BCL6 is a context-dependent mediator of the glioblastoma response to irradiation therapy.

Glioblastoma is a rapidly fatal brain cancer that does not respond to therapy. Previous research showed that the transcriptional repressor protein BCL6 is upregulated by chemo and radiotherapy in glioblastoma, and inhibition of BCL6 enhances the effectiveness of these therapies. Therefore, BCL6 is a promising target to improve the efficacy of current glioblastoma treatment. BCL6 acts as a transcriptional repressor in germinal centre B cells and as an oncogene in lymphoma and other cancers. However, in glioblastoma, BCL6 induced by therapy may not be able to repress transcription. Using a BCL6 inhibitor, the whole proteome response to irradiation was compared with and without BCL6 activity. Acute high dose irradiation caused BCL6 to switch from repressing the DNA damage response to promoting stress response signalling. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) enabled comparison of BCL6 partner proteins between untreated and irradiated glioblastoma cells. BCL6 was associated with transcriptional coregulators in untreated glioblastoma including the known partner NCOR2. However, this association was lost in response to acute irradiation, where BCL6 unexpectedly associated with synaptic and plasma membrane proteins. These results reveal the activity of BCL6 under therapy-induced stress is context-dependent, and potentially altered by the intensity of that stress.

Aryl-functionalised α,α'-Trehalose 6,6'-Glycolipid Induces Mincle-independent Pyroptotic Cell Death.

α,α'-Trehalose 6,6'-glycolipids have long been known for their immunostimulatory properties. The adjuvanticity of α,α'-trehalose 6,6'-glycolipids is mediated by signalling through the macrophage inducible C-type lectin (Mincle) and the induction of an inflammatory response. Herein, we present an aryl-functionalised trehalose glycolipid, AF-2, that leads to the release of cytokines and chemokines, including IL-6, MIP-2 and TNF-α, in a Mincle-dependent manner. Furthermore, plate-coated AF-2 also leads to the Mincle-independent production of IL-1ß, which is unprecedented for this class of glycolipid. Upon investigation into the mode of action of plate-coated AF-2, it was observed that the treatment of WT and Mincle-/- bone marrow derived macrophages (BMDM), murine RAW264.7 cells, and human monocytes with AF-2 led to lytic cell death, as evidenced using Sytox Green and lactate dehydrogenase assays, and confocal and scanning electron microscopy. The requirement for functional Gasdermin D and Caspase-1 for IL-1ß production and cell death by AF-2 confirmed pyroptosis as the mode of action of AF-2. The inhibition of NLRP3 and K+ efflux reduced AF-2 mediated IL-1ß production and cell death, and allowed us to conclude that AF-2 leads to Capase-1 dependent NLRP3 inflammasome-mediated cell death. The unique mode of action of plate-coated AF-2 was surprising and highlights how the physical presentation of Mincle ligands can lead to dramatically different immunological outcomes.

The Big Picture of Glioblastoma Malignancy: A Meta-Analysis of Glioblastoma Proteomics to Identify Altered Biological Pathways.

Glioblastoma is a highly malignant cancer with no effective treatment. It is vital to elucidate the mechanisms which drive glioblastoma in order to identify therapeutic targets. The differences in protein expression between glioblastoma, grade I-III glioma, and normal brain tissue reflect the functional alterations driving malignancy. However, proteomic analysis of glioblastoma has been hampered by the heterogeneity of glioblastoma and the variety of methodology used in its study. To reduce these inconsistencies, we performed a meta-analysis of the literature published since 2015, including 14 datasets from eight papers comparing the whole proteome of glioblastoma to normal brain or grade I-III glioma. We found that 154 proteins were commonly upregulated and 116 proteins were commonly downregulated in glioblastoma compared to normal brain. Meanwhile, 240 proteins were commonly upregulated and 125 proteins were commonly downregulated in glioblastoma compared to grade I-III glioma. Functional enrichment analysis revealed upregulation of proteins involved in mRNA splicing and the immune system and downregulation of proteins involved in synaptic signaling and glucose and glutamine metabolism. The identification of these altered biological pathways provides a basis for deeper investigation in the pursuit of an effective treatment for glioblastoma.

The oncogene BCL6 is up-regulated in glioblastoma in response to DNA damage, and drives survival after therapy.

The prognosis for people with the high-grade brain tumor glioblastoma is very poor, due largely to low cell death in response to genotoxic therapy. The transcription factor BCL6, a protein that normally suppresses the DNA damage response during immune cell maturation, and a known driver of B-cell lymphoma, was shown to mediate the survival of glioblastoma cells. Expression was observed in glioblastoma tumor specimens and cell lines. When BCL6 expression or activity was reduced in these lines, increased apoptosis and a profound loss of proliferation was observed, consistent with gene expression signatures suggestive of anti-apoptotic and pro-survival signaling role for BCL6 in glioblastoma. Further, treatment with the standard therapies for glioblastoma-ionizing radiation and temozolomide-both induced BCL6 expression in vitro, and an in vivo orthotopic animal model of glioblastoma. Importantly, inhibition of BCL6 in combination with genotoxic therapies enhanced the therapeutic effect. Together these data demonstrate that BCL6 is an active transcription factor in glioblastoma, that it drives survival of cells, and that it increased with DNA damage, which increased the survival rate of therapy-treated cells. This makes BCL6 an excellent therapeutic target in glioblastoma-by increasing sensitivity to standard DNA damaging therapy, BCL6 inhibitors have real potential to improve the outcome for people with this disease.

The coadministration of trehalose dibehenate and monosodium urate crystals promotes an antitumor phenotype in human-derived myeloid cells.

Trehalose dibehenate (TDB), a ligand for the macrophage-inducible C-type lectin, has shown promise as an adjuvant for preventative vaccines and also as an anticancer agent in murine assays. The potential for TDB to affect the antitumor immune response of human myeloid cells, however, has not been studied. We investigated the effect of the adjuvants TDB and monosodium urate (MSU) crystals on the protumor or antitumor immune phenotype of human monocytes, macrophages and monocyte-derived dendritic cells (Mo-DCs). TDB treatment alone led to an inflammatory response in all three cell types, which was most pronounced when using human monocytes, with MSU augmenting this response. TDB also decreased cell surface markers associated with a protumorigenic phenotype, with MSU showing some ability to augment this response. Notably, a significant reduction in CD115 was observed for all antigen-presenting cells upon TDB or MSU + TDB treatment. The potential to increase the antigen-presenting capabilities of the myeloid cells was also observed upon treatment with TDB and MSU + TDB, as indicated by the upregulation of cell surface markers such as CD86 for all three cell types and a favorable ratio of interleukin (IL)-12p40 to IL-10 for monocytes stimulated with MSU + TDB. There was no significant production of IL-12p40 by Mo-DC; however, in a mixed lymphocyte assay, MSU + TDB costimulation of Mo-DC led to a significant increase in CD4+ T-cell numbers and in the IL-12p40-to-IL-10 ratio. Taken together, these findings show for the first time the potential of MSU + TDB costimulation to favor a tumor-suppressive phenotype in human-derived myeloid cells.

MSU Crystals Enhance TDB-Mediated Inflammatory Macrophage IL-1ß Secretion.

The tumour microenvironment predominantly consists of macrophages with phenotypes ranging from pro-inflammatory (M1-like) to anti-inflammatory (M2-like). Trehalose-6,6'-dibehenate (TDB) displays moderate anti-tumour activity and stimulates M1-like macrophages via the macrophage inducible C-type lectin (Mincle) resulting in IL-1ß production. In this study, we examined if monosodium urate (MSU), a known vaccine adjuvant, can boost IL-1ß production by TDB-stimulated macrophages. We investigated the effect of MSU/TDB co-treatment on IL-1ß production by GM-CSF (M1-like) and M-CSF/IL-4 (M2-like) differentiated mouse bone marrow macrophages (BMMs) and found that MSU/TDB co-treatment of GM-CSF BMMs significantly enhanced IL-1ß production in a Mincle-dependent manner. Western blot analysis showed that increased IL-1ß production by GM-CSF BMMs was associated with the induction of pro-IL-1ß expression by TDB rather than MSU. Flow cytometry analysis showed that MSU/TDB co-stimulation of GM-CSF BMMs led to greater expansion of CD86high/MHC IIhigh and CD86low/MHC IIlow subpopulations; however, only the latter showed increased production of IL-1ß. Together, these findings provide evidence of the potential to use MSU/TDB co-treatment to boost IL-1ß-mediated anti-tumour activity in M1-like tumour-associated macrophages.

Perfluorocarbon emulsions radiosensitise brain tumors in carbogen breathing mice with orthotopic GL261 gliomas.

BACKGROUND: Tumour hypoxia limits the effectiveness of radiation therapy. Delivering normobaric or hyperbaric oxygen therapy elevates pO2 in both tumour and normal brain tissue. However, pO2 levels return to baseline within 15 minutes of stopping therapy. AIM: To investigate the effect of perfluorocarbon (PFC) emulsions on hypoxia in subcutaneous and intracranial mouse gliomas and their radiosensitising effect in orthotopic gliomas in mice breathing carbogen (95%O2 and 5%CO2). RESULTS: PFC emulsions completely abrogated hypoxia in both subcutaneous and intracranial GL261 models and conferred a significant survival advantage orthotopically (Mantel Cox: p = 0.048) in carbogen breathing mice injected intravenously (IV) with PFC emulsions before radiation versus mice receiving radiation alone. Carbogen alone decreased hypoxia levels substantially and conferred a smaller but not statistically significant survival advantage over and above radiation alone. CONCLUSION: IV injections of PFC emulsions followed by 1h carbogen breathing, radiosensitises GL261 intracranial tumors.

High Dose Ascorbate Causes Both Genotoxic and Metabolic Stress in Glioma Cells.

We have previously shown that exposure to high dose ascorbate causes double stranded breaks (DSBs) and a build-up in S-phase in glioblastoma (GBM) cell lines. Here we investigated whether or not this was due to genotoxic stress as well as metabolic stress generated by exposure to high dose ascorbate, radiation, ascorbate plus radiation and H2O2 in established and primary GBM cell lines. Genotoxic stress was measured as phosphorylation of the variant histone protein, H2AX, 8-oxo-7,8-dihydroguanine (8OH-dG) positive cells and cells with comet tails. Metabolic stress was measured as a decrease in NADH flux, mitochondrial membrane potential (by CMXRos), ATP levels (by ATP luminescence) and mitochondrial superoxide production (by mitoSOX). High dose ascorbate, ascorbate plus radiation, and H2O2 treatments induced both genotoxic and metabolic stress. Exposure to high dose ascorbate blocked DNA synthesis in both DNA damaged and undamaged cell of ascorbate sensitive GBM cell lines. H2O2 treatment blocked DNA synthesis in all cell lines with and without DNA damage. DNA synthesis arrest in cells with damaged DNA is likely due to both genotoxic and metabolic stress. However, arrest in DNA synthesis in cells with undamaged DNA is likely due to oxidative damage to components of the mitochondrial energy metabolism pathway.

The Mincle ligand trehalose dibehenate differentially modulates M1-like and M2-like macrophage phenotype and function via Syk signaling.

INTRODUCTION: Macrophages play a significant role in the progression of diseases, such as cancer, making them a target for immune-modulating agents. Trehalose dibehenate (TDB) is known to activate M1-like macrophages via Mincle, however, the effect of TDB on M2-like macrophages, which are found in the tumor microenvironment, has not been studied. METHODS: qRT-PCR, flow cytometry, cytokine ELISA, and Western Blotting were used to study the effect of TDB on GM-CSF and M-CSF/IL-4 derived bone marrow macrophages (BMMs) from C57BL/6 and Mincle-/- mice. RESULTS: TDB treatment up-regulated M1 markers over M2 markers by GM-CSF BMMs, whereas M-CSF/IL-4 BMMs down-regulated marker gene expression overall. TDB treatment resulted in Mincle-independent down-regulation of CD11b, CD115, and CD206 expression by GM-CSF macrophages and CD115 in M-CSF/IL-4 macrophages. GM-CSF BMMs produced of significant levels of proinflammatory cytokines (IL-1ß, IL-6, TNF-α), which was Mincle-dependent and further enhanced by LPS priming. M-CSF BMMs produced little or no cytokines in response to TDB regardless of LPS priming. Western blot analysis confirmed that the absence of cytokine production was associated with a lack of activation of the Syk kinase pathway. CONCLUSION: This study illustrates that TDB has the potential to differentially regulate M1- and M2-like macrophages in the tumor environment.

Th2 responses are primed by skin dendritic cells with distinct transcriptional profiles.

The dendritic cell signals required for the in vivo priming of IL-4-producing T cells are unknown. We used RNA sequencing to characterize DCs from skin LN of mice exposed to two different Th2 stimuli: the helminth parasite Nippostrongylus brasiliensis (Nb) and the contact sensitizer dibutyl phthalate (DBP)-FITC. Both Nb and DBP-FITC induced extensive transcriptional changes that involved multiple DC subsets. Surprisingly, these transcriptional changes were highly distinct in the two models, with only a small number of genes being similarly regulated in both conditions. Pathway analysis of expressed genes identified no shared pathways between Nb and DBP-FITC, but revealed a type-I IFN (IFN-I) signature unique to DCs from Nb-primed mice. Blocking the IFN-I receptor at the time of Nb treatment had little effect on DC migration and antigen transport to the LN, but inhibited the up-regulation of IFN-I-induced markers on DCs and effectively blunted Th2 development. In contrast, the response to DBP-FITC was not affected by IFN-I receptor blockade, a finding consistent with the known dependence of this response on the innate cytokine TSLP. Thus, the priming of Th2 responses is associated with distinct transcriptional signatures in DCs in vivo, reflecting the diverse environments in which Th2 immune responses are initiated.

Mitochondrial Transfer from Astrocytes to Neurons following Ischemic Insult: Guilt by Association?

Intercellular mitochondrial transfer has been shown in tumor models, following lung injury and in xenotransplants of leukemic cells, but trafficking between cells in the brain remains unexplored. A suggestion that mitochondria move from astrocytes to neurons in a model of ischemia in a recent article in Nature by Hayakawa et al. (2016) should be interpreted with caution.

Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations.

BACKGROUND: The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. METHODS: We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. RESULTS: We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. CONCLUSION: We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.

Targeted inhibition of dominant PI3-kinase catalytic isoforms increase expression of stem cell genes in glioblastoma cancer stem cell models.

Cancer stem cells (CSC) exhibit therapy resistance and drive self-renewal of the tumour, making cancer stem cells an important target for therapy. The PI3K signalling pathway has been the focus of considerable research effort, including in glioblastoma (GBM), a cancer that is notoriously resistant to conventional therapy. Different isoforms of the catalytic sub-unit have been associated with proliferation, migration and differentiation in stem cells and cancer stem cells. Blocking these processes in CSC would improve patient outcome. We examined the effect of isoform specific PI3K inhibitors in two models of GBM CSC, an established GBM stem cell line 08/04 and a neurosphere formation model. We identified the dominant catalytic PI3K isoform for each model, and inhibition of the dominant isoform blocked AKT phosphorylation, as did pan-PI3K/mTOR inhibition. Analysis of SOX2, OCT4 and MSI1 expression revealed that inhibition of the dominant p110 subunit increased expression of cancer stem cell genes, while pan-PI3K/mTOR inhibition caused a similar, though not identical, increase in cancer stem cell gene expression. This suggested that PI3K inhibition enhanced, rather than blocked, CSC activity. Careful analysis of the response to specific isoform inhibition will be necessary before specific subunit inhibitors can be successfully deployed against GBM CSC.

Horizontal transfer of mitochondria between mammalian cells: beyond co-culture approaches.

Current dogma holds that genes are the property of individual mammalian cells and partition between daughter cells during cell division. However, and rather unexpectedly, recent research has demonstrated horizontal cell-to-cell transfer of mitochondria and mitochondrial DNA in several mammalian cell culture systems. Furthermore, unequivocal evidence that mitochondrial DNA transfer occurs in vivo has now been published. While these studies show horizontal transfer of mitochondrial DNA in pathological settings, it is also possible that intercellular mitochondrial transfer is a fundamental physiological process with a role in development and tissue homeostasis.

N,N-Bis(glycityl)amines as anti-cancer drugs.

A series of N,N-bis(glycityl)amines with promising anti-cancer activity were prepared via the reductive amination of pentoses and hexoses, and subsequently screened for their ability to selectively inhibit the growth of cancerous versus non-cancerous cells. For the first time, we show that this class of compounds possesses anti-proliferative activity, and, while the selective killing of brain cancer (LN18) cells versus matched (SVG-P12) cells was modest, several of the amines, including d-arabinitylamine 1a and d-fucitylamine 1g, exhibited low micromolar IC50 values for HL60 cells. Moreover, these two amines showed good selectivity towards HL60 cells when compared to non-cancerous HEK-293 cells. The compounds also showed low micromolar inhibition of the leukaemic cell line, THP-1. The modes of action of amines 1a and 1g were then determined using yeast chemical genetics, whereby it was established that both compounds affect similar but distinct sets of biochemical pathways. Notably purine nucleoside monophosphate biosynthesis was identified as an enriched mechanism. The rapid synthesis of the amines and their unique mode of action thus make them attractive targets for further development as anti-cancer drugs.

A combination of tyrosine kinase inhibitors, crizotinib and dasatinib for the treatment of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Despite the advances in surgery, radiotherapy and chemotherapy, patient survival averages only 14.6 months. In most GBM tumors, tyrosine kinases show increased activity and/or expression and actively contribute to the development, recurrence and onset of treatment resistance; making their inhibition an appealing therapeutic strategy. We compared the cytotoxicity of 12 tyrosine kinase inhibitors in vitro. A combination of crizotinib and dasatinib emerged as the most cytotoxic across established and primary human GBM cell lines. The combination treatment induced apoptotic cell death and polyploidy. Furthermore, the combination treatment led to the altered expression and localization of several tyrosine kinase receptors such as Met and EGFR and downstream effectors as such as SRC. Furthermore, the combination treatment reduced the migration and invasion of GBM cells and prevented endothelial cell tube formation in vitro. Overall, our study demonstrated the broad specificity of a combination of crizotinib and dasatinib across multiple GBM cell lines. These findings provide insight into the development of alternative therapy for the treatment of GBM.

Post transcriptional control of the epigenetic stem cell regulator PLZF by sirtuin and HDAC deacetylases.

BACKGROUND: The transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) is critical for the regulation of normal stem cells maintenance by establishing specific epigenetic landscape. We have previously shown that CBP/p300 acetyltransferase induces PLZF acetylation in order to increase its deoxynucleotidic acid (DNA) binding activity and to enhance its epigenetic function (repression of PLZF target genes). However, how PLZF is inactivated is not yet understood. RESULTS: In this study, we demonstrate that PLZF is deacetylated by both histone deacetylase 3 and the NAD+ dependent deacetylase silent mating type information regulation 2 homolog 1 (SIRT1). Unlike other PLZF-interacting deacetylases, these two proteins interact with the zinc finger domain of PLZF, where the activating CBP/p300 acetylation site was previously described, inducing deacetylation of lysines 647/650/653. Overexpression of histone deacetylase 3 (HDAC3) and SIRT1 is associated with loss of PLZF DNA binding activity and decreases PLZF transcriptional repression. As a result, the chromatin status of the promoters of PLZF target genes, involved in oncogenesis, shift from a heterochromatin to an open euchromatin environment leading to gene expression even in the presence of PLZF. CONCLUSIONS: Consequently, SIRT1 and HDAC3 mediated-PLZF deacetylation provides for rapid control and fine-tuning of PLZF activity through post-transcriptional modification to regulate gene expression and cellular homeostasis.

Enhanced immunosuppression by therapy-exposed glioblastoma multiforme tumor cells.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an extremely short time to relapse following standard treatment. Since recurrent GBM is often resistant to subsequent radiotherapy and chemotherapy, immunotherapy has been proposed as an alternative treatment option. Although it is well established that GBM induces immune suppression, it is currently unclear what impact prior conventional therapy has on the ability of GBM cells to modulate the immune environment. In this study, we investigated the interaction between immune cells and glioma cells that had been exposed to chemotherapy or irradiation in vitro. We demonstrate that treated glioma cells are more immunosuppressive than untreated cells and form tumors at a faster rate in vivo in an animal model. Cultured supernatant from in vitro-treated primary human GBM cells were also shown to increase suppression, which was independent of accessory suppressor cells or T regulatory cell generation, and could act directly on CD4(+) and CD8(+) T cell proliferation. While a number of key immunosuppressive cytokines were overexpressed in the treated cells, including IL-10, IL-6 and GM-CSF, suppression could be alleviated in a number of treated GBM lines by inhibition of prostaglandin E2. These results reveal for the first time that conventional therapies can alter immunosuppressive pathways in GBM tumor cells, a finding with important implications for the combination of immunotherapy with standard treatment.

Radiosensitisation by pharmacological ascorbate in glioblastoma multiforme cells, human glial cells, and HUVECs depends on their antioxidant and DNA repair capabilities and is not cancer specific.

We previously showed that 5 mM ascorbate radiosensitized early passage radioresistant glioblastoma multiforme (GBM) cells derived from one patient tumor. Here we investigate the sensitivity of a panel of cell lines to 5 mM ascorbate and 6 Gy ionizing radiation, made up of three primary human GBM cells, three GBM cell lines, a human glial cell line, and primary human vascular endothelial cells. The response of different cells lines to ascorbate and/or radiation was determined by measuring viability, colony-forming ability, generation and repair of double-stranded DNA breaks (DSBs), cell cycle progression, antioxidant capacity and generation of reactive oxygen species. Individually, radiation and ascorbate both decreased viability and clonogenicity by inducing DNA damage, but had differential effects on cell cycle progression. Radiation led to G2/M arrest in most cells whereas ascorbate caused accumulation in S phase, which was moderately associated with poor DSB repair. While high dose ascorbate radiosensitized all cell lines in clonogenic assays, the sensitivity to radiation, high dose ascorbate, and combined treatment varied between cell lines. Normal glial cells were similar to GBM cells with respect to free radical scavenging potential and effect of treatment on DNA damage and repair, viability, and clonogenicity. Both GBM cells and normal cells coped equally poorly with oxidative stress caused by radiation and/or high dose ascorbate, dependent primarily on their antioxidant and DSB repair capacity.