Updated EANO guideline on rational molecular testing of gliomas, glioneuronal, and neuronal tumors in adults for targeted therapy selection - Update 1.

The standard of care for adult patients with gliomas, glioneuronal and neuronal tumors consists of combinations of surgery, radiotherapy, and chemotherapy. For many systemic cancers, targeted treatments are a major part of the standard treatment, however, the predictive significance of most of the targets for treatment in systemic cancer are less well established in central nervous system (CNS) tumors . In 2023 the EANO Guideline Committee presented evidence based recommendations for rational testing of molecular targets for targeted treatments. From all targets reviewed, only testing for BRAF V600E mutations was of proven clinical benefit; despite regulatory approvals for tumor agnostic treatment of NTRK gene fusions and high Tumor Mutational Burden (TMB) for patients with adult brain tumors, the evidence of clinical benefit for patients was still limited . This guideline has a modular structure, allowing regular updating of individual sections and adding new ones. The present version (Update 1) presents a review of the rationale of testing for PTEN, H3F3A, MTAP, RET and IDH, and presents an update of the text on TMB high and mismatch repair deficiency. It also presents an overview of therapeutic yield of routine next generation sequencing for mutations and fusion detection. The supplement accompanying this version contains the in depth review of all targets, whereas in the main manuscript the final recommendations of the revised and new targets are presented. Updates will be made on a regular basis.

Preoperative prediction of diffuse glioma type and grade in adults: a gadolinium-free MRI-based decision tree.

OBJECTIVES: To develop a gadolinium-free MRI-based diagnosis prediction decision tree (DPDT) for adult-type diffuse gliomas and to assess the added value of gadolinium-based contrast agent (GBCA) enhanced images. MATERIALS AND METHODS: This study included preoperative grade 2-4 adult-type diffuse gliomas (World Health Organization 2021) scanned between 2010 and 2021. The DPDT, incorporating eleven GBCA-free MRI features, was developed using 18% of the dataset based on consensus readings. Diagnosis predictions involved grade (grade 2 vs. grade 3/4) and molecular status (isocitrate dehydrogenase (IDH) and 1p/19q). GBCA-free diagnosis was predicted using DPDT, while GBCA-enhanced diagnosis included post-contrast images. The accuracy of these predictions was assessed by three raters with varying experience levels in neuroradiology using the test dataset. Agreement analyses were applied to evaluate the prediction performance/reproducibility. RESULTS: The test dataset included 303 patients (age (SD): 56.7 (14.2) years, female/male: 114/189, low-grade/high-grade: 54/249, IDH-mutant/wildtype: 82/221, 1p/19q-codeleted/intact: 34/269). Per-rater GBCA-free predictions achieved ≥ 0.85 (95%-CI: 0.80-0.88) accuracy for grade and ≥ 0.75 (95%-CI: 0.70-0.80) for molecular status, while GBCA-enhanced predictions reached ≥ 0.87 (95%-CI: 0.82-0.90) and ≥ 0.77 (95%-CI: 0.71-0.81), respectively. No accuracy difference was observed between GBCA-free and GBCA-enhanced predictions. Group inter-rater agreement was moderate for GBCA-free (0.56 (95%-CI: 0.46-0.66)) and substantial for GBCA-enhanced grade prediction (0.68 (95%-CI: 0.58-0.78), p = 0.008), while substantial for both GBCA-free (0.75 (95%-CI: 0.69-0.80) and GBCA-enhanced (0.77 (95%-CI: 0.71-0.82), p = 0.51) molecular status predictions. CONCLUSION: The proposed GBCA-free diagnosis prediction decision tree performed well, with GBCA-enhanced images adding little to the preoperative diagnostic accuracy of adult-type diffuse gliomas. KEY POINTS: Question Given health and environmental concerns, is there a gadolinium-free imaging protocol to preoperatively evaluate gliomas comparable to the gadolinium-enhanced standard practice? Findings The proposed gadolinium-free diagnosis prediction decision tree for adult-type diffuse gliomas performed well, and gadolinium-enhanced MRI demonstrated only limited improvement in diagnostic accuracy. Clinical relevance Even inexperienced raters effectively classified adult-type diffuse gliomas using the gadolinium-free diagnosis prediction decision tree, which, until further validation, can be used alongside gadolinium-enhanced images to respect standard practice, despite this study showing that gadolinium-enhanced images hardly improved diagnostic accuracy.

Gemcitabine therapeutically disrupts essential SIRT1-mediated p53 repression in atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (ATRTs) are highly malignant embryonal tumors of the central nervous system with a dismal prognosis. Using a newly developed and validated patient-derived ATRT culture and xenograft model, alongside a panel of primary ATRT models, we found that ATRTs are selectively sensitive to the nucleoside analog gemcitabine. Gene expression and protein analyses indicate that gemcitabine treatment causes the degradation of sirtuin 1 (SIRT1), resulting in cell death through activation of nuclear factor κB (NF-κB) and p53. Furthermore, we discovered that gemcitabine-induced loss of SIRT1 results in a nucleus-to-cytoplasm translocation of the sonic hedgehog (SHH) signaling activator GLI2, explaining the observed additional gemcitabine sensitivity in SHH-subtype ATRT. Treatment of ATRT xenograft-bearing mice with gemcitabine resulted in a >30% increase in median survival and yielded long-term survivors in two independent patient-derived xenograft models. These findings demonstrate that ATRTs are highly sensitive to gemcitabine treatment and may form part of a future multimodal treatment strategy for ATRTs.

cIMPACT-NOW Update 8: Clarifications on molecular risk parameters and recommendations for WHO grading of meningiomas.

Meningiomas are the most frequent primary intracranial tumors. Hence, they constitute a major share of diagnostic specimens in neuropathology practice. The 2021 WHO Classification of Central Nervous System Tumors ("CNS5") has introduced the first molecular grading parameters for meningioma with oncogenic variants in the TERT promoter and homozygous deletion of CDKN2A/B as markers for CNS WHO grade 3. However, after publication of the new classification volume, clarifications were requested, not only on novel but also on long-standing questions in meningioma grading that were beyond the scope of the WHO "blue book". In addition, more recent research into possible new molecular grading parameters could not yet be implemented in the 2021 classification but constitute a compelling body of literature. Hence, the cIMPACT-NOW Steering Committee convened a working group to provide such clarification and assess the evidence of possible novel molecular criteria. As a result, this cIMPACT-NOW update provides guidance for more standardized morphological evaluation and interpretation, most prominently pertaining to brain invasion, identifies scenarios in which advanced molecular testing is recommended, proposes to assign CNS WHO grade 2 for cases with CNS WHO grade 1 morphology but chromosomal arm 1p deletion in combination with 22q deletion and/or NF2 oncogenic variants, and discusses areas in which the current evidence is not yet sufficient to result in new recommendations.

The impact of ATP-binding cassette transporters in the diseased brain: Context matters.

ATP-binding cassette (ABC) transporters facilitate the movement of diverse molecules across cellular membranes, including those within the CNS. While most extensively studied in microvascular endothelial cells forming the blood-brain barrier (BBB), other CNS cell types also express these transporters. Importantly, disruptions in the CNS microenvironment during disease can alter transporter expression and function. Through this comprehensive review, we explore the modulation of ABC transporters in various brain pathologies and the context-dependent consequences of these changes. For instance, downregulation of ABCB1 may exacerbate amyloid beta plaque deposition in Alzheimer's disease and facilitate neurotoxic compound entry in Parkinson's disease. Upregulation may worsen neuroinflammation by aiding chemokine-mediated CD8 T cell influx into multiple sclerosis lesions. Overall, ABC transporters at the BBB hinder drug entry, presenting challenges for effective pharmacotherapy. Understanding the context-dependent changes in ABC transporter expression and function is crucial for elucidating the etiology and developing treatments for brain diseases.

Malignant glioma in L-2-Hydroxyglutaric Aciduria: thorough molecular characterization of a case and literature review.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the L2HGDH gene. L-2-HGA patients have a significantly increased lifetime risk of central nervous system (CNS) tumors. Here, we present a 16-year-old girl with L-2-HGA who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. Histologically, the tumor had a high-grade diffuse glioma phenotype. DNA sequencing revealed the inactivating homozygous germline L2HGDH mutation as well as inactivating mutations in TP53, BCOR and NF1. Genome-wide DNA-methylation analysis was unable to classify the tumor with high confidence. More detailed analysis revealed that this tumor clustered amongst IDH-wildtype gliomas by methylation profiling and did not show the glioma CpG island methylator phenotype (G-CIMP) in contrast to IDH-mutant diffuse gliomas with accumulated levels of D-2-HG, the stereoisomer of L-2-HD. These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis of the tumor was diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Due to rapid tumor progression the patient died nine months after initial diagnosis. In this manuscript, we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of L-2-HGA-associated CNS tumors.

RT-PCR assay to detect FGFR3::TACC3 fusions in formalin-fixed, paraffin-embedded glioblastoma samples.

Background: One targeted treatment option for isocitrate dehydrogenase (IDH)-wild-type glioblastoma focuses on tumors with fibroblast growth factor receptor 3::transforming acidic coiled-coil-containing protein 3 (FGFR3::TACC3) fusions. FGFR3::TACC3 fusion detection can be challenging, as targeted RNA next-generation sequencing (NGS) is not routinely performed, and immunohistochemistry is an imperfect surrogate marker. Fusion status can be determined using reverse transcription polymerase chain reaction (RT-PCR) on fresh frozen (FF) material, but sometimes only formalin-fixed, paraffin-embedded (FFPE) tissue is available. Aim: To develop an RT-PCR assay to determine FGFR3::TACC3 status in FFPE glioblastoma samples. Methods: Twelve tissue microarrays with 353 historical glioblastoma samples were immunohistochemically stained for FGFR3. Samples with overexpression of FGFR3 (n = 13) were subjected to FGFR3::TACC3 RT-PCR on FFPE, using 5 primer sets for the detection of 5 common fusion variants. Fusion-negative samples were additionally analyzed with NGS (n = 6), FGFR3 Fluorescence In Situ Hybridization (n = 6), and RNA sequencing (n = 5). Results: Using RT-PCR on FFPE material of the 13 samples with FGFR3 overexpression, we detected an FGFR3::TACC3 fusion in 7 samples, covering 3 different fusion variants. For 5 of these FF was available, and the presence of the fusion was confirmed through RT-PCR on FF. With RNA sequencing, 1 additional sample was found to harbor an FGFR3::TACC3 fusion (variant not covered by current RT-PCR for FFPE). The frequency of FGFR3::TACC3 fusion in this cohort was 9/353 (2.5%). Conclusions: RT-PCR for FGFR3::TACC3 fusions can successfully be performed on FFPE material, with a specificity of 100% and (due to limited primer sets) a sensitivity of 83.3%. This assay allows for the identification of potential targeted treatment options when only formalin-fixed tissue is available.

Protocol for investigating data quality and reporting outcomes of pediatric gliomas in population-based cancer registry research.

Cancer registry data on pediatric gliomas come with inherent limitations as inclusion criteria and registration practices of these tumors differ between registries due to specific guidelines that are lacking. These limitations can lead to biased estimates in incidence and survival outcomes. Here, we present a protocol to investigate data quality and comparability for retrospective population-based pediatric glioma studies. We describe steps for obtaining institutional permissions, dealing with data quality issues, regrouping tumors, and reporting tumors in a clinically relevant manner. For complete details on the use and execution of this protocol, please refer to Hoogendijk et al.1.

The immunological landscape of peripheral blood in glioblastoma patients and immunological consequences of age and dexamethasone treatment.

Background: Glioblastomas manipulate the immune system both locally and systemically, yet, glioblastoma-associated changes in peripheral blood immune composition are poorly studied. Age and dexamethasone administration in glioblastoma patients have been hypothesized to limit the effectiveness of immunotherapy, but their effects remain unclear. We compared peripheral blood immune composition in patients with different types of brain tumor to determine the influence of age, dexamethasone treatment, and tumor volume. Methods: High-dimensional mass cytometry was used to characterise peripheral blood mononuclear cells of 169 patients with glioblastoma, lower grade astrocytoma, metastases and meningioma. We used blood from medically-refractory epilepsy patients and healthy controls as control groups. Immune phenotyping was performed using FlowSOM and t-SNE analysis in R followed by supervised annotation of the resulting clusters. We conducted multiple linear regression analysis between intracranial pathology and cell type abundance, corrected for clinical variables. We tested correlations between cell type abundance and survival with Cox-regression analyses. Results: Glioblastoma patients had significantly fewer naive CD4+ T cells, but higher percentages of mature NK cells than controls. Decreases of naive CD8+ T cells and alternative monocytes and an increase of memory B cells in glioblastoma patients were influenced by age and dexamethasone treatment, and only memory B cells by tumor volume. Progression free survival was associated with percentages of CD4+ regulatory T cells and double negative T cells. Conclusion: High-dimensional mass cytometry of peripheral blood in patients with different types of intracranial tumor provides insight into the relation between intracranial pathology and peripheral immune status. Wide immunosuppression associated with age and pre-operative dexamethasone treatment provide further evidence for their deleterious effects on treatment with immunotherapy.

AllergoOncology: Biomarkers and refined classification for research in the allergy and glioma nexus-A joint EAACI-EANO position paper.

Epidemiological studies have explored the relationship between allergic diseases and cancer risk or prognosis in AllergoOncology. Some studies suggest an inverse association, but uncertainties remain, including in IgE-mediated diseases and glioma. Allergic disease stems from a Th2-biased immune response to allergens in predisposed atopic individuals. Allergic disorders vary in phenotype, genotype and endotype, affecting their pathophysiology. Beyond clinical manifestation and commonly used clinical markers, there is ongoing research to identify novel biomarkers for allergy diagnosis, monitoring, severity assessment and treatment. Gliomas, the most common and diverse brain tumours, have in parallel undergone changes in classification over time, with specific molecular biomarkers defining glioma subtypes. Gliomas exhibit a complex tumour-immune interphase and distinct immune microenvironment features. Immunotherapy and targeted therapy hold promise for primary brain tumour treatment, but require more specific and effective approaches. Animal studies indicate allergic airway inflammation may delay glioma progression. This collaborative European Academy of Allergy and Clinical Immunology (EAACI) and European Association of Neuro-Oncology (EANO) Position Paper summarizes recent advances and emerging biomarkers for refined allergy and adult-type diffuse glioma classification to inform future epidemiological and clinical studies. Future research is needed to enhance our understanding of immune-glioma interactions to ultimately improve patient prognosis and survival.

Next generation sequencing of high-grade adult-type diffuse glioma in the Netherlands: interlaboratory variation in the primary diagnostic and recurrent setting.

PURPOSE: Next generation sequencing (NGS) is an important tool used in clinical practice to obtain the required molecular information for accurate diagnostics of high-grade adult-type diffuse glioma (HGG). Since individual centers use either in-house produced or standardized panels, interlaboratory variation could play a role in the practice of HGG diagnosis and treatment. This study aimed to investigate the current practice in NGS application for both primary and recurrent HGG. METHODS: This nationwide Dutch survey used the expertise of (neuro)pathologists and clinical scientists in molecular pathology (CSMPs) by sending online questionnaires on clinical and technical aspects. Primary outcome was an overview of panel composition in the different centers for diagnostic practice of HGG. Secondary outcomes included practice for recurrent HGG and future perspectives. RESULTS: Out of twelve neuro-oncology centers, the survey was filled out by eleven (neuro)pathologists and seven CSMPs. The composition of the diagnostic NGS panels differed in each center with numbers of genes ranging from 12 to 523. Differences are more pronounced when tests are performed to find therapeutic targets in the case of recurrent disease: about half of the centers test for gene fusions (60%) and tumor mutational burden (40%). CONCLUSION: Current notable interlaboratory variations as illustrated in this study should be reduced in order to refine diagnostics and improve precision oncology. In-house developed tests, standardized panels and routine application of broad gene panels all have their own advantages and disadvantages. Future research would be of interest to study the clinical impact of variation in diagnostic approaches.

The Epigenetic Evolution of Glioma Is Determined by the IDH1 Mutation Status and Treatment Regimen.

Tumor adaptation or selection is thought to underlie therapy resistance in glioma. To investigate longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 132 matched initial and recurrent tumors from patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) glioma. IDHwt gliomas showed a stable epigenome over time with relatively low levels of global methylation. The epigenome of IDHmut gliomas showed initial high levels of genome-wide DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. Integration of epigenomics, gene expression, and functional genomics identified HOXD13 as a master regulator of IDHmut astrocytoma evolution. Furthermore, relapse of IDHmut tumors was accompanied by histologic progression that was associated with survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment varied between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neoangiogenesis and T-cell infiltration upon treatment of IDHmut gliomas. This study provides one of the largest cohorts of paired longitudinal glioma samples with epigenomic, transcriptomic, and genomic profiling and suggests that treatment of IDHmut glioma is associated with epigenomic evolution toward an IDHwt-like phenotype. SIGNIFICANCE: Standard treatments are related to loss of DNA methylation in IDHmut glioma, resulting in epigenetic activation of genes associated with tumor progression and alterations in the microenvironment that resemble treatment-naïve IDHwt glioma.

Longitudinal characteristics of T2-FLAIR mismatch in IDH-mutant astrocytomas: Relation to grade, histopathology, and overall survival in the GLASS-NL cohort.

Background: The T2-FLAIR mismatch sign is defined by signal loss of the T2-weighted hyperintense area with Fluid-Attenuated Inversion Recovery (FLAIR) on magnetic resonance imaging, causing a hypointense region on FLAIR. It is a highly specific diagnostic marker for IDH-mutant astrocytoma and is postulated to be caused by intercellular microcystic change in the tumor tissue. However, not all IDH-mutant astrocytomas show this mismatch sign and some show the phenomenon in only part of the lesion. The aim of the study is to determine whether the T2-FLAIR mismatch phenomenon has any prognostic value beyond initial noninvasive molecular diagnosis. Methods: Patients initially diagnosed with histologically lower-grade (2 or 3) IDH-mutant astrocytoma and with at least 2 surgical resections were included in the GLASS-NL cohort. T2-FLAIR mismatch was determined, and the growth pattern of the recurrent tumor immediately before the second resection was annotated as invasive or expansive. The relation between the T2-FLAIR mismatch sign and tumor grade, microcystic change, overall survival (OS), and other clinical parameters was investigated both at first and second resection. Results: The T2-FLAIR mismatch sign was significantly related to Grade 2 (80% vs 51%), longer post-resection median OS (8.3 vs 5.2 years), expansive growth, and lower age at second resection. At first resection, no relation was found between the mismatch sign and OS. Microcystic change was associated with areas of T2-FLAIR mismatch. Conclusions: T2-FLAIR mismatch in IDH-mutant astrocytomas is correlated with microcystic change in the tumor tissue, favorable prognosis, and Grade 2 tumors at the time of second resection.

BCRP drives intrinsic chemoresistance in chemotherapy-naïve breast cancer brain metastasis.

Although initially successful, treatments with chemotherapy often fail because of the recurrence of chemoresistant metastases. Since these tumors develop after treatment, resistance is generally thought to occur in response to chemotherapy. However, alternative mechanisms of intrinsic chemoresistance in the chemotherapy-naïve setting may exist but remain poorly understood. Here, we study drug-naïve murine breast cancer brain metastases (BCBMs) to identify how cancer cells growing in a secondary site can acquire intrinsic chemoresistance without cytotoxic agent exposure. We demonstrate that drug-naïve murine breast cancer cells that form cancer lesions in the brain undergo vascular mimicry and concomitantly express the adenosine 5'-triphosphate-binding cassette transporter breast cancer resistance protein (BCRP), a common marker of brain endothelial cells. We reveal that expression of BCRP by the BCBM tumor cells protects them against doxorubicin and topotecan. We conclude that BCRP overexpression can cause intrinsic chemoresistance in cancer cells growing in metastatic sites without prior chemotherapy exposure.

Sex-related incidence and survival differences in pediatric high-grade glioma subtypes: A population-based cohort study.

Not much is known on sex differences in incidence, survival, and treatment characteristics for midline and hemispheric pHGGs. This population-based study confirms previously reported study results that found worse survival outcomes for malignant diffuse gliomas in girls in the age group 0-9 years. Additionally, in our study we pinpoint this difference to girls with midline pHGGs aged 0-4 years. We provide insight in the possible underlying mechanisms contributing to sex survival differences in pHGG patients. With first line treatment having no impact on the higher risk of dying for girls, but age and tumor characteristics having a neutralizing effect. The results of this population-based study serve as a basis for future pre-clinical and clinical studies to further unravel the underlying mechanisms responsible for the survival gap between sexes in midline pHGG.

PET Imaging and Protein Expression of Prostate-Specific Membrane Antigen in Glioblastoma: A Multicenter Inventory Study.

Upregulation of prostate-specific membrane antigen (PSMA) in neovasculature has been described in glioblastoma multiforme (GBM), whereas vasculature in nonaffected brain shows hardly any expression of PSMA. It is unclear whether PSMA-targeting tracer uptake on PET is based on PSMA-specific binding to neovasculature or aspecific uptake in tumor. Here, we quantified uptake of various PSMA-targeting tracers in GBM and correlated this with PSMA expression in tumor biopsy samples from the same patients. Methods: Fourteen patients diagnosed with de novo (n = 8) or recurrent (n = 6) GBM underwent a preoperative PET scan after injection of 1.5 MBq/kg [68Ga]Ga-PSMA-11 (n = 7), 200 MBq of [18F]DCFpyl (n = 3), or 200 MBq of [18F]PSMA-1007 (n = 4). Uptake in tumor and tumor-to-background ratios, with contralateral nonaffected brain as background, were determined. In a subset of patients, PSMA expression levels from different regions in the tumor tissue samples (n = 40), determined using immunohistochemistry (n = 35) or RNA sequencing (n = 13), were correlated with tracer uptake on PET. Results: Moderate to high (SUVmax, 1.3-20.0) heterogeneous uptake was found in all tumors irrespective of the tracer type used. Uptake in nonaffected brain was low, resulting in high tumor-to-background ratios (6.1-359.0) calculated by dividing SUVmax of tumor by SUVmax of background. Immunohistochemistry showed variable PSMA expression on endothelial cells of tumor microvasculature, as well as on dispersed individual cells (of unknown origin), and granular staining of the neuropil. No correlation was found between in vivo uptake and PSMA expression levels (for immunohistochemistry, r = -0.173, P = 0.320; for RNA, r = -0.033, P = 0.915). Conclusion: Our results indicate the potential use of various PSMA-targeting tracers in GBM. However, we found no correlation between PSMA expression levels on immunohistochemistry and uptake intensity on PET. Whether this may be explained by methodologic reasons, such as the inability to measure functionally active PSMA with immunohistochemistry, tracer pharmacokinetics, or the contribution of a disturbed blood-brain barrier to tracer retention, should still be investigated.

Screening of predicted synergistic multi-target therapies in glioblastoma identifies new treatment strategies.

Background: IDH-wildtype glioblastoma (GBM) is a highly malignant primary brain tumor with a median survival of 15 months after standard of care, which highlights the need for improved therapy. Personalized combination therapy has shown to be successful in many other tumor types and could be beneficial for GBM patients. Methods: We performed the largest drug combination screen to date in GBM, using a high-throughput effort where we selected 90 drug combinations for their activity onto 25 patient-derived GBM cultures. 43 drug combinations were selected for interaction analysis based on their monotherapy efficacy and were tested in a short-term (3 days) as well as long-term (18 days) assay. Synergy was assessed using dose-equivalence and multiplicative survival metrics. Results: We observed a consistent synergistic interaction for 15 out of 43 drug combinations on patient-derived GBM cultures. From these combinations, 11 out of 15 drug combinations showed a longitudinal synergistic effect on GBM cultures. The highest synergies were observed in the drug combinations Lapatinib with Thapsigargin and Lapatinib with Obatoclax Mesylate, both targeting epidermal growth factor receptor and affecting the apoptosis pathway. To further elaborate on the apoptosis cascade, we investigated other, more clinically relevant, apoptosis inducers and observed a strong synergistic effect while combining Venetoclax (BCL targeting) and AZD5991 (MCL1 targeting). Conclusions: Overall, we have identified via a high-throughput drug screening several new treatment strategies for GBM. Moreover, an exceptionally strong synergistic interaction was discovered between kinase targeting and apoptosis induction which is suitable for further clinical evaluation as multi-targeted combination therapy.