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Purpose: Oncolytic virotherapy with herpes simplex virus-1 (HSV) has shown promise for the treatment of pediatric and adult brain tumors; however, completed and ongoing clinical trials have utilized intratumoral/peritumoral oncolytic HSV (oHSV) inoculation due to intraventricular/intrathecal toxicity concerns. Intratumoral delivery requires an invasive neurosurgical procedure, limits repeat injections, and precludes direct targeting of metastatic and leptomeningeal disease. To address these limitations, we determined causes of toxicity from intraventricular oHSV and established methods for mitigating toxicity to treat disseminated brain tumors in mice. Experimental Design: HSV-sensitive CBA/J mice received intraventricular vehicle, inactivated oHSV, or treatment doses (1×107 plaque-forming units) of oHSV, and toxicity was assessed by weight loss and IHC. Protective strategies to reduce oHSV toxicity, including intraventricular low-dose oHSV or interferon inducer polyinosinic-polycytidylic acid (poly I:C) prior to oHSV treatment dose, were evaluated and then utilized to assess intraventricular oHSV treatment of multiple models of disseminated CNS disease. Results: A standard treatment dose of intraventricular oHSV damaged ependymal cells via virus replication and induction of CD8+ T cells, whereas vehicle or inactivated virus resulted in no toxicity. Subsequent doses of intraventricular oHSV caused little additional toxicity. Interferon induction with phosphorylation of eukaryotic initiation factor-2α (eIF2α) via intraventricular pretreatment with low-dose oHSV or poly I:C mitigated ependyma toxicity. This approach enabled the safe delivery of multiple treatment doses of clinically relevant oHSV G207 and prolonged survival in disseminated brain tumor models. Conclusions: Toxicity from intraventricular oHSV can be mitigated, resulting in therapeutic benefit. These data support the clinical translation of intraventricular G207.

Body weights of CBA/J, BALB/c and C57BL/6 mice (N = 5/group) after inoculation with 1x107 PFU of IVT oHSV.

IHC staining of the ependymal lining for CD8+ cells and confirmation of CD8 depletion.

Interferon β (IFN β) levels over time in the CSF after a single 50 µg IVT dose of poly I:C or saline (N = 2 mice/group/time point).

Comparison of the protective effect of different pretreatment low doses of IVT oHSV from toxicity after a subsequent treatment dose.

Effects of poly I:C alone and combined with oHSV in vitro and in vivo.

Toxicity from IVT oHSV in the left lateral ventricle and third and fourth ventricles at different time points.

Time to recover baseline body weight in mice pretreated with low-dose oHSV prior to a standard treatment dose.

Toxicity with repeated doses of IVT oHSV.

Purpose:Oncolytic virotherapy with herpes simplex virus-1 (HSV) has shown promise for the treatment of pediatric and adult brain tumors; however, completed and ongoing clinical trials have utilized intratumoral/peritumoral oncolytic HSV (oHSV) inoculation due to intraventricular/intrathecal toxicity concerns. Intratumoral delivery requires an invasive neurosurgical procedure, limits repeat injections, and precludes direct targeting of metastatic and leptomeningeal disease. To address these limitations, we determined causes of toxicity from intraventricular oHSV and established methods for mitigating toxicity to treat disseminated brain tumors in mice.Experimental Design:HSV-sensitive CBA/J mice received intraventricular vehicle, inactivated oHSV, or treatment doses (1×107 plaque-forming units) of oHSV, and toxicity was assessed by weight loss and IHC. Protective strategies to reduce oHSV toxicity, including intraventricular low-dose oHSV or interferon inducer polyinosinic-polycytidylic acid (poly I:C) prior to oHSV treatment dose, were evaluated and then utilized to assess intraventricular oHSV treatment of multiple models of disseminated CNS disease.Results:A standard treatment dose of intraventricular oHSV damaged ependymal cells via virus replication and induction of CD8+ T cells, whereas vehicle or inactivated virus resulted in no toxicity. Subsequent doses of intraventricular oHSV caused little additional toxicity. Interferon induction with phosphorylation of eukaryotic initiation factor-2α (eIF2α) via intraventricular pretreatment with low-dose oHSV or poly I:C mitigated ependyma toxicity. This approach enabled the safe delivery of multiple treatment doses of clinically relevant oHSV G207 and prolonged survival in disseminated brain tumor models.Conclusions:Toxicity from intraventricular oHSV can be mitigated, resulting in therapeutic benefit. These data support the clinical translation of intraventricular G207.

Background Chromosomal translocation has been detected in many human cancers including gliomas and is considered a driving force in tumorigenesis. Co-deletion of chromosome arms 1p and 19q is a hallmark for oligodendrogliomas. On the molecular level, 1p/19q co-deletion results from t(1;19)(q10;p10), which leads to the concomitant formation of a hybrid chromosome containing the 1q and 19p arms. A method to generate 1p/19q co-deletion is lacking, which hinders the investigation of how 1p/19q co-deletion contributes to gliomagenesis. Methods We hypothesized that chromosomal translocation, such as t(1;19)(q10;p10) resulting in the 1p/19q co-deletion, may be induced by simultaneously introducing DNA double-strand breaks (DSBs) into chromosomes 1p and 19q using CRISPR/Cas9. We developed a CRISPR/Cas9-based strategy to induce t(1;19)(q10;p10) and droplet digital PCR (ddPCR) assays to detect the hybrid 1q/19p and 1p/19q chromosomes. Results After translocation induction, we detected both 1p/19q and 1q/19p hybrid chromosomes by PCR amplification of the junction regions in HEK 293T, and U-251 and LN-229 glioblastoma cells. Sequencing analyses of the PCR products confirmed DNA sequences matching both chromosomes 1 and 19. Furthermore, the 1p/19q hybrid chromosome was rapidly lost in all tested cell lines. The 1q/19p hybrid chromosome also become undetectable over time likely due to cell survival disadvantage. Conclusion We demonstrated that t(1;19)(q10;p10) may be induced by CRISPR/Cas9-mediated genomic editing. This method represents an important step toward engineering the 1p/19q co-deletion to model oligodendrogliomas. This method may also be generalizable to engineering other cancer-relevant translocations, which may facilitate the understanding of translocation roles in cancer progression.

Hypocretin 1 and hypocretin 2 (orexin A and B) regulate sleep, wakefulness and emotion. Tumour necrosis factor alpha (TNF‐α) is an important neuroinflammation mediator. Here, we examined the effects of TNF‐α treatment on hypocretin expression in vivo and behaviour in mice. TNF‐α decreased hypocretin 1 and hypocretin 2 expression in a dose‐dependent manner in cultured hypothalamic neurons. TNF‐α decreased mRNA stability of prepro‐hypocretin, the single precursor of hypocretin 1 and hypocretin 2. Mice challenged with TNF‐α demonstrated decreased expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in hypothalamus. In response to TNF‐α, prepro‐hypocretin mRNA decay was increased in hypothalamus. TNF‐α neutralizing antibody restored the expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in vivo in TNF‐α challenged mice, supporting hypocretin system can be impaired by increased TNF‐α through decreasing hypocretin expression. Repeated TNF‐α challenge induced muscle activity during rapid eye movement sleep and sleep fragmentation, but decreased learning, cognition and memory in mice. TNF‐α neutralizing antibody blocked the effects of TNF‐α; in contrast, hypocretin receptor antagonist enhanced the effects of TNF‐α. The data support that TNF‐α is involved in the regulation of hypocretin expression, sleep and cognition. The findings shed some lights on the role of neuroinflammation in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.

The optimization of cell culture modes and basal media is very important to improve the production of therapeutic monoclonal antibodies (mAbs) by Chinese hamster ovary (CHO) cells, but the underlying cellular mechanism has not been well investigated. The objective of this study was to elucidate the interaction between the intracellular proteome and key process parameters by employing comparative proteomics. Three cell culture operations and four basal media were characterized in the production of anti-HER2 mAb using CHO DG44. The representative host cell proteins that demonstrated significant responses to various culture modes and media, including the enzymes involved in carbohydrate metabolism, transcription, translation, and post translational modification, were described. The fed-batch culture in a 2-L bioreactor produced mAb with a titer of 2411 mg/L using Dynamis medium. The purified mAb showed strong and specific targeting to HER2+ MDA-MB-361 cell and similar anti-cancer toxicity to the FDA approved Trastuzumab. Moreover, this proteomics study also indicated several key considerations for future rationally engineering a mAb production process, which could benefit biopharmaceutical manufacturing.

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody–drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of &gt, 98% to meningioma CH157-MN cells but a low binding rate of &lt, 5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of &lt, 10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.

Simple Summary Interactions of integrins with the extracellular matrix play a key role in cancer cell migration, invasion, and growth. However, whether integrin engagement promotes cancer progression through metabolic reprogramming has not been completely understood. This study investigates the role and mechanism of integrin αvβ3 engagement with its ligand in metabolic reprogramming. The data support that integrin αvβ3 plays an important role in increased glucose uptake and aerobic glycolysis, as well as in decreased mitochondrial oxidative phosphorylation, in glioblastoma cells. In addition, the data imply that focal adhesion kinase (FAK) and protein arginine methyltransferase 5 (PRMT5) are likely downstream effectors of integrin αvβ3, and regulate metabolic shift toward glycolysis. These findings provide new insight into how cancer cells regulate their metabolism based on microenvironmental cues transmitted by integrin and extracellular matrix proteins, and how the signals eventually translate to metabolic modifications coupled with changes in cell behavior, including migration, invasion, and growth. Abstract Metabolic reprogramming promotes glioblastoma cell migration and invasion. Integrin αvβ3 is one of the major integrin family members in glioblastoma multiforme cell surface mediating interactions with extracellular matrix proteins that are important for glioblastoma progression. The role of αvβ3 integrin in regulating metabolic reprogramming and its mechanism of action have not been determined in glioblastoma cells. Integrin αvβ3 engagement with osteopontin promotes glucose uptake and aerobic glycolysis, while inhibiting mitochondrial oxidative phosphorylation. Blocking or downregulation of integrin αvβ3 inhibits glucose uptake and aerobic glycolysis and promotes mitochondrial oxidative phosphorylation, resulting in decreased migration and growth in glioblastoma cells. Pharmacological inhibition of focal adhesion kinase (FAK) or downregulation of protein arginine methyltransferase 5 (PRMT5) blocks metabolic shift toward glycolysis and inhibits glioblastoma cell migration and invasion. These results support that integrin αvβ3 and osteopontin engagement plays an important role in promoting the metabolic shift toward glycolysis and inhibiting mitochondria oxidative phosphorylation in glioblastoma cells. The metabolic shift in cell energy metabolism is coupled to changes in migration, invasion, and growth, which are mediated by downstream FAK and PRMT5 in glioblastoma cells.

Pulmonary edema associated with increased vascular permeability is a severe complication of Pseudomonas (P.) aeruginosa-induced acute lung injury. The mechanisms underlying P aeruginosa-induced vascular permeability are not well understood. In the present study, we investigated the role of neuronal Wiskott Aldrich syndrome protein (N-WASP) in modulating P aeruginosa-induced vascular permeability. Using lung microvascular endothelial and alveolar epithelial cells, we demonstrated that N-WASP downregulation attenuated P aeruginosa-induced actin stress fiber formation and prevented paracellular permeability. P aeruginosa-induced dissociation between VE-cadherin and β-catenin, but increased association between N-WASP and VE-cadherin, suggesting a role for N-WASP in promoting P aeruginosa-induced adherens junction rupture. P aeruginosa increased N-WASP-Y256 phosphorylation, which required the activation of Rho GTPase and focal adhesion kinase. Increased N-WASP-Y256 phosphorylation promotes N-WASP and integrin αVβ6 association as well as TGF-β-mediated permeability across alveolar epithelial cells. Inhibition of N-WASP-Y256 phosphorylation by N-WASP-Y256F overexpression blocked N-WASP effects in P aeruginosa-induced actin stress fiber formation and increased paracellular permeability. In vivo, N-WASP knockdown attenuated the development of pulmonary edema and improved survival in a mouse model of P aeruginosa pneumonia. Together, our data demonstrate that N-WASP plays an essential role in P aeruginosa-induced vascular permeability and pulmonary edema through the modulation of actin cytoskeleton dynamics.

Dynamic regulation of histone modification enzymes such as PRMT1 (protein arginine methyltransferase 1) determines the ordered epigenetic transitions in hematopoiesis. Sorting cells according to the expression levels of histone modification enzymes may further define subpopulations in hematopoietic lineages with unique differentiation potentials that are presently defined by surface markers. We discovered a vital near infrared dye, E84, that fluoresces brightly following binding to PRMT1 and excitation with a red laser. The staining intensity as measured by flow cytometry is correlated with the PRMT1 expression level. Importantly, E84 staining has no apparent negative effect on the proliferation of the labeled cells. Given that long-term hematopoietic stem cells (LT-HSCs) produce low levels of PRMT1, we used E84 to sort LT-HSCs from mouse bone marrow. We found that SLAM (the signalling lymphocyte activation molecule family) marker–positive LT-HSCs were enriched in the E84low cell fraction. We then performed bone marrow transplantations with E84high or E84low Lin−Sca1+Kit+ (LSK) cells and showed that whole blood cell lineages were successfully reconstituted 16 weeks after transplanting 200 E84low LSK cells. Thus, E84 is a useful new tool to probe the role of PRMT1 in hematopoiesis and leukemogenesis. Developing E84 and other small molecules to label histone modification enzymes provides a convenient approach without modifying gene loci to study the interaction between hematopoietic stem/progenitor cell epigenetic status and differentiation state.

Pediatric high-grade brain tumors and adult glioblastoma are associated with significant morbidity and mortality. Oncolytic herpes simplex virus-1 (oHSV) is a promising approach to target brain tumors; oHSV G207 and M032 (encodes human interleukin-12) are currently in phase I clinical trials in children with malignant supratentorial brain tumors and adults with glioblastoma, respectively. We sought to compare the sensitivity of patient-derived pediatric malignant brain tumor and adult glioblastoma xenografts to these clinically-relevant oHSV. In so doing we found that pediatric brain tumors were more sensitive to the viruses and expressed significantly more nectin-1 (CD111) than adult glioblastoma. Pediatric embryonal and glial tumors were 74-fold and 14-fold more sensitive to M002 and 16-fold and 6-fold more sensitive to G207 than adult glioblastoma, respectively. Of note, pediatric embryonal tumors were more sensitive than glial tumors. Differences in sensitivity may be due in part to nectin-1 expression, which predicted responses to the viruses. Treatment with oHSV resulted in prolonged survival in both pediatric and adult intracranial patient-dervied tumor xenograft models. Our results suggest that pediatric brain tumors are ideal targets for oHSV and that brain tumor expression of nectin-1 may be a useful biomarker to predict patient response to oHSV.

Summary: Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Despite advances in conventional chemotherapy, surgical techniques, and radiation, outcomes for patients with relapsed, refractory, or metastatic soft tissue sarcomas are dismal. Survivors often suffer from lasting morbidity from current treatments. New targeted therapies with less toxicity, such as those that harness the immune system, and immunocompetent murine sarcoma models to test these therapies are greatly needed. We characterized two new serendipitous murine models of undifferentiated sarcoma (SARC-28 and SARC-45) and tested their sensitivity to virotherapy with oncolytic herpes simplex virus 1 (HSV-1). Both models expressed high levels of the primary HSV entry molecule nectin-1 (CD111) and were susceptible to killing by interleukin-12 (IL-12) producing HSV-1 M002 in vitro and in vivo. M002 resulted in a significant intratumoral increase in effector CD4+ and CD8+ T cells and activated monocytes, and a decrease in myeloid-derived suppressor cells (MDSCs) in immunocompetent mice. Compared to parent virus R3659 (no IL-12 production), M002 resulted in higher CD8:MDSC and CD8:T regulatory cell (Treg) ratios, suggesting that M002 creates a more favorable immune tumor microenvironment. These data provide support for clinical trials targeting sarcomas with oncolytic HSV-1. These models provide an exciting opportunity to explore combination therapies for soft tissue sarcomas that rely on an intact immune system to reach full therapeutic potential. Keywords: oncolytic, HSV, murine sarcoma, immunotherapy, M002, herpes simplex virus

Primary malignant central nervous system (CNS) tumors are the leading cause of childhood cancer-related death and morbidity. While advances in surgery, radiation, and chemotherapy have improved the survival rates in children with malignant brain tumors, mortality persists in certain subpopulations and current therapies are associated with extreme morbidity. This is especially true for children with malignant infratentorial tumors. Accordingly, G207, a genetically engineered herpes simplex virus (HSV-1) capable of selectively targeting cancer cells has emerged as a promising therapeutic option for this patient population. Herein, we demonstrate that cerebellar inoculation of G207 was systemically non-toxic in an immunocompetent, HSV-1 sensitive mouse strain (CBA/J). Mice had neither abnormal brain/organ pathology nor evidence of G207 replication by immunohistochemistry at days 7 and 30 after cerebellar G207 inoculation. While a minute amount viral DNA was recovered in the cerebellum and brainstem of mice at day 7, no viral DNA persisted at day 30. Critically, G207 delivered to the cerebellum was able to target/treat the highly aggressive MYC-overexpressed group 3 murine medulloblastoma increasing survival vs controls. These results provide critical safety and efficacy data to support the translation of G207 for pediatric clinical trials in intractable cerebellar malignancies.

Antiangiogenic therapy can rapidly reduce vascular permeability and cerebral edema but high doses of bevacizumab may induce selective pressure to promote resistance. This trial evaluated the efficacy of low dose bevacizumab in combination with lomustine (CCNU) compared to standard dose bevacizumab in patients with recurrent glioblastoma. Patients (N = 71) with recurrent glioblastoma who previously received radiation and temozolomide were randomly assigned 1:1 to receive bevacizumab monotherapy (10 mg/kg) or low dose bevacizumab (5 mg/kg) in combination with lomustine (90 mg/m(2)). The primary end point was progression-free survival (PFS) based on a blinded, independent radiographic assessment of post-contrast T1-weighted and non-contrast T2/FLAIR weighted magnetic resonance imaging (MRI) using RANO criteria. For 69 evaluable patients, median PFS was not significantly longer in the low dose bevacizumab + lomustine arm (4.34 months, CI 2.96-8.34) compared to the bevacizumab alone arm (4.11 months, CI 2.69-5.55, p = 0.19). In patients with first recurrence, there was a trend towards longer median PFS time in the low dose bevacizumab + lomustine arm (4.96 months, CI 4.17-13.44) compared to the bevacizumab alone arm (3.22 months CI 2.5-6.01, p = 0.08). The combination of low dose bevacizumab plus lomustine was not superior to standard dose bevacizumab in patients with recurrent glioblastoma. Although the study was not designed to exclusively evaluate patients at first recurrence, a strong trend towards improved PFS was seen in that subgroup for the combination of low dose bevacizumab plus lomustine. Further studies are needed to better identify such subgroups that may most benefit from the combination treatment.

INTRODUCTION. IDH1 mutation has been identified as a early genetic event driving low grade gliomas (LGGs) and it has been proven to exerts a powerful epigenetic effect. Cells containing IDH1 mutation are refractory to epigenetical reprogramming to iPSC induced by expression of Yamanaka transcription factors, a feature that we employed to study early genetic amplifications or deletions in gliomagenesis. METHODS. We made iPSC clones from freshly surgically resected IDH1 mutant LGGs by forced expression of Yamanaka transcription factors. We sequenced the IDH locus and analyzed the genetic composition of multiple iPSC clones by array-based comparative genomic hybridization (aCGH). RESULTS. We hypothesize that the primary cell pool isolated from LGG tumor contains a heterogeneous population consisting tumor cells at various stages of tumor progression including cells with early genetic lesions if any prior to acquisition of IDH1 mutation. Because cells containing IDH1 mutation are refractory to reprogramming, we predict that iPSC clones should originate only from LGG cells without IDH1 mutation, i.e. cells prior to acquisition of IDH1 mutation. As expected, we found that none of the iPSC clones contains IDH1 mutation. Further analysis by aCGH of the iPCS clones reveals that they contain regional chromosomal amplifications which are also present in the primary LGG cells. CONCLUSIONS. These results indicate that there exists a subpopulation of cells harboring gene amplification but without IDH1 mutation in the LGG primary cell pool. Further analysis of TCGA LGG database demonstrates that these regional chromosomal amplifications are also present in some cases of low grade gliomas indicating they are reoccurring lesions in glioma albeit at a low frequency. Taken together, these data suggest that regional chromosomal alterations may exist prior to the acquisition of IDH mutations in at least some cases of LGGs.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults,1 and a majority of patients eventually develop recurrence (at a median 8 months) after receiving trimodality therapy.2 In general, patients with recurrent GBM have a poor prognosis and have been treated with various modalities including surgery, chemotherapy, and/or salvage reirradiation.3,4 In 2009 bevacizumab (BVZ), a humanized monoclonal antibody that targets vascular endothelial growth factor (VEGF), was approved by the FDA as a single agent for recurrent GBM, with rationale for approval based largely on randomized phase II studies showing efficacy (6-month progression-free survival [PFS] of 40%–50%) and corticosteroid-sparing effects.5,6 More recently, 2 prospective clinical trials have reported the safety and efficacy of concurrent stereotactic radiosurgery (SRS) with BVZ for the treatment of recurrent high-grade gliomas.7,8 Both trials reported acceptable toxicity profiles, and the authors concluded that this treatment for recurrent GBM was well tolerated; radiographic responses and survival suggest that this is an active regimen for recurrent malignant gliomas. The results of these trials, combined with the results of a large institutional experience utilizing hypofractionated radiotherapy for recurrent malignant glioma,9 have prompted activation of the Radiation Therapy Oncology Group (RTOG) 1205 study, which is a prospective, randomized phase II trial comparing single agent BVZ versus hypofractionated radiotherapy concurrent with BVZ for recurrent GBM.10 Despite these initial reports and the ongoing multicenter randomized trial, a number of clinical questions remain (and will likely persist even after results from RTOG 1205 are reported) due to differences in radiation technique, dose/fractionation, usage of temozolomide, reirradiation sequence (as first versus subsequent salvage therapies), and heterogeneous patient populations between trials. The purpose of this report is to add to the growing body of literature describing the technique, toxicity, and efficacy of concurrent BVZ with SRS for recurrent glioma in an effort to further tailor therapy for those patients most likely to benefit from this treatment. Though the underlying rationale for usage of BVZ concurrent with hypofractionated SRS at our institution is congruent with the rationale for previously reported trials, we have treated a patient population that is slightly more inclusive than those in the aforementioned clinical trials, especially inclusion of patients with larger targets and/or with the addition of chemotherapy (temozolomide or CCNU) with BVZ and SRS.

Objectives: This case report describes two cases of high-dose methotrexate–induced nephrotoxicity: death in the case of conventional supportive care and successful renal function recovery in a patient treated with glucarpidase and continuous dialysis. Methods: High dose methotrexate is widely used for management of adult and pediatric malignancies. However, high-dose methotrexate–induced renal nephrotoxicity may cause severe, even lethal complications. Here we present examples of such outcomes. Results: We present one case of lethal high-dose methotrexate nephrotoxicity in a patient treated with conventional rescue therapy. We contrast this outcome with another patient with high-dose methotrexate–induced anuric acute kidney injury, who has recovered renal function following therapy with glucarpidase and continuous dialysis. Conclusions: This is only the second reported case of high-dose methotrexate–induced anuric acute kidney injury, and the only one with a reported clinical outcome. This first report of recovery from high-dose methotrexate–induced anuric acute kidney injury after glucarpidase administration supports available evidence pointing to the effectiveness of this therapy.

Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however, the efficacy of γ(1)34.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric human cytolomegalovirus (HCMV)/HSV-1 virus, which expresses the human CMV protein kinase R evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared with the γ(1)34.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia. Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

Myofibroblasts are effector cells in fibrotic disorders that synthesize and remodel the extracellular matrix (ECM). This study investigated the role of the Src kinase pathway in myofibroblast activation in vitro and fibrogenesis in vivo. The profibrotic cytokine, transforming growth factor β1 (TGF-β1), induced rapid activation of Src kinase, which led to myofibroblast differentiation of human lung fibroblasts. The Src kinase inhibitor AZD0530 (saracatinib) blocked TGF-β1-induced Src kinase activation in a dose-dependent manner. Inhibition of Src kinase significantly reduced α-smooth muscle actin (α-SMA) expression, a marker of myofibroblast differentiation, in TGF-β1-treated lung fibroblasts. In addition, the induced expression of collagen and fibronectin and three-dimensional collagen gel contraction were also significantly inhibited in AZD0530-treated fibroblasts. The therapeutic efficiency of Src kinase inhibition in vivo was tested in the bleomycin murine lung fibrosis model. Src kinase activation and collagen accumulation were significantly reduced in the lungs of AZD0530-treated mice when compared with controls. Furthermore, the total fibrotic area and expression of α-SMA and ECM proteins were significantly decreased in lungs of AZD0530-treated mice. These results indicate that Src kinase promotes myofibroblast differentiation and activation of lung fibroblasts. Additionally, these studies provide proof-of-concept for targeting the noncanonical TGF-β signaling pathway involving Src kinase as an effective therapeutic strategy for lung fibrosis.

Src family kinases (SFKs) are highly expressed and active in clinical glioblastoma multiforme (GBM) specimens. SFKs inhibitors have been demonstrated to inhibit proliferation and migration of glioma cells. However, the role of SFKs in glioma stem cells (GSCs), which are important for treatment resistance and recurrence, has not been reported. Here, we examined the expression pattern of individual members of SFKs and their functional role in CD133⁺ GSCs in comparison to primary glioma cells. We found that Fyn, c-Src and Yes were robustly expressed in GSCs while Lck was absent. Knockdown of c-Src, Yes or treatment with the SFK inhibitor dasatinib inhibited the migration of GSCs, but had no impact on their growth or self-renewal. These results suggest that SFKs represent an effective target for GSC migration but not for their growth.

Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of ω-NG,N'G-symmetric dimethylarginine residues on histones as well as other proteins. These modifications play an important role in cell differentiation and tumor cell growth. However, the role of PRMT5 in human glioma cells has not been characterized. In this study, we assessed protein expression profiles of PRMT5 in control brain, WHO grade II astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme (GBM) by immunohistochemistry. PRMT5 was low in glial cells in control brain tissues and low grade astrocytomas. Its expression increased in parallel with malignant progression, and was highly expressed in GBM. Knockdown of PRMT5 by small hairpin RNA caused alterations of p-ERK1/2 and significantly repressed the clonogenic potential and viability of glioma cells. These findings indicate that PRMT5 is a marker of malignant progression in glioma tumors and plays a pivotal role in tumor growth.

Triple negative breast cancer (TNBC) is characterized by rapid growth, metastasis, and high recurrence. The severe adverse effects and drug resistance associated with standard cytotoxic chemotherapies minimize their clinical benefits. The anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) had been developed and tested to treat TNBC, but no significant efficiency was demonstrated in clinical trials. The goal of this study was to develop a novel antibody-drug conjugate (ADC) to treat TNBC. Specifically, we established a platform to conjugate anti EGFR mAb with mertansine, which inhibits cell division by blocking tubulin polymerization, and also improved the conjugation efficiency by optimizing the linker and drug-to-antibody ratio. Flow cytometry and live cell confocal laser scanning microscopy analysis confirmed that the anti-EGFR ADC can specifically bind to multiple TNBC subtypes, is effectively internalized, and release potent drug intracellularly within 1 hour after mixing. The IC50 study showed that the anti-EGFR ADC killed >90% TNBC cells with dose of 5 nM. The TNBC xenograft mouse model also showed that the constructed ADC had high anticancer efficacy and successfully inhibited tumor growth. Moreover, the live-animal imaging using In Vivo Imaging System and the tissue microarray samples with immunohistochemical analysis of TNBC and organs indicated that our ADC specifically target TNBC in vivo. Further evaluations, such as maximal tolerated dose (MTD), pharmacokinetics (PK), and biodistribution, are needed. To summarize, we developed an EGFR-targeted ADC, which could provide an effective targeted therapy to treat TNBC and improve the life quality and survival rate of patients with TNBC. Note: This abstract was not presented at the meeting. Citation Format: Yingnan Si, Nghi Dang, Seulhee Kim, Lufang Zhou, Xiaosi Han, Runhua Liu, Xiaoguang (Margaret) Liu. Antibody drug conjugate to treat triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 537.

Background Low-grade gliomas cause significant neurological morbidity by brain invasion. There is no universally accepted objective technique available for detection of enlargement of low-grade gliomas in the clinical setting; subjective evaluation by clinicians using visual comparison of longitudinal radiological studies is the gold standard. The aim of this study is to determine whether a computer-assisted diagnosis (CAD) method helps physicians detect earlier growth of low-grade gliomas. Methods and findings We reviewed 165 patients diagnosed with grade 2 gliomas, seen at the University of Alabama at Birmingham clinics from 1 July 2017 to 14 May 2018. MRI scans were collected during the spring and summer of 2018. Fifty-six gliomas met the inclusion criteria, including 19 oligodendrogliomas, 26 astrocytomas, and 11 mixed gliomas in 30 males and 26 females with a mean age of 48 years and a range of follow-up of 150.2 months (difference between highest and lowest values). None received radiation therapy. We also studied 7 patients with an imaging abnormality without pathological diagnosis, who were clinically stable at the time of retrospective review (14 May 2018). This study compared growth detection by 7 physicians aided by the CAD method with retrospective clinical reports. The tumors of 63 patients (56 + 7) in 627 MRI scans were digitized, including 34 grade 2 gliomas with radiological progression and 22 radiologically stable grade 2 gliomas. The CAD method consisted of tumor segmentation, computing volumes, and pointing to growth by the online abrupt change-of-point method, which considers only past measurements. Independent scientists have evaluated the segmentation method. In 29 of the 34 patients with progression, the median time to growth detection was only 14 months for CAD compared to 44 months for current standard of care radiological evaluation (p < 0.001). Using CAD, accurate detection of tumor enlargement was possible with a median of only 57% change in the tumor volume as compared to a median of 174% change of volume necessary to diagnose tumor growth using standard of care clinical methods (p < 0.001). In the radiologically stable group, CAD facilitated growth detection in 13 out of 22 patients. CAD did not detect growth in the imaging abnormality group. The main limitation of this study was its retrospective design; nevertheless, the results depict the current state of a gold standard in clinical practice that allowed a significant increase in tumor volumes from baseline before detection. Such large increases in tumor volume would not be permitted in a prospective design. The number of glioma patients (n = 56) is a limitation; however, it is equivalent to the number of patients in phase II clinical trials. Conclusions The current practice of visual comparison of longitudinal MRI scans is associated with significant delays in detecting growth of low-grade gliomas. Our findings support the idea that physicians aided by CAD detect growth at significantly smaller volumes than physicians using visual comparison alone. This study does not answer the questions whether to treat or not and which treatment modality is optimal. Nonetheless, early growth detection sets the stage for future clinical studies that address these questions and whether early therapeutic interventions prolong survival and improve quality of life., Hassan Fathallah-Shaykh and colleagues reveal an improved approach for detection of growth in low-grade gliomas., Author summary Why was this study done? Low-grade gliomas constitute 15% of all adult brain tumors and cause significant neurological morbidity by brain invasion. There is no universally accepted objective technique available for detection of enlargement of low-grade gliomas in the clinical setting. The current gold standard is subjective evaluation by clinicians using visual comparison of 2D images from longitudinal radiological studies. To improve visual evaluation, a computer-assisted diagnostic procedure that digitizes the tumor and generates volumetric measures could enhance detection of tumor growth by directing the attention of the physician to a change in volume. What did the researchers do and find? We studied the longitudinal radiological studies of 63 patients with a median follow-up period of 33.6 months, and compared detection of growth by 7 physicians aided by the computer-assisted diagnostic procedure to detection of growth by the standard method (visual comparison). The computer-assisted method helped physicians detect growth at significantly earlier times and at significantly smaller tumor volumes than the current standard method. Physicians aided by the computer-assisted method diagnosed tumor growth in 13 of 22 glioma patients labeled as clinically stable by the standard method. Having tumor volume measurements and time series, we were able to identify tumors with nonlinear and nonhomogeneous growth. What do these findings mean? Current clinical practice is associated with significant delays in detecting growth of low-grade gliomas. Earlier growth detection and detection at smaller tumor volumes are desirable because smaller tumors are associated with smaller fields of radiation, optimal surgical resections, and longer survival with less neurological morbidity. Early growth detection holds the potential of lowering the morbidity, and perhaps mortality, of patients with low-grade gliomas, a possibility that needs to be tested in prospective studies. In the future, there could be a policy of classifying and treating patients with low-grade gliomas based on tumor growth rates and direction of tumor growth. The decision to treat would be determined by the rate of growth and proximity to critical areas of the brain, once they have been measured.

Neural stem cells (NSCs) have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes, and therefore represent a promising donor tissue source for treating neurodegenerative diseases and repairing injuries of the nervous system. However, it remains unclear how canonical microRNAs (miRNAs), the subset of miRNAs requiring the Drosha-Dgcr8 microprocessor and the type III RNase Dicer for biogenesis, regulate NSCs. In this study, we established and characterized Dgcr8-/- NSCs from conditionally Dgcr8-disrupted mouse embryonic brain. RNA-seq analysis demonstrated that disruption of Dgcr8 in NSCs causes a complete loss of canonical miRNAs and an accumulation of pri-miRNAs. Dgcr8-/- NSCs can be stably propagated in vitro, but progress through the cell cycle at reduced rates. When induced for differentiation, Dgcr8-/- NSCs failed to differentiate into neurons, astrocytes, or oligodendrocytes under permissive conditions. Compared to Dgcr8+/- NSCs, Dgcr8-/- NSCs exhibit significantly increased DNA damage. Comparative RNA-seq analysis and gene set enrichment analysis (GSEA) revealed that Dgcr8-/- NSCs significantly downregulate genes associated with neuronal differentiation, cell cycle progression, DNA replication, protein translation, and DNA damage repair. Furthermore, we discovered that Dgcr8-/- NSCs significantly downregulate genes responsible for cholesterol biosynthesis and demonstrated that Dgcr8-/- NSCs contain lower levels of cholesterol. Together, our data demonstrate that canonical miRNAs play essential roles in enabling lineage specification, protecting DNA against damage, and promoting cholesterol biosynthesis in NSCs.

TGF-β1 induces an increase in paracellular permeability and actin stress fiber formation in lung microvascular endothelial and alveolar epithelial cells via small Rho GTPase. The molecular mechanism involved is not fully understood. Neuronal Wiskott-Aldrich syndrome protein (N-WASP) has an essential role in actin structure dynamics. We hypothesized that N-WASP plays a critical role in these TGF-β1-induced responses. In these cell monolayers, we demonstrated that N-WASP down-regulation by short hairpin RNA prevented TGF-β1-mediated disruption of the cortical actin structure, actin stress filament formation, and increased permeability. Furthermore, N-WASP down-regulation blocked TGF-β1 activation mediated by IL-1β in alveolar epithelial cells, which requires actin stress fiber formation. Control short hairpin RNA had no effect on these TGF-β1-induced responses. TGF-β1-induced phosphorylation of Y256 of N-WASP via activation of small Rho GTPase and focal adhesion kinase mediates TGF-β1-induced paracellular permeability and actin cytoskeleton dynamics. In vivo, compared with controls, N-WASP down-regulation increases survival and prevents lung edema in mice induced by bleomycin exposure-a lung injury model in which TGF-β1 plays a critical role. Our data indicate that N-WASP plays a crucial role in the development of TGF-β1-mediated acute lung injury by promoting pulmonary edema via regulation of actin cytoskeleton dynamics.-Wagener, B. M., Hu, M., Zheng, A., Zhao, X., Che, P., Brandon, A., Anjum, N., Snapper, S., Creighton, J., Guan, J.-L., Han, Q., Cai, G.-Q., Han, X., Pittet, J.-F., Ding, Q. Neuronal Wiskott-Aldrich syndrome protein regulates TGF-β1-mediated lung vascular permeability.

Rationale Tumor necrosis factor-alpha (TNF-α) is a potent pro-inflammatory mediator and its expression is up-regulated in chronic obstructive pulmonary disease (COPD). Tristetraprolin (TTP) is implicated in regulation of TNF-α expression; however, whether TTP is involved in cigarette smoke-induced TNF-α expression has not been determined. Methods TTP expression was examined by western blot analysis in murine alveolar macrophages and alveolar epithelial cells challenged without or with cigarette smoke extract (CSE). TNF-α mRNA stability, and the decay of TNF-α mRNA, were determined by real-time quantitative RT-PCR. TNF-α protein levels were examined at the same time in these cells. To identify the molecular mechanism involved, a construct expressing the human beta-globin reporter mRNA containing the TNF-α 3’-untranslated region was generated to characterize the TTP targeted site within TNF-α mRNA. Results CSE induced TTP down-regulation in alveolar macrophages and alveolar epithelial cells. Reduced TTP expression resulted in significantly increased TNF-α mRNA stability. Importantly, increased TNF-α mRNA stability due to impaired TTP function resulted in significantly increased TNF-α levels in these cells. Forced TTP expression abrogated the increased TNF-α mRNA stability and expression induced by CSE. By using the globin reporter construct containing TNF-α mRNA 3’-untranslated region, the data indicate that TTP directly targets the adenine- and uridine-rich region (ARE) of TNF-α mRNA and negatively regulates TNF-α expression at the post-transcriptional level. Conclusion The data demonstrate that cigarette smoke exposure reduces TTP expression and impairs TTP function, resulting in significantly increased TNF-α mRNA stability and excessive TNF-α expression in alveolar macrophages and epithelial cells. The data suggest that TTP is a novel post-transcriptional regulator and limits excessive TNF-α expression and inflammatory response induced by cigarette smoke.

Tumor surveillance is a common practice in oncology; its goal is timely detection of cancer progression, which is typically diagnosed by Visual Comparison (VC) or bidirectional measurement of longitudinal radiological images. Low-grade gliomas (LGG) can cause significant neurological morbidity by progressive brain invasion over time. We analyzed the longitudinal magnetic resonance imaging (MRI) studies of patients, diagnosed with grade 2 gliomas without any radiation therapy at the initial diagnosis, who had at least 4 MRI studies. Forty-eight patients met the inclusion criteria, including 13 oligodendrogliomas, 24 astrocytomas and 11 mixed gliomas. Thirty-four patients had clinical progression (CP) and 14 patients were considered clinically stable (CS) by VC at the time of the study. The computer assisted diagnosis (CAD) method consisted of segmentation of the fluid-attenuated inversion recovery (FLAIR) images, computing tumor volumes, and determining progression by the online abrupt change of point method, which considered only past MRIs. In the CP group, CAD detected progression at earlier times than the clinical diagnosis in 26/34 patients. In the CS group, CAD detected progression in 10/14 patients. Six physicians from the departments of neurology (neuro-oncology), radiology, and radiation oncology reviewed and agreed with the progression determined by the CAD method including the study subjects in the CS group. CAD was able to diagnose tumor progression when the tumor size grew at an average of 74% (range 14% to 364%) since the base line compared to 320% (range 25% to 2019%) in the VC method. The average time to progression was only 17 months when CAD was used compared to 50 months when VC method was used. Early detection of tumor progression is critical for LGG patients because it can optimize therapeutic options with significantly lower volume of brain resection or irradiation

As systemic cancer therapies have improved, the natural history and importance of treating brain metastases continues to evolve. Historically, most patients with brain metastases have been managed with whole brain radiation therapy (WBRT) with surgical resection or radiosurgery added for patients with single or few metastases. Because the potential late toxicity of WBRT is increasingly recognized when systemic tumor is more effectively controlled, there has been increased interest in the use of focal therapies such as radiosurgery with deferred WBRT even for patients with larger numbers of metastases. Although WBRT in combination with radiosurgery or surgical resection significantly reduces central nervous system recurrences at the treated site and elsewhere in the brain, it is not clear whether a patient's quality of life is more affected by tumor recurrence or by treatment with WBRT. In our practice, most patients with fewer than 7 to 10 tumors are treated with radiosurgery alone, with WBRT initially deferred because of concerns about its late toxicity. The ongoing technical improvements in radiosurgery have made this transition away from WBRT clinically feasible. This approach also allows patients to begin systemic therapy sooner, rather than waiting 2 to 4 weeks to complete WBRT. For patients with large or very symptomatic tumors, surgical resection is performed, followed by postoperative radiosurgery to the resection cavity, again initially deferring WBRT for many patients. This focal-only approach in the postoperative setting is associated with a higher rate of subdural dissemination and needs further prospective study, as some would argue that tumor progression is the major determinant of loss of function. Ultimately, better survival will require better systemic therapy that both controls extracranial disease and penetrates the brain to reduce intracranial recurrences. Unfortunately, many clinical trials of novel agents exclude patients with brain metastases.

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia. DOI: http://dx.doi.org/10.7554/eLife.07938.001, eLife digest The many different cell types in an adult animal all develop from a single fertilized egg. The development of cells into more specialized cell types is called ‘differentiation’. Proteins and other molecules from both inside and outside of the cells regulate the differentiation process. RNA is a molecule that is similar to DNA, and performs several important roles inside cells. Perhaps most importantly, RNA molecules act as messengers and carry genetic instructions during gene expression. RBM15 is an RNA-binding protein that is found throughout nature, and is involved in a number of developmental processes. Previous research has linked the incorrect control of RBM15 with an increased risk of certain cancers, including megakaryocytic leukemia. However, it is not clear what role RNA-binding proteins such as RBM15 play during differentiation. Now, Zhang, Tran, Su et al. have investigated the role of RBM15 during the development of large cells found in human bone marrow (called megakaryocytes). First, the experiments demonstrated that an enzyme called PRMT1 modifies RBM15. This enzyme adds a chemical mark called a methyl group at a specific site (an arginine amino acid) on the RNA-binding protein. Next, Zhang, Tran, Su et al. showed that the addition of this methyl group earmarks RBM15 for destruction. This means that an increase in PRMT1 levels reduces the amount of RBM15 in cells, while decreases in PRMT1 have the opposite effect. Further experiments showed that RBM15 normally processes the RNA messengers that carry the genetic instructions needed for the differentiation of bone marrow cells. An excess of PRMT1 enzyme leads to a lack of this RNA-binding protein. This in turn interferes with the differentiation process, and can contribute to the development of cancers such as megakaryocytic leukemia. Future work will therefore explore whether targeting PRMT1 with drugs could represent an effective treatment for these kinds of cancers. DOI: http://dx.doi.org/10.7554/eLife.07938.002

Background Glioblastoma (GBM) is the most common malignant brain tumor, and glioma stem cells (GSCs) are considered a major source of treatment resistance for glioblastoma. Identifying new compounds that inhibit the growth of GSCs and understanding their underlying molecular mechanisms are therefore important for developing novel therapy for GBM. Methods We investigated the potential inhibitory effect of isorhapontigenin (ISO), an anticancer compound identified in our recent investigations, on anchorage-independent growth of patient-derived glioblastoma spheres (PDGS) and its mechanism of action. Results ISO treatment resulted in significant anchorage-independent growth inhibition, accompanied with cell cycle G0-G1 arrest and cyclin D1 protein downregulation in PDGS. Further studies established that cyclin D1 was downregulated by ISO at transcription levels in a SOX2-dependent manner. In addition, ISO attenuated SOX2 expression by specific induction of miR-145, which in turn suppressed 3'UTR activity of SOX2 mRNA without affecting its mRNA stability. Moreover, ectopic expression of exogenous SOX2 rendered D456 cells resistant to induction of cell cycle G0-G1 arrest and anchorage-independent growth inhibition upon ISO treatment, whereas inhibition of miR-145 resulted in D456 cells resistant to ISO inhibition of SOX2 and cyclin D1 expression. In addition, overexpression of miR-145 mimicked ISO treatment in D456 cells. Conclusions ISO induces miR-145 expression, which binds to the SOX2 mRNA 3'UTR region and inhibits SOX2 protein translation. Inhibition of SOX2 leads to cyclin D1 downregulation and PDGS anchorage-independent growth inhibition. The elucidation of the miR-145/SOX2/cyclin D1 axis in PDGS provides a significant insight into understanding the anti-GBM effect of ISO compound.

Acute disseminated encephalomyelitis (ADEM) is a disease characterized by inflammation and destruction of myelin. Acute hemorrhagic leukoencephalitis (AHLE) is a severe form of ADEM known for its particularly poor outcome. We present a case of a young Caucasian female who presented with drowsiness and slurred speech followed by rapid brainstem involvement resembling rhomboencephalitis. Despite multiple diagnostic tests and empiric therapy with immunosuppressants, immunoglobulins, and antimicrobials, she lost most brainstem reflexes within a few weeks and ultimately passed away. Magnetic resonance imaging (MRI) showed progression of lesions from the brainstem to eventually involve bilateral cerebral hemispheres. Autopsy and microscopic examination of the brain revealed several hemorrhagic lesions throughout the brain and rendered a diagnosis of AHLE. AHLE was initially described in 1941 and is thought to be autoimmune related, possibly related to cross reactivity between the immune system and CNS tissues like myelin. While a definitive inciting pathogen was not discovered, this case emphasizes the importance of considering AHLE in the differential diagnosis of patients with rapid loss of neurologic function and highlights an atypical presentation of ADEM/AHLE.

Background Childhood medulloblastoma is associated with significant morbidity and mortality that is compounded by neurotoxicity for the developing brain caused by current therapies, including surgery, craniospinal radiation, and chemotherapy. Innate therapeutic resistance of some aggressive pediatric medulloblastoma has been attributed to a subpopulation of cells, termed cancer-initiating cells or cancer stemlike cells (CSCs), marked by the surface protein CD133 or CD15. Brain tumors characteristically contain areas of pathophysiologic hypoxia, which has been shown to drive the CSC phenotype leading to heightened invasiveness, angiogenesis, and metastasis. Novel therapies that target medulloblastoma CSCs are needed to improve outcomes and decrease toxicity. We hypothesized that oncolytic engineered herpes simplex virus (oHSV) therapy could effectively infect and kill pediatric medulloblastoma cells, including CSCs marked by CD133 or CD15. Methods Using 4 human pediatric medulloblastoma xenografts, including 3 molecular subgroup 3 tumors, which portend worse patient outcomes, we determined the expression of CD133, CD15, and the primary HSV-1 entry molecule nectin-1 (CD111) by fluorescence activated cell sorting (FACS) analysis. Infectability and cytotoxicity of clinically relevant oHSVs (G207 and M002) were determined in vitro and in vivo by FACS, immunofluorescent staining, cytotoxicity assays, and murine survival studies. Results We demonstrate that hypoxia increased the CD133+ cell fraction, while having the opposite effect on CD15 expression. We established that all 4 xenografts, including the CSCs, expressed CD111 and were highly sensitive to killing by G207 or M002. Conclusions Pediatric medulloblastoma, including Group 3 tumors, may be an excellent target for oHSV virotherapy, and a clinical trial in medulloblastoma is warranted.

Bevacizumab is widely used for treatment of high-grade gliomas and other malignancies. Because bevacizumab has been shown to be associated with neurocognitive decline, this study is designed to investigate whether prolonged treatment with bevacizumab is also associated with brain atrophy. We identified 12 high-grade glioma patients who received bevacizumab for 12 months at the first recurrence and 13 matched controls and blindly compared the volumes of the contralateral hemispheres and contralateral ventricle in these two groups at baseline and after 12 ± 2 months of the baseline scan by two independent analyses. The volumes of the contralateral hemispheres and ventricles did not differ significantly between the two groups at baseline. Whereas, in the control group the volumes of the contralateral hemisphere changed subtly from baseline to follow-up (p = 0.23), in the bevacizumab-treated group the volumes significantly decreased from baseline to follow-up (p = 0.03). There was significant increase in the contralateral ventricle volume from base line to follow-up scans in both the control group (p = 0.01) and in the bevacizumab group (p = 0.005). Both the absolute and the percentage changes of contralateral hemisphere volumes and contralateral ventricular volumes between the two patient groups were statistically significant (p < 0.05). Results of this study demonstrate prolonged treatment with bevacizumab is associated with atrophy of the contralateral brain hemisphere.

Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of ω-NG,N′G-symmetric dimethylarginine residues on histones as well as other proteins. The modification play an important role in cell differentiation and tumor cell growth. However, the role of PRMT5 in human glioma cells has not been characterized. In this study, we assessed protein expression profiles of PRMT5 in control brain, WHO grade II astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme (GBM) by immunohistochemistry. PRMT5 was low in glial cells in control brain tissues and low grade astrocytomas. Its expression increased in parallel with malignant progression, and was highly expressed in GBM. Knockdown of PRMT5 by small hairpin RNA caused alterations of p-ERK1/2 and significantly repressed the clonogenic potential and viability of glioma cells. These findings indicate that PRMT5 is a marker of malignant progression in glioma tumors and plays a pivotal role in tumor growth.

S100A4 expression is associated with poor clinical outcomes of patients with pancreatic cancer. The effects of loss or gain of S100A4 were examined in pancreatic cancer cell lines. S100A4 downregulation remarkably reduces cell migration and invasion, inhibits proliferation and induces apoptosis in pancreatic tumor cells. S100A4 downregulation results in significant cell growth inhibition and apoptosis in response to TGF-β1, supporting a non-canonical role of S100A4 in pancreatic cancer. The role of S100A4 in tumor progression was studied by using an orthotopic human pancreatic cancer xenograft mouse model. Tumor mass is remarkably decreased in animals injected with S100A4-deficient pancreatic tumor cells. P27Kip1 expression and cleaved caspase-3 are increased, while cyclin E expression is decreased, in S100A4-deficient pancreatic tumors in vivo. S100A4-deficient tumors have lower expression of vascular endothelial growth factor, suggesting reduced angiogenesis. Biochemical assays revealed that S100A4 activates Src and focal adhesion kinase (FAK) signaling events and inhibition of both kinases is required to maximally block the tumorigenic potential of pancreatic cancer cells. These findings support that S100A4 plays an important role in pancreatic cancer progression in vivo and S100A4 promotes tumorigenic phenotypes of pancreatic cancer cells through the Src-FAK mediated dual signaling pathway.

Reiterative transcription is a reaction catalyzed by RNA polymerase, in which nucleotides are repetitively added to the 3′ end of a nascent transcript due to upstream slippage of the transcript without movement of the DNA template. In Escherichia coli , the expression of several operons is regulated through mechanisms in which high intracellular levels of UTP promote reiterative transcription that adds extra U residues to the 3′ end of a nascent transcript during transcription initiation. Immediately following the addition of one or more extra U residues, the nascent transcripts are released from the transcription initiation complex, thereby reducing the level of gene expression. Therefore, gene expression can be regulated by internal UTP levels, which reflect the availability of external pyrimidine sources. The magnitude of gene regulation by these mechanisms varies considerably, even when control mechanisms are analogous. These variations apparently are due to differences in promoter sequences. One of the operons regulated (in part) by UTP-sensitive reiterative transcription in E. coli is the carAB operon, which encodes the first enzyme in the pyrimidine nucleotide biosynthetic pathway. In this study, we used the carAB operon to examine the effects of nucleotide sequence at and near the transcription start site and spacing between the start site and −10 region of the promoter on reiterative transcription and gene regulation. Our results indicate that these variables are important determinants in establishing the extent of reiterative transcription, levels of productive transcription, and range of gene regulation.

Cytokines can influence the interactions between members of the integrin cell adhesion receptor family and the extracellular matrix thereby potentially affecting cell function and promoting cell adhesion, growth and migration of malignant astrocytoma tumor cells. As malignant astrocytoma cells synthesize TGF-beta1 in vivo, we analysed the effects of TGF-beta1 on signaling events associated with integrin receptor ligation, focusing on the effects on paxillin, a phosphorylated adaptor protein, that acts as a scaffold for signaling molecules recruited to focal adhesions. TGF-beta1-stimulation of primary astrocytes and serum-starved U-251MG malignant astrocytoma cells attached to fibronectin induced a substantial increase in the levels of paxillin protein (fivefold increase at 2.0 ng/ml) in a dose- and time-dependent manner compared to the levels observed on plating onto fibronectin in the absence of stimulation. In the astrocytoma cells, this resulted in an increase in the pool of tyrosine-phosphorylated paxillin, although it did not appear to alter the extent of phosphorylation of the paxillin molecules. In contrast, in primary astrocytes the protein levels were upregulated in the absence of a parallel increase in phosphorylation. The TGF-beta1-stimulated increase in paxillin levels required ligation of the fibronectin receptor, as it was not induced when the cells were plated onto vitronectin, collagen or laminin. The increase in the pool of paxillin on TGF-beta1 stimulation of the fibronectin-plated astrocytoma cells was associated with an increase in translation, but was not associated with an increase in the steady-state levels of paxillin mRNA. Stimulation with TGF-beta1 on a fibronectin substrate increased subsequent attachment and spreading of U-251MG cells onto fibronectin and, to a lesser extent, vitronectin, but not collagen. Our results indicate that physiologic levels of TGF-beta1 stimulate the expression of paxillin protein at the level of translation through a process that requires engagement of the fibronectin receptor, and promotes attachment and spreading of malignant astrocytoma cells on fibronectin.

In Escherichia coli , expression of the carAB operon is subject to cumulative repression, which occurs by ArgR-mediated repression at a downstream promoter, P2, and by pyrimidine-mediated regulation at an upstream promoter, P1. In this study, we show that pyrimidine-mediated regulation occurs in part through a mechanism involving UTP-sensitive reiterative transcription (i.e., repetitive addition of U residues to the 3′ end of a nascent transcript due to transcript-template slippage). In this case, reiterative transcription occurs at the end of a run of three T · A base pairs in the initially transcribed region of the carAB P1 promoter. The sequence of this region is 5′-GTTTGC (nontemplate strand). In the proposed regulatory mechanism, increased intracellular levels of UTP promote reiterative transcription, which results in the synthesis of transcripts with the sequence GUUUU n (where n = 1 to >30). These transcripts are not extended downstream to include structural gene sequences. In contrast, lower levels of UTP enhance normal template-directed addition of a G residue at position 5 of the nascent transcript. This addition precludes reiterative transcription and permits normal transcript elongation capable of producing translatable carAB transcripts. Thus, carAB expression, which is necessary for pyrimidine nucleotide (and arginine) biosynthesis, increases in proportion to the cellular need for UTP. The proposed mechanism appears to function independently of a second pyrimidine-mediated control mechanism that involves the regulatory proteins CarP and integration host factor.

Malignant gliomas comprise a significant number of new cases of brain cancer diagnosed in the United States each year. Despite recent therapeutic advances, they remain associated with high morbidity and mortality rates. The current standard of care for newly diagnosed malignant gliomas includes surgical resection followed by radiotherapy with concomitant and adjuvant Temozolomide. Bevacizumab, a humanized anti-VEGF (vascular endothelial growth factor) monoclonal antibody, has recently gained FDA approval for use in the treatment of recurrent glioblastoma multiforme (GBM), based on clinical trials that revealed both efficacy and favorable side-effects. Anti-angiogenic therapy has raised new hopes, new basic and clinical questions, and has uncovered new insights into the biological and clinical behavior of these tumors. Here, we review the medical, social, and economic significance of gliomas, discuss briefly the evolution of therapeutic modalities leading to anti-angiogenesis, depict the basic mechanisms of angiogenesis, detail data from Bevacizumab clinical trials, and address new clinical issues leading to the revision of the Macdonald criteria. We close with unresolved questions and potential future directions.

2005 Background: Antiangiogenic therapy can rapidly reduce vascular permeability, but high doses of bevacizumab (BEV) may induce selective pressure to promote resistance. This trial evaluated the e...