Survival signalling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults and one of the most aggressive cancers in man. Despite technological advances in surgical management, combined regimens of radiotherapy with new generation chemotherapy, the median survival for these patients is 14.6 months. This is largely due to a highly deregulated tumour genome with opportunistic deletion of tumour suppressor genes, amplification and/or mutational hyper-activation of receptor tyrosine kinase receptors. The net result of these genetic changes is augmented survival pathways and systematic defects in the apoptosis signalling machinery. The only randomised, controlled phase II trial conducted targeting the epidermal growth factor receptor (EGFR) signalling with the small molecule inhibitor, erlotinib, has showed no therapeutic benefit. Survival signalling and apoptosis resistance in GBMs can be viewed as two sides of the same coin. Targeting increased survival is unlikely to be efficacious without at the same time targeting apoptosis resistance. We have critically reviewed the literature regarding survival and apoptosis signalling in GBM, and highlighted experimental, preclinical and recent clinical trials attempting to target these pathways. Combined therapies simultaneously targeting apoptosis and survival signalling defects might shift the balance from tumour growth stasis to cytotoxic therapeutic responses that might be associated with greater therapeutic benefits

Cancer stem cells as mediators of treatment resistance in brain tumors: Status and controversies

Malignant primary brain tumors are characterized by a short median survival and an almost 100% tumorrelated mortality. Despite the addition of new chemotherapy regimes, the overall survival has improved marginally, and radiotherapy is only transiently effective, illustrating the profound impact of treatment resistance on prognosis. Recent studies suggest that a small subpopulation of cancer stem cells (CSCs) has the capacity to repopulate tumors and drive malignant progression and mediate radio- and chemoresistance. This implies that future therapies should turn from the elimination of the rapidly dividing, but differentiated tumor cells, to specifically targeting the minority of tumor cells that repopulate the tumor. Although there exists some support for the CSC hypothesis, there remain many uncertainties regarding theoretical, technical, and interpretational aspects of the data supporting it. If correct, the CSC hypothesis could have profound implications for the way tumors are classified and treated. In this review of the literature, we provide original data and hypotheses supporting alternative explanations and outline some of the therapeutic implications that can be derived.publishedVersio

Therapeutic potential and challenges of natural killer cells in treatment of solid tumors

Natural killer (NK) cells are innate lymphoid cells that hold tremendous potential for effective immunotherapy for a broad range of cancers. Due to the mode of NK cell killing, requiring one-to-one target engagement and site-directed release of cytolytic granules, the therapeutic potential of NK cells has been most extensively explored in hematological malignancies. However, their ability to precisely kill antibody coated cells, cancer stem cells, and genotoxically altered cells, while maintaining tolerance to healthy cells makes them appealing therapeutic effectors for all cancer forms, including metastases. Due to their release of pro-inflammatory cytokines, NK cells may potently reverse the anti-inflammatory tumor microenvironment (TME) and augment adaptive immune responses by promoting differentiation, activation, and/or recruitment of accessory immune cells to sites of malignancy. Nevertheless, integrated and coordinated mechanisms of subversion of NK cell activity against the tumor and its microenvironment exist. Although our understanding of the receptor ligand interactions that regulate NK cell functionality has evolved remarkably, the diversity of ligands and receptors is complex, as is their mechanistic foundations in regulating NK cell function. In this article, we review the literature and highlight how the TME manipulates the NK cell phenotypes, genotypes, and tropism to evade tumor recognition and elimination. We discuss counter strategies that may be adopted to augment the efficacy of NK cell anti-tumor surveillance, the clinical trials that have been undertaken so far in solid malignancies, critically weighing the challenges and opportunities with this approach.publishedVersio

Combining NK cells and mAb9.2.27 to combat NG2-dependent and anti-inflammatory signals in glioblastoma

Glioblastoma is a deadly brain cancer with limited treatment options. Targeting chondroitin sulfate proteoglycan 4 (CSPG4, best known as NG2) with the monoclonal antibody mAb9.2.27 and activated natural killer (NK) cells abrogated the tumor growth and prolonged the survival of glioblastoma-bearing animals by favoring the establishment of a pro-inflammatory microenvironment. The combination of NK cells and mAb9.2.27 recruited ED1+CCR2low macrophages that stimulated ED1+ED2lowMHCIIhigh microglial cells to exert robust cytotoxicity. Our findings demonstrate the therapeutic potential of targeting salient tumor associated-antigens.publishedVersio

NK cells with KIR2DS2 immunogenotype have a functional activation advantage to efficiently kill glioblastoma and prolong animal survival

Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig–like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2+ NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2− immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2− NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell–mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2+ donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56+CD16+ NK cells from a KIR2DS2+ donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2− NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals.publishedVersio

A reproducible brain tumour model established from human glioblastoma biopsies

Background: Establishing clinically relevant animal models of glioblastoma multiforme (GBM) remains a challenge, and many commonly used cell line-based models do not recapitulate the invasive growth patterns of patient GBMs. Previously, we have reported the formation of highly invasive tumour xenografts in nude rats from human GBMs. However, implementing tumour models based on primary tissue requires that these models can be sufficiently standardised with consistently high take rates. Methods: In this work, we collected data on growth kinetics from a material of 29 biopsies xenografted in nude rats, and characterised this model with an emphasis on neuropathological and radiological features. Results: The tumour take rate for xenografted GBM biopsies were 96% and remained close to 100% at subsequent passages in vivo, whereas only one of four lower grade tumours engrafted. Average time from transplantation to the onset of symptoms was 125 days ± 11.5 SEM. Histologically, the primary xenografts recapitulated the invasive features of the parent tumours while endothelial cell proliferations and necrosis were mostly absent. After 4-5 in vivo passages, the tumours became more vascular with necrotic areas, but also appeared more circumscribed. MRI typically revealed changes related to tumour growth, several months prior to the onset of symptoms. Conclusions: In vivo passaging of patient GBM biopsies produced tumours representative of the patient tumours, with high take rates and a reproducible disease course. The model provides combinations of angiogenic and invasive phenotypes and represents a good alternative to in vitro propagated cell lines for dissecting mechanisms of brain tumour progression.publishedVersio

Gamma knife surgery as monotherapy with clinically relevant doses prolongs survival in a Human GBM Xenograft Model

Object. Gamma knife surgery (GKS) may be used for recurring glioblastomas (GBMs). However, patients have then usually undergone multimodal treatment, which makes it difficult to specifically validate GKS independent of established treatments. Thus, we developed an experimental brain tumor model to assess the efficacy and radiotoxicity associated with GKS. Methods. GBM xenografts were implanted intracerebrally in nude rats, and engraftment was confirmed with MRI. The rats were allocated to GKS, with margin doses of 12Gy or 18Gy, or to no treatment. Survival time was recorded, tumor sections were examined, and radiotoxicity was evaluated in a behavioral open field test. Results. In the first series, survival from the time of implantation was 96 days in treated rats and 72 days in controls ( < 0.001). In a second experiment, survival was 72 days in the treatment group versus 54 days in controls ( < 0.006). Polynuclear macrophages and fibrosis was seen in groups subjected to GKS. Untreated rats with GBM xenografts displayed less mobility than GKS-treated animals in the open field test 4 weeks after treatment ( = 0.04). Conclusion.GKS administered with clinically relevant doses prolongs survival in rats harboringGBMxenografts, and the associated toxicity is mild.publishedVersio

Targeting the NG2/CSPG4 Proteoglycan Retards Tumour Growth and Angiogenesis in Preclinical Models of GBM and Melanoma

Aberrant expression of the progenitor marker Neuron-glia 2 (NG2/CSPG4) or melanoma proteoglycan on cancer cells and angiogenic vasculature is associated with an aggressive disease course in several malignancies including glioblastoma multiforme (GBM) and melanoma. Thus, we investigated the mechanism of NG2 mediated malignant progression and its potential as a therapeutic target in clinically relevant GBM and melanoma animal models. Xenografting NG2 overexpressing GBM cell lines resulted in increased growth rate, angiogenesis and vascular permeability compared to control, NG2 negative tumours. The effect of abrogating NG2 function was investigated after intracerebral delivery of lentivirally encoded shRNAs targeting NG2 in patient GBM xenografts as well as in established subcutaneous A375 melanoma tumours. NG2 knockdown reduced melanoma proliferation and increased apoptosis and necrosis. Targeting NG2 in two heterogeneous GBM xenografts significantly reduced tumour growth and oedema levels, angiogenesis and normalised vascular function. Vascular normalisation resulted in increased tumour invasion and decreased apoptosis and necrosis. We conclude that NG2 promotes tumour progression by multiple mechanisms and represents an amenable target for cancer molecular therapy

Sequential bortezomib and temozolomide treatment promotes immunological responses in glioblastoma patients with positive clinical outcomes: A phase 1B study

Background Glioblastoma (GBM) is an aggressive malignant brain tumor where median survival is approximately 15 months after best available multimodal treatment. Recurrence is inevitable, largely due to O6 methylguanine DNA methyltransferase (MGMT) that renders the tumors resistant to temozolomide (TMZ). We hypothesized that pretreatment with bortezomib (BTZ) 48 hours prior to TMZ to deplete MGMT levels would be safe and tolerated by patients with recurrent GBM harboring unmethylated MGMT promoter. The secondary objective was to investigate whether 26S proteasome blockade may enhance differentiation of cytotoxic immune subsets to impact treatment responses measured by radiological criteria and clinical outcomes. Methods Ten patients received intravenous BTZ 1.3 mg/m2 on days 1, 4, and 7 during each 4th weekly TMZ‐chemotherapy starting on day 3 and escalated from 150 mg/m2 per oral 5 days/wk via 175 to 200 mg/m2 in cycles 1, 2, and 3, respectively. Adverse events and quality of life were evaluated by CTCAE and EQ‐5D‐5L questionnaire, and immunological biomarkers evaluated by flow cytometry and Luminex enzyme‐linked immunosorbent assay. Results Sequential BTZ + TMZ therapy was safe and well tolerated. Pain and performance of daily activities had greatest impact on patients' self‐reported quality of life and were inversely correlated with Karnofsky performance status. Patients segregated a priori into three groups, where group 1 displayed stable clinical symptoms and/or slower magnetic resonance imaging radiological progression, expanded CD4+ effector T‐cells that attenuated cytotoxic T‐lymphocyte associated protein‐4 and PD‐1 expression and secreted interferon γ and tumor necrosis factor α in situ and ex vivo upon stimulation with PMA/ionomycin. In contrast, rapidly progressing group 2 patients exhibited tolerised T‐cell phenotypes characterized by fourfold to sixfold higher interleukin 4 (IL‐4) and IL‐10 Th‐2 cytokines after BTZ + TMZ treatment, where group 3 patients exhibited intermediate clinical/radiological responses. Conclusion Sequential BTZ + TMZ treatment is safe and promotes Th1‐driven immunological responses in selected patients with improved clinical outcomes (Clinicaltrial.gov (NCT03643549)).publishedVersio

Expression of the progenitor marker NG2/CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O6-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling