Neuronal Wiskott-Aldrich syndrome protein regulates Pseudomonas aeruginosa-induced lung vascular permeability through the modulation of actin cytoskeletal dynamics.

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 ß-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 αVß6 association as well as TGF-ß-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.

Molecular mechanism of tumour necrosis factor alpha regulates hypocretin (orexin) expression, sleep and behaviour.

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.

Diagnosing growth in low-grade gliomas with and without longitudinal volume measurements: A retrospective observational study.

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.

Characterization of iPSCs derived from low grade gliomas revealed early regional chromosomal amplifications during gliomagenesis.

INTRODUCTION: IDH1 mutation has been identified as an 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 iPSC 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.

Neuronal Wiskott-Aldrich syndrome protein regulates TGF-ß1-mediated lung vascular permeability.

TGF-ß1 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-ß1-induced responses. In these cell monolayers, we demonstrated that N-WASP down-regulation by short hairpin RNA prevented TGF-ß1-mediated disruption of the cortical actin structure, actin stress filament formation, and increased permeability. Furthermore, N-WASP down-regulation blocked TGF-ß1 activation mediated by IL-1ß in alveolar epithelial cells, which requires actin stress fiber formation. Control short hairpin RNA had no effect on these TGF-ß1-induced responses. TGF-ß1-induced phosphorylation of Y256 of N-WASP via activation of small Rho GTPase and focal adhesion kinase mediates TGF-ß1-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-ß1 plays a critical role. Our data indicate that N-WASP plays a crucial role in the development of TGF-ß1-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-ß1-mediated lung vascular permeability.

A randomized phase II trial of standard dose bevacizumab versus low dose bevacizumab plus lomustine (CCNU) in adults with recurrent glioblastoma.

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.

Prolonged treatment with bevacizumab is associated with brain atrophy: a pilot study in patients with high-grade gliomas.

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.

Transcription start site sequence and spacing between the -10 region and the start site affect reiterative transcription-mediated regulation of gene expression in Escherichia coli.

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.

Therapeutic targeting of SRC kinase in myofibroblast differentiation and pulmonary fibrosis.

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 ß1 (TGF-ß1), induced rapid activation of Src kinase, which led to myofibroblast differentiation of human lung fibroblasts. The Src kinase inhibitor AZD0530 (saracatinib) blocked TGF-ß1-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-ß1-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-ß signaling pathway involving Src kinase as an effective therapeutic strategy for lung fibrosis.

Role of MRI in primary brain tumor evaluation.

The NCCN Clinical Practice Guidelines in Oncology for Central Nervous System Cancers use radiologic presentation in the initial evaluation of patients with primary brain tumors and in the determination of response to therapy. The dominant modality use is MRI because of its superior image resolution, speed of acquisition, and high safety profile for patients. The interpretation of MRI is a critical aspect of patient care and evaluation. This article reviews the predominant aspects of MRI for brain tumors, the standard sequences, the criteria to consider in determining treatment response, and advanced aspects currently available. The proper integration of this essential imaging modality into patient care ensures timely disease evaluation and guides the use of therapeutic tools.

Survival analysis in patients with newly diagnosed primary glioblastoma multiforme using pre- and post-treatment peritumoral perfusion imaging parameters.

The objective of this study was to evaluate if peritumoral (PT) perfusion parameters obtained from dynamic susceptibility weighted contrast enhanced perfusion MRI can predict overall survival (OS) and progression free survival (PFS) in patients with newly diagnosed glioblastoma multiforme (GBM). Twenty-eight newly diagnosed GBM patients, who were treated with resection followed by concurrent chemoradiation and adjuvant chemotherapy, were included in this study. Evaluated perfusion parameters were pre- and post-treatment PT relative cerebral blood volume (rCBV) and relative cerebral blood flow (rCBF). Proportional hazard analysis was used to assess the relationship OS, PFS and perfusion parameters. Kaplan-Meier survival estimates and log-rank test were used to characterize and compare the patient groups with high and low perfusion parameter values in terms of OS and PFS. Pretreatment PT rCBV and rCBF were not associated with OS and PFS whereas there was statistically significant association of both posttreatment PT rCBV and rCBF with OS and posttreatment rCBV with PFS (association of PFS and posttreatment rCBF was not statistically significant). Neither the Kaplan-Meier survival estimates nor the log-rank test demonstrated any differences in OS between high and low pretreatment PT rCBV values and rCBF values; however, high and low post-treatment PT rCBV and rCBF values did demonstrate statistically significant difference in OS and PFS. Our study found posttreatment, not pretreatment, PT perfusion parameters can be used to predict OS and PFS in patients with newly diagnosed GBM.

Expression of PRMT5 correlates with malignant grade in gliomas and plays a pivotal role in tumor growth in vitro.

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.

Generation of chromosome 1p/19q co-deletion by CRISPR/Cas9-guided genomic editing.

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.

Safety and Efficacy of Intraventricular Immunovirotherapy with Oncolytic HSV-1 for CNS Cancers.

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.

Integrin αvß3 Engagement Regulates Glucose Metabolism and Migration through Focal Adhesion Kinase (FAK) and Protein Arginine Methyltransferase 5 (PRMT5) in Glioblastoma Cells.

Metabolic reprogramming promotes glioblastoma cell migration and invasion. Integrin αvß3 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 αvß3 integrin in regulating metabolic reprogramming and its mechanism of action have not been determined in glioblastoma cells. Integrin αvß3 engagement with osteopontin promotes glucose uptake and aerobic glycolysis, while inhibiting mitochondrial oxidative phosphorylation. Blocking or downregulation of integrin αvß3 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 αvß3 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.

Antibody-Drug Conjugate to Treat Meningiomas.

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 >98% to meningioma CH157-MN cells but a low binding rate of <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 <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.

Methylation of dual-specificity phosphatase 4 controls cell differentiation.

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.

Management of brain metastases.

OPINION STATEMENT: 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.

Safety and efficacy of oncolytic HSV-1 G207 inoculated into the cerebellum of mice.

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.

Enhanced Sensitivity of Patient-Derived Pediatric High-Grade Brain Tumor Xenografts to Oncolytic HSV-1 Virotherapy Correlates with Nectin-1 Expression.

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.