Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor type in the central nervous system in adults. Resistance to chemotherapy remains one of the major obstacles in GBM treatment. Identifying and overcoming the mechanisms of therapy resistance is instrumental to develop novel therapeutic approaches for patients with GBM. To determine the major drivers of temozolomide (TMZ) sensitivity, we performed shRNA screenings in GBM lines with different O6-methylguanine-DNA methyl-transferase (MGMT) status. We then evaluated dianhydrogalactitol (Val-083), a small alkylating molecule that induces interstrand DNA crosslinking, as a potential treatment to bypass TMZ-resistance mechanisms. We found that loss of mismatch repair (MMR) components and MGMT expression are mutually exclusive mechanisms driving TMZ resistance in vitro Treatment of established GBM cells and tumorsphere lines with Val-083 induces DNA damage and cell-cycle arrest in G2-M phase, independently of MGMT or MMR status, thus circumventing conventional resistance mechanisms to TMZ. Combination of TMZ and Val-083 shows a synergic cytotoxic effect in tumor cells in vitro, ex vivo, and in vivo We propose this combinatorial treatment as a potential approach for patients with GBM.

MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas.

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.

Role of MSK1 in the Induction of NF-κB by the Chemokine CX3CL1 in Microglial Cells.

Microglial cells are essential mediators of neuroinflammatory processes involved in several pathologies. Moreover, the chemokine fractalkine (CX3CL1) is essential in the crosstalk between neurons and microglia. However, the exact roles of CX3CL1, CX3CL1 receptor (CX3CR1) and microglia signalling are not fully understood in neuroinflammation. In addition, the findings reported on this subject are controversial. In this work, we investigated whether CX3CL1 induced pro-inflammatory signalling activation through NF-κB pathway. We were able to show that CX3CL1 activates the pro-inflammatory pathway mediated by the transcription factor NF-κB as an early response in microglial cells. On the other side, CX3CR1-deficient microglia showed impaired NF-κB axis. Phospho-kinase assay proteome profiles indicated that CX3CL1 induced several kinases such as MAPK's (ERK and JNK), SRC-family tyrosine kinases (YES, FGR, LCK and LYN) and most interesting and also related to NF-κB, the mitogen- and stress-activated kinase-1 (MSK1). Knockdown of MSK1 with short interfering RNAs decreased partially MSK1 protein levels (about 50%), enough to decrease the mRNA levels of Il-1ß, Tnf-α and iNos triggered by stimulation with CX3CL1. These results indicate the relevance of CX3CL1 in the activation of the pro-inflammatory NF-κB signalling pathway through MSK1 in microglial cells.

Role of Microvesicles in the Spread of Herpes Simplex Virus 1 in Oligodendrocytic Cells.

Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in the neurons of sensory ganglia. In some cases, the virus spreads into the central nervous system, causing encephalitis or meningitis. Cells infected with several different types of viruses may secrete microvesicles (MVs) containing viral proteins and RNAs. In some instances, extracellular microvesicles harboring infectious virus have been found. Here we describe the features of shedding microvesicles released by the human oligodendroglial HOG cell line infected with HSV-1 and their participation in the viral cycle. Using transmission electron microscopy, we detected for the first time microvesicles containing HSV-1 virions. Interestingly, the Chinese hamster ovary (CHO) cell line, which is resistant to infection by free HSV-1 virions, was susceptible to HSV-1 infection after being exposed to virus-containing microvesicles. Therefore, our results indicate for the first time that MVs released by infected cells contain virions, are endocytosed by naive cells, and lead to a productive infection. Furthermore, infection of CHO cells was not completely neutralized when virus-containing microvesicles were preincubated with neutralizing anti-HSV-1 antibodies. The lack of complete neutralization and the ability of MVs to infect nectin-1/HVEM-negative CHO-K1 cells suggest a novel way for HSV-1 to spread to and enter target cells. Taken together, our results suggest that HSV-1 could spread through microvesicles to expand its tropism and that microvesicles could shield the virus from neutralizing antibodies as a possible mechanism to escape the host immune response.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in neurons. Extracellular vesicles are a heterogeneous group of membrane vesicles secreted by most cell types. Microvesicles, which are extracellular vesicles which derive from the shedding of the plasma membrane, isolated from the supernatant of HSV-1-infected HOG cells were analyzed to find out whether they were involved in the viral cycle. The importance of our investigation lies in the detection, for the first time, of microvesicles containing HSV-1 virions. In addition, virus-containing microvesicles were endocytosed into CHO-K1 cells and were able to actively infect these otherwise nonpermissive cells. Finally, the infection of CHO cells with these virus-containing microvesicles was not completely neutralized by anti-HSV-1 antibodies, suggesting that these extracellular vesicles might shield the virus from neutralizing antibodies as a possible mechanism of immune evasion.