. Moreover, insertion of a high-affinity ligand (LewisY) for DC-SIGN resulted in a four-fold increase of uptake by monocyte-derived DCs. In conclusion, we show that the glycocalyx composition of EVs is a key factor of efficient DC targeting and that modification of the EV glycocalyx potentiates EVs as anti-cancer vaccine

Reducing severe fatigue in patients with diffuse glioma: a study protocol for an RCT on the effect of blended cognitive behavioural therapy

Background: Fatigue is the most frequent and burdensome symptom of patients with diffuse glioma. It is closely linked to decreased health-related quality of life and symptoms such as depression and sleep disturbances. Currently, there is no evidence-based treatment that targets severe fatigue in patients with brain tumours. Cognitive behavioural therapy is aimed at fatigue-maintaining beliefs and behaviour. This therapy has been proven effective in reducing severe fatigue in cancer survivors and patients with multiple sclerosis. A blended therapy program combines sessions with a therapist with therapist-guided web-based therapy modules. The aim of this randomized controlled trial is to determine the efficacy of blended cognitive behavioural therapy in treating severe fatigue in patients with diffuse glioma. Methods: We will include a maximum of 100 patients with diffuse glioma with clinically and radiologically stable disease and severe fatigue (i.e. Checklist Individual Strength, subscale fatigue severity ≥ 35). Patients will be randomized to blended cognitive behavioural therapy or a waiting list condition. The 12-week intervention GRIP on fatigue consists of five patient-therapist sessions and five to eight individualized web-based therapy modules supported by email contact. The primary outcome measure is fatigue severity. Secondary outcome measures include sleep quality, health-related quality of life, depression, anxiety, functional impairment and subjective and objective cognitive functioning. Primary and secondary outcome measures will be assessed at baseline and after 14 and 24 weeks. Magnetoencephalography and MRI will be used to evaluate potential biomarkers for intervention success. This trial has a Bayesian design: we will conduct multiple interim analyses to test for efficacy or futility of the trial. This is the first trial within the GRIP trial platform: a platform developing four to five different interventions for the most common symptoms in patients with diffuse glioma. Discussion: The results of the GRIP on fatigue trial will provide information about the efficacy of this intervention on fatigue in patients with diffuse glioma. Multiple other outcomes and possible predictors of treatment success will also be explored. Trial registration: Netherlands Trial Register NL8711. Registered on 14 June 2020

Reducing severe fatigue in patients with diffuse glioma: a study protocol for an RCT on the effect of blended cognitive behavioural therapy

BACKGROUND: Fatigue is the most frequent and burdensome symptom of patients with diffuse glioma. It is closely linked to decreased health-related quality of life and symptoms such as depression and sleep disturbances. Currently, there is no evidence-based treatment that targets severe fatigue in patients with brain tumours. Cognitive behavioural therapy is aimed at fatigue-maintaining beliefs and behaviour. This therapy has been proven effective in reducing severe fatigue in cancer survivors and patients with multiple sclerosis. A blended therapy program combines sessions with a therapist with therapist-guided web-based therapy modules. The aim of this randomized controlled trial is to determine the efficacy of blended cognitive behavioural therapy in treating severe fatigue in patients with diffuse glioma. METHODS: We will include a maximum of 100 patients with diffuse glioma with clinically and radiologically stable disease and severe fatigue (i.e. Checklist Individual Strength, subscale fatigue severity ≥ 35). Patients will be randomized to blended cognitive behavioural therapy or a waiting list condition. The 12-week intervention GRIP on fatigue consists of five patient-therapist sessions and five to eight individualized web-based therapy modules supported by email contact. The primary outcome measure is fatigue severity. Secondary outcome measures include sleep quality, health-related quality of life, depression, anxiety, functional impairment and subjective and objective cognitive functioning. Primary and secondary outcome measures will be assessed at baseline and after 14 and 24 weeks. Magnetoencephalography and MRI will be used to evaluate potential biomarkers for intervention success. This trial has a Bayesian design: we will conduct multiple interim analyses to test for efficacy or futility of the trial. This is the first trial within the GRIP trial platform: a platform developing four to five different interventions for the most common symptoms in patients with diffuse glioma. DISCUSSION: The results of the GRIP on fatigue trial will provide information about the efficacy of this intervention on fatigue in patients with diffuse glioma. Multiple other outcomes and possible predictors of treatment success will also be explored. TRIAL REGISTRATION: Netherlands Trial Register NL8711 . Registered on 14 June 2020

WEE1 Kinase Targeting Combined with DNA-Damaging Cancer Therapy Catalyzes Mitotic Catastrophe

WEE1 kinase is a key molecule in maintaining G₂-cell-cycle checkpoint arrest for premitotic DNA repair. Whereas normal cells repair damaged DNA during G₁-arrest, cancer cells often have a deficient G₁-arrest and largely depend on G₂-arrest. The molecular switch for the G₂-M transition is held by WEE1 and is pushed forward by CDC25. WEE1 is overexpressed in various cancer types, including glioblastoma and breast cancer. Preclinical studies with cancer cell lines and animal models showed decreased cancer cell viability, reduced tumor burden, and improved survival after WEE1 inhibition by siRNA or small molecule inhibitors, which is enhanced by combination with conventional DNA-damaging therapy, such as radiotherapy and/or cytostatics. Mitotic catastrophe results from premature entry into mitosis with unrepaired lethal DNA damage. As such, cancer cells become sensitized to conventional therapy by WEE1 inhibition, in particular those with insufficient G₁-arrest due to deficient p53 signaling, like glioblastoma cells. One WEE1 inhibitor has now reached clinical phase I studies. Dose-limiting toxicity consisted of hematologic events, nausea and/or vomiting, and fatigue. The combination of DNA-damaging cancer therapy with WEE1 inhibition seems to be a rational approach to push cancer cells in mitotic catastrophe. Its safety and efficacy are being evaluated in clinical studie

The TICking clock of EGFR therapy resistance in glioblastoma: Target Independence or target Compensation

Targeted therapy against driver mutations responsible for cancer progression has been shown to be effective in many tumor types. For glioblastoma (GBM), the epidermal growth factor receptor (EGFR) gene is the most frequently mutated oncogenic driver and has therefore been considered an attractive target for therapy. However, so far responses to EGFR-pathway inhibitors have been disappointing. We performed an exhaustive analysis of the mechanisms that might account for therapy resistance against EGFR inhibition. We define two major mechanisms of resistance and propose modalities to overcome them. The first resistance mechanism concerns target independence. In this case, cells have lost expression of the EGFR protein and experience no negative impact of EGFR targeting. Loss of extrachromosomally encoded EGFR as present in double minute DNA is a frequent mechanism for this type of drug resistance. The second mechanism concerns target compensation. In this case, cells will counteract EGFR inhibition by activation of compensatory pathways that render them independent of EGFR signaling. Compensatory pathway candidates are platelet-derived growth factor β (PDGFβ), Insulin-like growth factor 1 (IGFR1) and cMET and their downstream targets, all not commonly mutated at the time of diagnosis alongside EGFR mutation. Given that both mechanisms make cells independent of EGFR expression, other means have to be found to eradicate drug resistant cells. To this end we suggest rational strategies which include the use of multi-target therapies that hit truncation mutations (mechanism 1) or multi-target therapies to co-inhibit compensatory proteins (mechanism 2)

RNA-Sequencing of Tumor-Educated Platelets, a Novel Biomarker for Blood-Based Sarcoma Diagnostics

Sarcoma is a heterogeneous group of rare malignancies arising from mesenchymal tissues. Recurrence rates are high and methods for early detection by blood-based biomarkers do not exist. Hence, development of blood-based liquid biopsies as disease recurrence monitoring biomarkers would be an important step forward. Recently, it has been shown that tumor-educated platelets (TEPs) harbor specific spliced ribonucleic acid(RNA)-profiles. These RNA-repertoires are potentially applicable for cancer diagnostics. We aim to evaluate the potential of TEPs for blood-based diagnostics of sarcoma patients. Fifty-seven sarcoma patients (active disease), 38 former sarcoma patients (cancer free for ≥3 years) and 65 healthy donors were included. RNA was isolated from platelets and sequenced. Quantified read counts were processed with self-learning particle-swarm optimization-enhanced thromboSeq analysis and subjected to analysis of variance (ANOVA) statistics. Highly correlating spliced platelet messenger RNAs (mRNAs) of sarcoma patients were compared to controls (former sarcoma + healthy donors) to identify a quantitative sarcoma-specific signature measure, the TEP-score. ANOVA analysis identified distinctive platelet RNA expression patterns of 2647 genes (false discovery rate <0.05) in sarcoma patients as compared to controls. The self-learning algorithm reached a diagnostic accuracy of 87% (validation set only; n = 53 samples, area under the curve (AUC): 0.93, 95% confidence interval (CI): 0.86-1). Our data indicates that TEP RNA-based liquid biopsies may enable for sarcoma diagnostics

The bivariate NRIP1/ZEB2 RNA marker permits non-invasive presymptomatic screening of pre-eclampsia

Using genome-wide transcriptome analysis by RNA sequencing of first trimester plasma RNA, we tested whether the identification of pregnancies at risk of developing pre-eclampsia with or without preterm birth or growth restriction is possible between weeks 9-14, prior to the appearance of clinical symptoms. We implemented a metaheuristic approach in the self-learning SVM algorithm for differential gene expression analysis of normal pregnancies (n = 108), affected pregnancies (n = 34) and non-pregnant controls (n = 19). Presymptomatic candidate markers for affected pregnancies were validated by RT-qPCR in first trimester samples (n = 34) from an independent cohort. PRKG1 was significantly downregulated in a subset of pregnancies with birth weights below the 10thpercentile as shared symptom. The NRIP1/ZEB2 ratio was found to be upregulated in pregnancies with pre-eclampsia or trisomy 21. Complementary quantitative analysis of both the linear and circular forms of NRIP1 permitted discrimination between pre-eclampsia and trisomy 21. Pre-eclamptic pregnancies showed an increase in linear NRIP1 compared to circular NRIP1, while trisomy 21 pregnancies did not

Circular RNA Sequencing of Maternal Platelets: A Novel Tool for the Identification of Pregnancy-Specific Biomarkers

BACKGROUND: In the first trimester of pregnancy, the maternal platelet is directly involved in a positive feedback mechanism that facilitates invasion of the extravillous trophoblast into the maternal spiral arteries. Dysfunctional trophoblast invasion with defective deep placentation is primordial in the etiology of the "great obstetrical syndromes." METHODS: In this proof-of-concept study, using transcriptome analysis of circular RNA (circRNA) following RNA sequencing of maternal platelets, we tested whether pregnancy-specific circRNA markers could be identified in the first trimester of normal pregnancies. Differential transcript expression analysis of circRNAs, as predicted by Accurate CircRNA Finder Suite, CircRNA Identifier (version 2), and Known and Novel Isoform Explorer, was done using thromboSeq.R with variation of multiple settings. Test performance was checked for (a) de novo circRNA identification using the novel platelet-specific Plt-circR4 as a positive control, (b) complete segregation of groups (pregnant vs nonpregnant) after heat map-dendrogram clustering, (c) identification of pregnancy-specific circRNA markers at a false discovery rate (FDR) 2), and 52 circRNAs were downregulated (logFC less than -2) in first-trimester platelet RNA. Of these, nuclear receptor-interacting protein 1 circRNA covering exons 2 and 3 of the 5'-untranslated region was pregnancy specific with upregulation in first-trimester maternal platelets compared to nonpregnant controls. CONCLUSION: CircRNA sequencing of first-trimester maternal platelets permits the identification of novel pregnancy-specific RNA biomarkers. Future use could include the assessment of maternal and fetal well-being