We describe a reproducible 3D migration assay to model the migratory and invasive potential of patient-derived glioblastoma gliomaspheres from newly diagnosed and recurrent tumors under clinically relevant hypoxic conditions. Uniform sized gliomaspheres are transferred onto a thin layer of extracellular matrix and co-cultured with magnetic iron oxide nanoparticles, which enable their centralized localization within culture wells via placement onto a magnetic plate holder. The inclusion of magnetic iron oxide nanoparticles on seeding facilitates precise, localized imaging of individual patient derived gliomaspheres via a gentle magnetic force and optimizes automated image processing pipelines by reducing positional variability. This assay supports detailed study of glioblastoma migratory behavior in a physiologically relevant microenvironment and allows direct comparison of invasive potential and migratory behavior between newly diagnosed and recurrent patient derived gliomaspheres. The method is compatible with live cell imaging and multiplexed analysis, offering a scalable platform for preclinical investigation of glioblastoma migration, invasive potential, and therapeutic response.

Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS).Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed.Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS.Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.

Medulloblastoma is the most common malignant paediatric brain tumour, representing 20% of all paediatric intercranial tumours. Current aggressive treatment protocols and the use of radiation therapy in particular are associated with high levels of toxicity and significant adverse effects, and long-term sequelae can be severe. Therefore, improving chemotherapy efficacy could reduce the current reliance on radiation therapy. Here, we demonstrated that systems-level analysis of basal apoptosis protein expression and their signalling interactions can differentiate between medulloblastoma cell lines that undergo apoptosis in response to chemotherapy, and those that do not. Combining computational predictions with experimental BH3 profiling, we identified a therapeutically-exploitable dependence of medulloblastoma cells on BCL-XL, and experimentally validated that BCL-XL targeting, and not targeting of BCL-2 or MCL-1, can potentiate cisplatin-induced cytotoxicity in medulloblastoma cell lines with low sensitivity to cisplatin treatment. Finally, we identified MCL-1 as an anti-apoptotic mediator whose targeting is required for BCL-XL inhibitor-induced apoptosis. Collectively, our study identifies that BCL-XL and MCL-1 are the key anti-apoptotic proteins in medulloblastoma, which mediate distinct protective roles. While BCL-XL has a first-line role in protecting cells from apoptosis basally, MCL-1 represents a second line of defence that compensates for BCL-XL upon its inhibition. We provide rationale for the further evaluation of BCL-XL and MCL-1 inhibitors in the treatment of medulloblastoma, and together with current efforts to improve the cancer-specificity of BCL-2 family inhibitors, these novel treatment strategies have the potential to improve the future clinical management of medulloblastoma.

Recent studies have demontrated that immune checkpoint receptors are expressed on the surface of oesophageal adenocarcinoma (OAC) cells and might confer a survival advantage. This study explores the role of PD-1 and TIGIT signalling in OAC cells in either promoting or inhibiting the survival of OAC cells under characteristic features of the tumour microenvironment including nutrient-deprivation and hypoxia. PD-1 and TIGIT are expressed in normal and pre-malignant oesophageal epithelial cells and this expression significantly decreases along the normal- Barrett's Oesophagus- OAC disease sequence. However, glucose-deprivation and hypoxia significantly upregulated PD-1 and TIGIT on the surface of OAC cells in vitro. PD-1 blockade decreased OAC cell proliferation under normoxia but enhanced proliferation and decreased cell death in OAC cells under hypoxia and glucose-deprivation. TIGIT blockade decreased proliferation and induced OAC cell death, an effect that was maintained under nutrient-deprivation and hypoxia. Basal respiration and glycolytic reserve were enhanced and GLUT1 was upregulated on the surface of a subpopulation of OAC cells following PD-1 blockade. In contrast, TIGIT blockade enhanced a glycolytic phenotype in OAC cells, yet decreased other metabolic parameters including oxidative phosphorylation and basal respiration. Interestingly, inhibition of oxidative phosphorylation significantly upregulated TIGIT expression and inhibition of oxidative phosphorylation and glycolysis significantly decreased PD-1 on the surface of a subpopulation of OAC cells in vitro. These findings suggest an immune-independent mechanism for PD-1 inhibitor resistance in hypoxic tumours and suggest that TIGIT might be a more effective therapeutic target in OAC compared with PD-1 for treating hypoxic tumours.

New, more effective therapeutics are required for the treatment of paediatric cancers. Current treatment protocols of cytotoxic treatments including chemotherapy trigger cancer-cell death by engaging the apoptosis pathway, and chemotherapy efficacy is frequently impeded by apoptosis dysregulation. Apoptosis dysregulation, through genetic or epigenetic mechanisms, is a feature of many cancer types, and contributes to reduced treatment response, disease progression and ultimately treatment resistance. Novel approaches are required to overcome dysregulated apoptosis signalling, increase the efficacy of cancer treatment and improve patient outcomes. Here, we provide an insight into current knowledge of how the apoptosis pathway is dysregulated in paediatric nervous system tumours, with a focus on TRAIL receptors, the BCL-2 proteins and the IAP family, and highlight preclinical evidence demonstrating that pharmacological manipulation of the apoptosis pathway can restore apoptosis signalling and sensitise cancer cells to treatment. Finally, we discuss the potential clinical implications of these findings.

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.

Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment resistance, 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 gliomaspheres. 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. Collectively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation.

Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood–brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases.

Background Immune checkpoint inhibitors (ICIs) reinvigorate anti-tumour immunity in oesophageal adenocarcinoma (OAC). However, emerging studies have identified novel immune-independent functions for immune checkpoints (ICs) in other solid tumour-types, whereby IC-intrinsic signalling in gastric cancer cells confers chemoresistance. This study explores immune-independent functions of ICs in OAC and if therapeutic blockade may enhance chemotherapy toxicity. Materials and Methods OAC cells were screened in vitro and in vivo (n=14 OAC human tissue biopsies) for a range of ICs (PD-1,TIGIT,TIM-3,LAG-3,A2aR,PD-L1,PD-L2,CD160) by flow cytometry. The phenotype of OAC cells expressing ICs was also assessed for features of stemness (ALDH, CD54), senescence (β-galactosidase) and invasiveness (vimentin) in the absence and presence of chemotherapy by flow cytometry. OAC cells were also treated with chemotherapy in the absence and presence of a MEK inhibitor to determine if MEK signalling regulated IC expression. Importantly, the effect of ICIs on the hallmarks of cancer in OAC cells was assessed which included: OAC cell viability (CCK-8 assay and western blot to assess Bcl-xL and Bcl-2 levels), proliferation (BrdU assay and ki67 expression by intracellular flow cytometry), chemo-sensitivity (annexin-V propidium iodide assay and cell cycle analysis by flow cytometry and expression of chemotherapy efflux and influx pumps by western blot: ATP7a, ATP7b, CTR1 and ABCB9), metabolism (seahorse), invasiveness and stemness characteristics (vimentin and aldefluor assay, respectively by flow cytometry) and DNA repair (γH2ax by flow cytometry to assess levels of DNA repair and the expression of DNA repair genes were quantified by qPCR: MLH1, SMUG1, PARP1, MMS19) was assessed in OAC cells. Results A subpopulation of stem-like, senescent and vimentin+ OAC cells were enriched for ICs, which was enhanced by FLOT and CROSS chemotherapy regimens. IC expression increased on the surface of OAC cells 48h post-chemotherapy treatment and was sustained up to 3 weeks post-treatment in vitro. Inhibition of pro-survivla MEK signalling reduced chemotherapy-induced upregulation of ICs. Blockade of PD-1, TIGIT, A2aR, TIM-3 and PD-L1 decreased proliferation, DNA repair, induced apoptosis and enhanced toxicity of FLOT in OAC cells. Blockade of TIGIT decreased pro-survival Bcl-xL factor, induced cell death and promoted a more glycolytic phenotype in OAC cells. Conclusions Several novel ICs have been identified as potential targets to enhance chemotherpay efficacy in OAC. Upregulation of ICs on OAC cells following chemotherapy may represent potential mechanisms of chemo-immune resistance for stem-like, senescent and vimentin+ aggressive cancer cell clones. Combining ICIs with chemotherapy may synergise with chemotherpay in OAC patients via immune-independent mechansims and boost response rates to current standards of care. Further studies are warranted through clincal trials to further establish synergistic ICI-chemotherapy combinations in OAC. Disclosure Information M. davern: None. C. Buckley: None. C. Fitzgerald: None. A.B. Heeran: None. N.E. Donlon: None. J. McGrath: None. R. O' Brien: None. F. O' Connell: None. B. Murphy: None. N. Lynam-Lennon: None. J.V. Reynolds: None. S.G. Maher: None. A.D. Sheppard: None. A. Bhardwaj: None. A. Bhardwaj: None. C. Butler: None. N. Ravi: None. J. Lysaght: None.

Background: Immune checkpoint inhibitors (ICIs) reinvigorate anti-tumour immunity in oesophageal adenocarcinoma (OAC). However, emerging studies have identified novel immune-independent functions for immune checkpoints (ICs) in other solid tumour-types, whereby IC signalling in gastric cancer cells confers chemoresistance. This study explores immune-independent functions of ICs in OAC and if therapeutic blockade may enhance treatment efficacy. Methods: OAC cells were screened in vitro and ex vivo for a range of ICs (PD-1, TIGIT, TIM-3, LAG-3, A2aR, PDL1, PD-L2, CD160) by flow cytometry. The phenotype of OAC cells expressing ICs was also assessed for features of stemness (ALDH, CD54), senescence (β-galactosidase) and invasiveness (vimentin) in the absence and presence of chemotherapy by flow cytometry. Importantly, the effect of ICIs on viability (CCK-8 assay and western blot to assess Bcl-xL), proliferation (BrdU assay), chemo-sensitivity (annexin-V propidium iodide assay), metabolism (seahorse), invasiveness and stemness characteristics (flow cytometry) was assessed in OAC cells. Results: A subpopulation of stem-like, senescent and vimentin+ cells were enriched for ICs, which was enhanced by FLOT and CROSS. Blockade of PD-1, TIGIT, A2aR, TIM-3 and PD-L1 decreased proliferation, induced apoptosis and enhanced toxicity of FLOT in OAC cells. Blockade of TIGIT decreased pro-survival Bcl-xL factor, induced cell death and promoted a more glycolytic phenotype in OAC cells. Conclusions: Several novel ICs have been identified as potential targets to enhance chemotherapy efficacy in OAC. Upregulation of ICs on OAC cells following chemotherapy may represent potential mechanisms of chemo-immune resistance for stem-like, senescent and vimentin+ aggressive cancer cell clones. Combination ICIs may be required to enhance efficacy of chemotherapy in OAC patients and warrants further investigation.

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation of CDKs are a feature of many tumours including glioblastoma (GBM), the most common and aggressive primary brain tumour. Patient resistance to the current standard of care, temozolomide and radiotherapy, is common and highlights the need to discover more effective treatment strategies. Additionally, glioma stem cells (GSCs), tumour initiating cells contribute to treatment resistance in GBM. Targeting GBM tumour cells and GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. The therapeutic efficacy of two CDK inhibitors (CKIs) was tested in a panel of ten low-passage GBM patient-derived gliomasphere cultures and semi-in vivo chick embryo xenograft models. Specifically, transcriptional inhibitors targeting CDK9/2 (CYC065) and CDK7 (THZ1) were used. Mechanism of cell death was examined following CKI treatment. Additionally, dependence on anti-apoptotic proteins was studied using genetic depletion and BH3 profiling. Fluorescence activated cell sorting (FACS) and light-sheet fluorescence microscopy (LSFM) were employed to study stem cell populations in recurrent GBM and evaluation of CKI efficacy against GSCs. We here demonstrate that CYC065 and THZ1 treatments cause loss of cell viability and induce caspase-dependent apoptosis in primary and recurrent patient-derived gliomaspheres while sparing primary cortical neurons. Importantly, apoptosis responses manifested across a range of time points that significantly correlated with the cell doubling time. Mechanistically, CYC065 and THZ1 downregulate the anti-apoptotic protein Mcl-1, which suffices to sensitise gliomasphere cultures to treatment-induced apoptosis in a Bim-dependent manner. Additionally, high expression levels of chemo- and radio-resistant GSCs were found in recurrent gliomaspheres. CKIs induced apoptosis in isolated CD133 and CD44 biomarker-positive cells while TMZ was ineffective, highlighting the potential of these drugs to overcome resistance to conventional chemotherapy. Additionally, using LSFM we shown that CD133, CD44 GSC biomarker-negative cells convert into GSC biomarker-positive cells and contribute to the enrichment in GSCs in recurrent GBM which could potentially explain the TMZ ineffectiveness. Finally, CKIs reduced proliferation and promoted apoptosis in chick embryo xenograft models of primary and recurrent GBM. Collectively, these data demonstrate that CYC065 and THZ1 display high anti-cancer activity in primary and recurrent GBM and provide scientific rationale for the further development of CDK inhibitors to potentiate their clinical utilization in the future.

Cyclin-dependent kinases (CDKs) are important regulatory enzymes in the normal physiological processes that drive cell-cycle transitions and regulate transcription. Virtually all cancers harbour genomic alterations that lead to the constitutive activation of CDKs, resulting in the proliferation of cancer cells. CDK inhibitors (CKIs) are currently in clinical use for the treatment of breast cancer, combined with endocrine therapy. In this review, we describe the potential of CKIs for the treatment of cancer with specific focus on glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. Despite intense effort to combat GBM with surgery, radiation and temozolomide chemotherapy, the median survival for patients is 15 months and the majority of patients experience disease recurrence within 6-8 months of treatment onset. Novel therapeutic approaches are urgently needed for both newly diagnosed and recurrent GBM patients. In this review, we summarise the current preclinical and clinical findings emphasising that CKIs could represent an exciting novel approach for GBM treatment.

Glioblastoma represents the most common primary malignancy of the central nervous system in adults and remains a largely incurable disease. The elucidation of disease subtypes based on mutational profiling, gene expression and DNA methylation has so far failed to translate into improved clinical outcomes. However, new knowledge emerging from the subtyping effort in the IDH-wild-type setting may provide directions for future precision therapies. Here, we review recent learnings in the field, and further consider how tumour microenvironment differences across subtypes may reveal novel contexts of vulnerability. We discuss recent treatment approaches and ongoing trials in the IDH-wild-type glioblastoma setting, and propose an integrated discovery stratagem incorporating multi-omics, single-cell technologies and computational approaches.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumour with a median overall survival of 15 months. To treat GBM, patients currently undergo a surgical resection followed by exposure to radiotherapy and concurrent and adjuvant temozolomide (TMZ) chemotherapy. However, this protocol often leads to treatment failure, with drug resistance being the main reason behind this. To date, many studies highlight the role of O-6-methylguanine-DNA methyltransferase (MGMT) in conferring drug resistance. The mechanism through which MGMT confers resistance is not well studied—particularly in terms of computational models. With only a few reasonable biological assumptions, we were able to show that even a minimal model of MGMT expression could robustly explain TMZ-mediated drug resistance. In particular, we showed that for a wide range of parameter values constrained by novel cell growth and viability assays, a model accounting for only stochastic gene expression of MGMT coupled with cell growth, division, partitioning and death was able to exhibit phenotypic selection of GBM cells expressing MGMT in response to TMZ. Furthermore, we found this selection allowed the cells to pass their acquired phenotypic resistance onto daughter cells in a stable manner (as long as TMZ is provided). This suggests that stochastic gene expression alone is enough to explain the development of chemotherapeutic resistance.

The synthesis and characterisation of a novel Pt(IV) monocarboxy precursor complex c,c,t-[Pt(DACH)(Ox)(OEt)(Suc)], which can be employed to synthesise mono functionalised Pt(IV) oxaliplatin-based complexes is reported. Reaction of c,c,t-[Pt(DACH)(Ox)(OEt)(Suc)] with a tumour penetrating peptide, TPP, afforded the corresponding novel monofunctionalised Pt(IV) peptide conjugate c,c,t-[Pt(DACH)(Ox)(OEt)(SucTPP)] in high purity. Conjugation of TPP to c,c,t-[Pt(DACH)(Ox)(OEt)(Suc)] was demonstrated to ameliorate its inherent cytotoxicity fifteen fold in the Pt sensitive HCT116 colorectal cancer cell line and two fold in the relatively resistant HT29 colorectal cancer cell line.

Background The evasion of apoptosis is a hallmark of cancer. Understanding this process holistically and overcoming apoptosis resistance is a goal of many research teams in order to develop better treatment options for cancer patients. Efforts are also ongoing to personalize the treatment of patients. Strategies to confirm the therapeutic efficacy of current treatments or indeed to identify potential novel additional options would be extremely beneficial to both clinicians and patients. In the past few years, system medicine approaches have been developed that model the biochemical pathways of apoptosis. These systems tools incorporate and analyse the complex biological networks involved. For their successful integration into clinical practice, it is mandatory to integrate systems approaches with routine clinical and histopathological practice to deliver personalized care for patients. Results We review here the development of system medicine approaches that model apoptosis for the treatment of cancer with a specific emphasis on the aggressive brain cancer, glioblastoma. Conclusions We discuss the current understanding in the field and present new approaches that highlight the potential of system medicine approaches to influence how glioblastoma is diagnosed and treated in the future.

GANT61-D is an important hedgehog pathway inhibitor and an interesting ligand candidate for metal coordination. The first examples of metal complexes of the potent hedgehog pathway inhibitor GANT61-D are described. The reaction of Ni(II), Pd(II), and Pt(II) precursors with the hedgehog pathway inhibitor GANT61-D gave [NiII(GANT61-D)(OH2)3(μ2-SO4)(μ3-SO4)] (1), [PdII(Cl)(GANT61-D)]Cl (2), [PtII(Cl)(GANT61-D)]Cl, and [PtII(CBDCA-2H)(GANT61-D)]. X-ray crystal structure analysis revealed that GANT61-D is a versatile N-donor ligand that can act as a bidentate ligand via the diaminopropane (DAP) N atoms or a tridentate ligand via the DAP N atoms and one dimethylaniline N atom. Protonation constants of the GANT61-D ligand in water and in a 60:40 (w/w) dimethyl sulfoxide-water solvent mixture were determined. Potentiometric and spectroscopic data on the NiII(GANT61-D) system indicate the formation of octahedral 1:1 species with medium stability in solution. 1 and 2 exhibited noteworthy in vitro cytotoxicity against medulloblastoma cancer cells.

Glioblastoma (GBM) is the most common primary brain tumor with no available cure. As previously described, seliciclib, a first-generation cyclin-dependent kinase (CDK) inhibitor, down-regulates the anti-apoptotic protein, Mcl-1, in GBM, thereby sensitizing GBM cells to the apoptosis-inducing effects of the death receptor ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we have assessed the efficacy of seliciclib when delivered in combination with the antibody against human death receptor 5, drozitumab, in clinically relevant patient-derived xenograft (PDX) models of GBM. A reduction in viability and significant levels of apoptosis were observed in vitro in human GBM neurospheres following treatment with seliciclib plus drozitumab. While the co-treatment strategy induced a similar effect in PDX models, the dosing regimen required to observe seliciclib-targeted responses in the brain, resulted in lethal toxicity in 45% of animals. Additional studies showed that the second-generation CDK inhibitor, CYC065, with improved potency in comparison to seliciclib, induced a significant decrease in the size of human GBM neurospheres in vitro and was well tolerated in vivo, upon administration at clinically relevant doses. This study highlights the continued need for robust pre-clinical assessment of promising treatment approaches using clinically relevant models.