Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.

Role of Klotho Protein in Tumor Genesis, Cancer Progression, and Prognosis in Patients with High-Grade Glioma.

BACKGROUND: Klotho, a single-pass transmembrane protein associated with premature aging, acts as a tumor suppressor gene by inhibiting insulin/insulin-like growth factor-1 and fibroblast growth factor pathways. Downregulated Klotho expression is reported in melanoma, mesothelioma, bladder, breast, gastric, cervix, lung, and kidney cancers and is associated with a poor prognosis. Klotho expression and Klotho promoter hypermethylation are predictive factors for patient prognosis. METHODS: To investigate the potential role of Klotho in glioblastoma-multiforme (GBM), 22 GBM samples were collected from the Sheba Tumor Bank and examined. RESULTS: We found that increased Klotho messenger ribonucleic acid (RNA) expression predicted longer survival (P = 0.03) of GBM patients. Methylation analysis was performed on bisulfite-treated deoxyribonucleic acid from the GBM patient samples using ionization time-of-flight mass spectrometry according to the Sequenom EpiTYPER protocols. Klotho promoter hypermethylation was detected in 65% of the GBM samples and correlated significantly with improved survival (P < 0.04). We found 3 major Klotho promotor hypermethylation sites located 585-579 bp, 540-533 bp, and 537-534 bp upstream of the transcription start site. Methylated deoxyribonucleic acid immunoprecipitation studies confirmed these results. Notably, the messenger RNA expression in these GBM samples revealed an unexpected linear correlation with methylation of these 3 hypermethylation sites identified in the Klotho promotor. Thus Klotho expression and methylation could predict prognosis in patients with GBM. CONCLUSIONS: Epigenetic regulation in GBM appears to be complicated. Specific CpG islands affect genes or micro RNAs that interact to control Klotho expression. The diverse effects of these islands may be due to unique factors of GBM.

Sustained Response to Imatinib in a Pediatric Patient with Concurrent Myeloproliferative Disease and Lymphoblastic Lymphoma Associated with a CCDC88C-PDGFRB Fusion Gene.

BACKGROUND: The WHO defined myeloid and lymphoid neoplasms (MLN) with eosinophilia associated with PDGFRB, PDGFRA, FGFR1 rearrangements as a new entity in 2016. PDGFRB-rearranged MLN sensitive to imatinib were described in adult patients. We report the first pediatric patient with PDGFRB-rearranged myeloproliferative disorder associated with T-lymphoblastic lymphoma bearing the t(5; 14)(q33;q32) translocation who was successfully treated with imatinib only. Methods/Aims: Analysis of bone marrow and peripheral blood cells by fluorescent in situ hybridization identified the PDGFRB partner as CCDC88C. Whole genome sequencing of the patient's DNA identified the exact junction site, confirmed by PCR amplification and Sanger sequencing. A real-time quantitative PCR assay was designed to quantify the fused CCDC88C-PDGFRB product. RESULTS: A 2.5-year-old boy was diagnosed with myeloproliferative disorder and eosinophilia associated with lymphoblastic lymphoma both bearing the CCDC88C-PDGFRB fusion. Imatinib therapy resulted in rapid clinical, hematological, and cytogenetic response. Molecular response to treatment was monitored by a real-time PCR assay specific for the CCDC88C- PDGFRB fusion. CONCLUSION: This is the first description of MLN with eosinophilia in the pediatric age group. Response to treatment with imatinib only was monitored by specific quantitative PCR assay with sustained remission lasting 5.5 years from diagnosis.

MutT homolog 1 counteracts the effect of anti-neoplastic treatments in adult and pediatric glioblastoma cells.

Glioblastoma, a fatal disease in both adult and pediatric patients, currently has limited treatment options that offer no more than temporary relief. Our experiments with adult and pediatric glioblastoma cell lines showed that radiation induces a dose-dependent increase in the level of MutT homolog 1 (MTH1) - an enzyme that hydrolyzes oxidized purine nucleoside triphosphates. Similarly, the combination of vorinostat, which is a histone deacetylase inhibitor, and ABT-888, which is a PARP-1 inhibitor, enhanced clonogenic death and increased the MTH1 level, relative to each treatment alone. This result suggests that the MTH1 level is directly related to the damage that is inflicted upon the cells, and its activity protects them against anti-neoplastic therapy. Indeed, the MTH1 inhibitor TH588 and MTH1 siRNA increased glioblastoma's response to both radiation and the combination of vorinostat and ABT-888. TH588 also inhibited glioblastoma's capacity for migration and invasion. In normal fibroblasts, low radiation doses and the combination of vorinostat and ABT-888 decreased the level of the enzyme. TH588 did not alter the fibroblasts' response to radiation and only mildly affected their response to the combination of vorinostat and ABT-888. In summary, the inhibition of MTH1 is required to better realize the therapeutic potential of anti-neoplastic treatments in glioblastoma.

Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.

Related to testes-specific, vespid and pathogenesis protein-1 is regulated by methylation in glioblastoma.

Related to testes-specific, vespid and pathogenesis protein-1 (RTVP-1), also known as glioma pathogenesis-related protein 1, is highly expressed and has oncogenic features in glioblastoma (GBM; World Health Organization class IV). Promoter methylation has been found to control RTVP-1 expression in prostate carcinoma, Wilms' tumor, acute myeloid leukemia and melanoma. In this bi-institutional study, the methylation status of RTVP-1 in astrocytic brain malignancies (GBM and oligodendroglioma) was examined. The RTVP-1 promoter was hypomethylated in GBM compared with non-tumor brain samples, but was hypermethylated in oligodendroglioma. RTVP-1 methylation correlated with RTVP-1 expression at the mRNA level. In GBM, hypermethylation of the RTVP-1 promoter was associated with improved overall survival although with no statistical significance.

Spotted fever group rickettsiae in ticks collected from wild animals in Israel.

We report molecular evidence for the presence of spotted fever group rickettsiae (SFGR) in ticks collected from roe deer, addax, red foxes, and wild boars in Israel. Rickettsia aeschlimannii was detected in Hyalomma marginatum and Hyalomma detritum while Rickettsia massiliae was present in Rhipicephalus turanicus ticks. Furthermore, a novel uncultured SFGR was detected in Haemaphysalis adleri and Haemaphysalis parva ticks from golden jackals. The pathogenicity of the novel SFGR for humans is unknown; however, the presence of multiple SFGR agents should be considered when serological surveillance data from Israel are interpreted because of significant antigenic cross-reactivity among Rickettsia. The epidemiology and ecology of SFGR in Israel appear to be more complicated than was previously believed.

Early diagnosis of severe Mediterranean spotted fever cases by nested-PCR detecting spotted fever Rickettsiae 17-kD common antigen gene.

We describe 3 cases of Mediterranean spotted fever (MSF) who presented with severe sepsis, in 2 of which the clinical diagnosis was unclear at presentation. In each case the diagnosis of MSF was made using a nested-PCR assay for Rickettsia conorii 17-kD protein gene. The nested-PCR based diagnosis preceded the serological results of MSF that were all negative at admission. The early diagnosis of MSF by specific PCR will facilitate an early institution of appropriate therapy, saving unnecessary tests and medications.

Fatal Rickettsia conorii subsp. israelensis infection, Israel.

Underdiagnosis of fatal spotted fever may be attributed to nonspecific clinical features and insensitive acute-phase serologic studies. We describe the importance of molecular and immunohistochemical methods in establishing the postmortem diagnosis of locally acquired Israeli spotted fever due to Rickettsia conorii subsp. israelensis in a traveler returning to Israel from India.

Clusters of Mediterranean spotted fever in Israel.

Mediterranean spotted fever (MSF) usually occurs as sporadic cases. We report five clusters of MSF in Israel. Each cluster consisted of two to three patients. In two clusters, one patient died while the other recovered. In the other three clusters the patients presented with a benign course of the disease. The diagnosis of MSF in the fatal cases was confirmed by nested-polymerase chain reaction (PCR) tests performed on samples obtained from internal organs. Rickettsial DNA was also found in a tick obtained from a dog owned by one of the patients. MSF was diagnosed in the recovered patients by serology. The diagnosis of MSF fever in one family member should raise the awareness to the possibility of other cases in the vicinity.

Polymerase chain reaction-based diagnosis of Mediterranean spotted fever in serum and tissue samples.

A nested polymerase chain reaction (PCR) assay has been developed and used in the diagnosis of fatal and benign cases of Mediterranean spotted fever (MSF). The test was based on specific primers derived from a Rickettsia conorii 17-kD protein gene. A positive signal was obtained from spotted fever group (SFG) and typhus group (TG) rickettsiae. Discrimination between SFG and TG rickettsiae was based on a restriction fragment length polymorphism test. Other gram-negative bacterial species tested did not generate a signal, attesting for the specificity of the assay. The SFG-specific DNA fragment was detected in four of 29 acute-phase sera from serologically confirmed patients with MSF, while acute-phase sera from 25 patients without MSF were PCR negative. Acute-phase sera samples (five of five) and tissue autopsies (six of seven) from fatal suspected cases of MSF were PCR positive. The results demonstrate that sera and tissue samples are suitable specimens for the nested PCR tests, especially in fatal cases.