The BCL-2 inhibitor obatoclax overcomes resistance to histone deacetylase inhibitors SAHA and LBH589 as radiosensitizers in patient-derived glioblastoma stem-like cells

Glioblastoma has shown resistance to histone deacetylase inhibitors (HDACi) as radiosensitizers in cultures with Bcl-XL over-expression. We study the efficacy of SAHA/RTx and LBH589/RTx when manipulating Bcl-2 family proteins using the Bcl-2 inhibitor Obatoclax in patient-derived glioblastoma stem-like cell (GSC) cultures. GSC cultures in general have a deletion in phosphatase and tensin homolog (PTEN). Synergy was determined by the Chou Talalay method. The effects on apoptosis and autophagy were studied by measuring caspase-3/7, Bcl-XL, Mcl-1 and LC3BI/II proteins. The relation between treatment response and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, recurrence and gene expression levels of the tumors were studied. Obatoclax synergized with SAHA and LBH589 and sensitized cells to HDACi/RTx. Over 50% of GSC cultures were responsive to Obatoclax with either single agent. Combined with HDACi/RTx treatment, Obatoclax increased caspase-3/7 and inhibited Bcl-2 family proteins Bcl-XL and Mcl-1 more effectively than other treatments. Genes predictive for treatment response were identified, including the F-box/WD repeat-containing protein-7, which was previously related to Bcl-2 inhibition and HDACi sensitivity. We emphasize the functional relation between Bcl-2 proteins and radiosensitization by HDACi and provide a target for increasing responsiveness in glioblastoma by using the Bcl-2 inhibitor Obatoclax

BMP4 and Temozolomide Synergize in the Majority of Patient-Derived Glioblastoma Cultures

One of the main causes of poor prognoses in patient with glioblastoma (GBM) is drug resistance to current standard treatment, which includes chemoradiation and adjuvant temozolomide (TMZ). In addition, the concept of cancer stem cells provides new insights into therapy resistance and management also in GBM and glioblastoma stem cell-like cells (GSCs), which might contribute to therapy resistance. Bone morphogenetic protein-4 (BMP4) stimulates astroglial differentiation of GSCs and thereby reduces their self-renewal capacity. Exposure of GSCs to BMP4 may also sensitize these cells to TMZ. A recent phase I trial has shown that local delivery of BMP4 is safe, but a large variation in survival is seen in these treated patients and in features of their cultured tumors. We wanted to combine TMZ and BMP4 (TMZ + BMP4) therapy and assess the inter-tumoral variability in response to TMZ + BMP4 in patient-derived GBM cultures. A phase II trial could then benefit a larger group of patients than those treated with BMP4 only. We first show that simultaneous treatment with TMZ + BMP4 is more effective than sequential treatment. Second, when applying our optimized treatment protocol, 70% of a total of 20 GBM cultures displayed TMZ + BMP4 synergy. This combination induces cellular apoptosis and does not inhibit cell proliferation. Comparative bulk RNA-sequencing indicates that treatment with TMZ + BMP4 eventually results in decreased MAPK signaling, in line with previous evidence that increased MAPK signaling is associated with resistance to TMZ. Based on these results, we advocate further clinical trial research to test patient benefit and validate pathophysiological hypothesis.</p

The heterogeneous sensitivity of pediatric brain tumors to different oncolytic viruses is predicted by unique gene expression profiles

Despite decades of research, the prognosis of high-grade pediatric brain tumors (PBTs) remains dismal; however, recent cases of favorable clinical responses were documented in clinical trials using oncolytic viruses (OVs). In the current study, we employed four different species of OVs: adenovirus Delta24-RGD, herpes simplex virus rQNestin34.5v1, reovirus R124, and the non-virulent Newcastle disease virus rNDV-F0-GFP against three entities of PBTs (high-grade gliomas, atypical teratoid/rhabdoid tumors, and ependymomas) to determine their in vitro efficacy. These four OVs were screened on 14 patient-derived PBT cell cultures and the degree of oncolysis was assessed using an ATP-based assay. Subsequently, the observed viral efficacies were correlated to whole transcriptome data and Gene Ontology analysis was performed. Although no significant tumor type-specific OV efficacy was observed, the analysis revealed the intrinsic biological processes that associated with OV efficacy. The predictive power of the identified expression profiles was further validated in vitro by screening additional PBTs. In summary, our results demonstrate OV susceptibility of multiple patient-derived PBT entities and the ability to predict in vitro responses to OVs using unique expression profiles. Such profiles may hold promise for future OV preselection with effective oncolytic potency in a specific tumor, therewith potentially improving OV responses.</p

Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells’ molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as “adaptive” (ADA) or “non-adaptive” (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor’s ability to survive. Depending on the tumor’s adaptability potential, subpopulations with acquired resistance mechanisms may arise.</p

Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers

Background: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). Methods: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. Results: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. Conclusion:GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.</p

Advancements, Challenges, and Future Directions in Tackling Glioblastoma Resistance to Small Kinase Inhibitors

Despite clinical intervention, glioblastoma (GBM) remains the deadliest brain tumor in adults. Its incurability is partly related to the establishment of drug resistance, both to standard and novel treatments. In fact, even though small kinase inhibitors have changed the standard clinical practice for several solid cancers, in GBM, they did not fulfill this promise. Drug resistance is thought to arise from the heterogeneity of GBM, which leads the development of several different mechanisms. A better understanding of the evolution and characteristics of drug resistance is of utmost importance to improve the current clinical practice. Therefore, the development of clinically relevant preclinical in vitro models which allow careful dissection of these processes is crucial to gain insights that can be translated to improved therapeutic approaches. In this review, we first discuss the heterogeneity of GBM, which is reflected in the development of several resistance mechanisms. In particular, we address the potential role of drug resistance mechanisms in the failure of small kinase inhibitors in clinical trials. Finally, we discuss strategies to overcome therapy resistance, particularly focusing on the importance of developing in vitro models, and the possible approaches that could be applied to the clinic to manage drug resistance

The multifaceted role of macrophages in oncolytic virotherapy

One of the cancer hallmarks is immune evasion mediated by the tumour microenvironment (TME). Oncolytic virotherapy is a form of immunotherapy based on the application of oncolytic viruses (OVs) that selectively replicate in and induce the death of tumour cells. Virotherapy confers reciprocal interaction with the host’s immune system. The aim of this review is to explore the role of macrophage-mediated responses in oncolytic virotherapy efficacy. The approach was to study current scientific literature in this field in order to give a comprehensive overview of the interactions of OVs and macrophages and their effects on the TME. The innate immune system has a central influence on the TME; tumour-associated macrophages (TAMs) generally have immunosuppressive, tumour-supportive properties. In the context of oncolytic virotherapy, macrophages were initially thought to predominantly contribute to anti-viral responses, impeding viral spread. However, macrophages have now also been found to mediate transport of OV particles and, after TME infiltration, to be subjected to a phenotypic shift that renders them pro-inflammatory and tumour-suppressive. These TAMs can present tumour antigens leading to a systemic, durable, adaptive anti-tumour immune response. After phagocytosis, they can recirculate carrying tissue-derived proteins, which potentially enables the monitoring of OV replication in the TME. Their role in therapeutic efficacy is therefore multifaceted, but based on research applying relevant, immunocompetent tumour models, macrophages are considered to have a central function in anti-cancer activity. These novel insights hold important clinical implications. When optimised, oncolytic virotherapy, mediating multifactorial inhibition of cancer immune evasion, could contribute to improved patient survival

The multifaceted role of macrophages in oncolytic virotherapy

One of the cancer hallmarks is immune evasion mediated by the tumour microenvironment (TME). Oncolytic virotherapy is a form of immunotherapy based on the application of oncolytic viruses (OVs) that selectively replicate in and induce the death of tumour cells. Virotherapy confers reciprocal interaction with the host’s immune system. The aim of this review is to explore the role of macrophage-mediated responses in oncolytic virotherapy efficacy. The approach was to study current scientific literature in this field in order to give a comprehensive overview of the interactions of OVs and macrophages and their effects on the TME. The innate immune system has a central influence on the TME; tumour-associated macrophages (TAMs) generally have immunosuppressive, tumour-supportive properties. In the context of oncolytic virotherapy, macrophages were initially thought to predominantly contribute to anti-viral responses, impeding viral spread. However, macrophages have now also been found to mediate transport of OV particles and, after TME infiltration, to be subjected to a phenotypic shift that renders them pro-inflammatory and tumour-suppressive. These TAMs can present tumour antigens l

Drug Repurposing, a Fast-Track Approach to Develop Effective Treatments for Glioblastoma

Glioblastoma (GBM) remains one of the most difficult tumors to treat. The mean overall survival rate of 15 months and the 5-year survival rate of 5% have not significantly changed for almost 2 decades. Despite progress in understanding the pathophysiology of the disease, no new effective treatments to combine with radiation therapy after surgical tumor debulking have become available since the introduction of temozolomide in 1999. One of the main reasons for this is the scarcity of compounds that cross the blood–brain barrier (BBB) and reach the brain tumor tissue in therapeutically effective concentrations. In this review, we focus on the role of the BBB and its importance in developing brain tumor treatments. Moreover, we discuss drug repurposing, a drug discovery approach to identify potential effective candidates with optimal pharmacokinetic profiles for central nervous system (CNS) penetration and that allows rapid implementation in clinical trials. Additionally, we provide an overview of repurposed candidate drug currently being investigated in GBM at the preclinical and clinical levels. Finally, we highlight the importance of phase 0 trials to confirm tumor drug exposure and we discuss emerging drug delivery technologies as an alternative route to maximize therapeutic efficacy of repurposed candidate drug

Transcriptional cdk inhibitors cyc065 and thz1 induce apoptosis in glioma stem cells derived from recurrent gbm

Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment re-sistance, temozolomide (TMZ) chemotherapy and radiotherapy, in glioblastoma (GBM), the most aggressive adult brain tumour. A major contributor to the uncontrolled tumour cell proliferation in GBM is the hyper activation of cyclin‐dependent kinases (CDKs). Due to resistance to standard of care, GBMs relapse in almost all patients. Targeting GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. TCGA‐GBM data analysis has shown that the GSC markers, CD133 and CD44 were significantly upregulated in GBM patient tumours compared to non‐tumour tissue. CD133 and CD44 stem cell markers were also expressed in gliomaspheres derived from recurrent GBM tumours. Light Sheet Florescence Microscopy (LSFM) further revealed heterogeneous expression of these GSC markers in glio-maspheres. Gliomaspheres from recurrent tumours were highly sensitive to transcriptional CDK inhibitors, CYC065 and THZ1 and underwent apoptosis while being resistant to TMZ. Apoptotic cell death in GSC subpopulations and non‐stem tumour cells resulted in sphere disruption. Collec-tively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation