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Circadian rhythms regulate a remarkable variety of physiologic functions in living organisms. Circadian disruption is associated with tumorigenesis and tumor progression through effects on cancer cell biological properties, including proliferation, DNA repair, apoptosis, metabolism, and stemness. Emerging evidence indicates that circadian clocks also play an influential role in the tumor microenvironment (TME). This review outlines the recent discoveries on how cancer cell clock components (including circadian clock and clock genes/proteins) regulate TME biology and, reciprocally, how TME clock components affect tumor growth, metastasis, and therapeutic response. An improved understanding of how clock components regulate the symbiosis between cancer cells and TME will inform the development of novel clock-oriented therapeutic strategies, including immunotherapy.

Objective. The objective of this research was to describe and analyze the role of psychological and behavioral factors on perceptions of COVID-19 in France and Quebec at three different times during the pandemic. Design. We conducted three qualitative and quantitative studies (Study 1 N = 255, Study 2 N = 230, Study 3 N = 143). Participants were asked to evaluate psychological and behavioral measures: at the beginning of lockdown (Study 1), during lockdown (Study 2), and during lockdown exit (Study 3). Results. Results of Study 1 show that perceptions of COVID-19 are organized around fear and a sense of threat. During the lockdown, participants mentioned for the first time the health practices to prevent the spread of COVID-19 (Study 2). Psychological and social impacts constitute a central theme in participants’ discourse (Study 2 and 3). Conclusions. The results show that perceptions of risk during a pandemic are socially constructed. Perceptions seem to be influenced by the political and health management of a territory and by the evolution of behavioral and psychological responses.

Purpose: The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. Experimental Design: To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. Results: We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. Conclusions: Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.

The distillery spent wash consists of extremely high COD values, high content of pollutants and has an objectionable dark colour. These properties mark the effluent as one of the challenging tasks ...

Background Lymphocyte antigen 6 complex, locus K (LY6K) is a putative oncogene in various cancers. Elevated expression of LY6K is correlated with poor patient prognosis in glioblastoma (GBM). The aim of this study is to advance our understanding of the mechanism by which LY6K contributes to GBM tumor biology. Methods Bioinformatic data mining was used to investigate LY6K expression in relation to GBM clinical outcome. To understand the role of LY6K in GBM, we utilized patient-derived glioma stemlike cells (GSCs) and U87 cells and employed immunoblotting, immunofluorescent staining, radiation treatment, and orthotopic GBM xenograft models. Results Our results show that increased expression of LY6K inversely correlates with GBM patient survival. LY6K promotes tumorigenicity in GBM cells both in vitro and in vivo. The mechanism underlying this tumorigenic behavior is enhancement of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Interestingly, we observed that tumor-promoting LY6K-ERK1/2 signaling is mediated by the interaction of LY6K with caveolin-1, rather than through oncogenic receptor tyrosine kinase–mediated signaling. Moreover, association of LY6K with the cell membrane is crucial for its tumorigenic functions. Finally, DNA methylation maintains LY6K silencing, and hypomethylation of the LY6K promoter increases its expression. In GSCs, ionizing radiation leads to demethylation of the LY6K promoter, thereby increasing LY6K expression and GSC resistance to radiation. Conclusions Our study highlights the importance of the contribution of LY6K to GBM tumor biology and suggests LY6K as a potential membrane target for treating GBM.

Glioblastoma (GBM) remains one of the most resistant and fatal forms of cancer. Previous studies have examined primary and recurrent GBM tumors, but it is difficult to study tumor evolution during therapy where resistance develops. To investigate this, we performed an in vivo single-cell RNA sequencing screen in a patient-derived xenograft (PDX) model. Primary GBM was modeled by mice treated with DMSO control, recurrent GBM was modeled by mice treated with temozolomide (TMZ), and during therapy GBM was modeled by mice euthanized after two of five TMZ treatments. Our analysis revealed the cellular population present during therapy to be distinct from primary and recurrent GBM. We found the Ribonucleotide Reductase gene family to exhibit a unique signature in our data due to an observed subunit switch to favor RRM2 during therapy. GBM cells were shown to rely on RRM2 during therapy causing RRM2-knockdown (KD) cells to be TMZ-sensitive. Using targeted metabolomics, we found RRM2-KDs to produce less dGTP and dCTP than control cells in response to TMZ (p). Supplementing RRM2-KDs with deoxycytidine and deoxyguanosine rescued TMZ-sensitivity, suggesting an RRM2-driven mechanism of chemoresistance, established by regulating the production of these nucleotides. In vivo, tumor-bearing mice treated with the RRM2-inhibitor, Triapine, in combination with TMZ, survived longer than mice treated with TMZ alone (p), indicating promising clinical opportunities in targeting RRM2. Our data present a novel understanding of RRM2 activity, and its alteration during therapeutic stress as response to TMZ-induced DNA damage.

Background Biological therapies have changed the landscape of pharmacological management of ulcerative colitis (UC). However, a large proportion of patients do not respond to biologics, lose their response over time, or present adverse drug events. This study aims to assess therapeutic response and treatment persistence to adalimumab, infliximab, and vedolizumab, 3 agents widely used in a tertiary referral center of Saguenay–Lac-Saint-Jean (Quebec, Canada). Methods We conducted a retrospective population-based study with a thorough review of patients’ medical charts. Adults at UC diagnosis, with current or past use of adalimumab, infliximab, or vedolizumab, were included in the study. Clinical data were collected in order to assess response phenotypes and persistence to treatment. Kaplan–Meier curves were performed to assess treatment persistence, and predictors for discontinuation were assessed using Cox regression analyses. Results A total of 134 patients were included in this study. For the cases exposed to adalimumab, infliximab, and vedolizumab, 56.9%, 62.5%, and 47.5% were responders, respectively. Mean persistence rates (95% CI) were 5.5 (4.3–6.6), 10.1 (8.7–11.5), and 3.6 (2.9–4.2) years for adalimumab, infliximab, and vedolizumab, respectively. Increased persistence rates were observed in biologic-naïve patients treated with infliximab in comparison to those with the previous exposition to 2 biologics, but no such effect was observed for adalimumab or vedolizumab. Overall, 61.9% of cases had adverse drug events and of these, 6 led to treatment discontinuation. Conclusion This study presents long-term treatment persistence data with adalimumab, infliximab, and vedolizumab, showing that more than half of cases treated with these biologics remained on treatment at least 24 months after initiation.

Background Longitudinal cohort studies provide important information about the clinical effectiveness of an intervention in the routine clinical setting, and are an opportunity to understand how a population presents for treatment and is managed. Methods INTEREST IN CD2 (NCT01753349) is a prospective, international, 3-year, longitudinal, observational study following the course of adult idiopathic cervical dystonia (CD) treated with botulinum neurotoxin type A (BoNT-A). The primary objective is to document long-term patient satisfaction with BoNT-A treatment. Here we report baseline data. Results This analysis includes 1036 subjects (67.4% of subjects were female; mean age was 54.7 years old; mean TWSTRS Total score was 31.7). BoNT-A injections were usually given in line with BoNT-A prescribing information. The most commonly injected muscles were splenius capitis (87.3%), sternocleidomastoid (82.6%), trapezius (64.3%), levator scapulae (40.9%) and semispinalis capitis (26.9%); 35.5% of subjects were injected using a guidance technique. Most subjects (87.8%) had been previously treated with BoNT-A (median interval between last pre-study injection and study baseline was 4 months); of these 84.8% reported satisfaction with BoNT-A treatment at peak effect during their previous treatment cycle and 51.5% remained satisfied at the end of the treatment. Analyses by geographical region revealed heterogeneity in the clinical characteristics and BoNT-A injection practice of CD subjects presenting for routine treatment. Conclusions These baseline analyses provide sizeable data regarding the epidemiology and clinical presentation of CD, and demonstrate an international heterogeneity of clinical practice. Future longitudinal analyses of the full 3-year study will explore how these factors impact treatment satisfaction. Electronic supplementary material The online version of this article (10.1007/s00415-017-8698-2) contains supplementary material, which is available to authorized users.

Background: Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Hormone receptor-positive (HR+) advanced BC cancer, in particular the inflammatory BC (IBC) represents a significant clinical challenge because of the development of endocrine resistance during either adjuvant or metastatic treatment translating to poor outcome. We aim to develop unique PDX tumor models and 3D spheroid/organoid cultures that recapture advanced IBC using pleural effusions or/and circulating tumor cells (CTCs) as tumor resource and that can help understand the mechanism of IBC metastasis for new drug development. Methods: Five samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from three advanced IBC patients were obtained from Northwestern Memorial Hospital to establish IBC PDX tumor model in immunodeficient NSG female mice via subcutaneous or breast fat pad xenografting and develop the derived 3D organoid/spheroid cultures for anti-cancer drug evaluation. STR profiling against with original patient tumor DNA was conducted to confirm the tumor authentication. Results: Three developed IBC PDX tumor models (one HR+-IBC and two triple negative (TN)-IBC) were demonstrated by pathology to have highly heterogeneous characteristics and metastatic features similar to the original patient tumor. NSG mice xenografted with original TN-IBC pleural effusion, or derived spheroid cultures with varying passage numbers (6 passages) manifested different inflammatory clinical symptoms. Mice xenografted with one case of original TN-IBC pleural effusion cells (~40% of tumor-associated macrophages and ~60% of tumor cells) developed palpable tumors and was shown to have the most IBC-like characteristics including reddish skin and ulcerative lesions. They were sacrificed 2 months after xenograft due to declining health. Mice xenografted with >6 passages or Citation Format: Wenan Qiang, Zhangfeng Zhong, Pamela Monahan, Kristy Tse, Youbin Zhang, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Demirkan Gursel, Reiner Bleher, Jian-Jun Wei, Charles D. James, Thomas V. O’Halloran, Massimo Cristofanilli. Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-06.

Background: This article describes the challenges in the discovery and optimization of mGlu2/4 heterodimer Positive Allosteric Modulators (PAMs). Methods: Initial forays based on VU0155041, a PAM of both the mGlu4 homodimer and the mGlu2/4 heterodimer, led to flat, intractable SAR that precluded advancement. Screening of a collection of 1,152 FDA approved drugs led to the discovery that febuxostat, an approved xanthine oxidase inhibitor, was a moderately potent PAM of the mGlu2/4 heterodimer (EC50 = 3.4 µM), but was peripherally restricted (rat Kp = 0.03). Optimization of this hit led to PAMs with improved potency (EC50s 2, an ~100-fold increase). Results: However, these new amide analogs of febuxostat proved to be either GIRK1/2 and GIRK1/4 activators (primary carboxamide congeners) or mGlu2 PAMs (secondary and tertiary amides) and not selective mGlu2/4 heterodimer PAMs. Conclusion: These results required the team to develop a new screening cascade paradigm, and exemplified the challenges in developing allosteric ligands for heterodimeric receptors.

BACKGROUND: Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. METHODS: Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. RESULTS: H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells increased cell response to chemotherapy. CONCLUSIONS: Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Background Advanced age is a major risk factor for the development of many diseases including those affecting the central nervous system. Wild-type isocitrate dehydrogenase glioblastoma (IDHwt GBM) is the most common primary malignant brain cancer and accounts for ≥90% of all adult GBM diagnoses. Patients with IDHwt GBM have a median age of diagnosis at 68–70 years of age, and increasing age is associated with an increasingly worse prognosis for patients with this type of GBM. Methods The Surveillance, Epidemiology, and End Results, The Cancer Genome Atlas, and the Chinese Glioma Genome Atlas databases were analyzed for mortality indices. Meta-analysis of 80 clinical trials was evaluated for log hazard ratio for aging to tumor survivorship. Results Despite significant advances in the understanding of intratumoral genetic alterations, molecular characteristics of tumor microenvironments, and relationships between tumor molecular characteristics and the use of targeted therapeutics, life expectancy for older adults with GBM has yet to improve. Conclusions Based upon the results of our analysis, we propose that age-dependent factors that are yet to be fully elucidated, contribute to IDHwt GBM patient outcomes.

Meningioma is the most common primary brain tumor with nearly thirty thousand new cases in the US every year. While most meningiomas grow slowly, hence categorized as benign, they can still impact brain structures and result in disability or lethality. Currently, there is no widely accepted chemotherapy for meningioma, and our understanding of the disease’s molecular characteristics is very limited. In this study, we aimed to identify druggable molecular targets for repurposing of chemotherapies against meningiomas. We analyzed previously published dataset of 493 meningioma patients by Felix Sahm et al (Lancet Oncology, 2017) for association with progression-free survival (PFS) and identified associations of methylation at individual CpG sites as detected by the Illumina 450k platform with PFS. Subsequently, we searched for candidate drugs targeting the pathways linked to poor patient prognosis. Our analyses indicated 981 genes for which methylation of mapped CpG sites was found to be consistently associated with shorter PFS (positive hazard ratios (HRs)) and with longer PFS (negative HRs) at FDR-adjusted p < 0.05. Using Cytoscape/Reactome FI app, we cross-referenced current cancer drugs that target these and identified docetaxel and raloxifene hydrochloride as potential candidates. In our vitro study, docetaxel caused apoptotic cell death in established and primary patient meningioma lines of various grades. IC50s of docetaxel in the sixteen meningioma cell lines tested ranged from 0.8nM to 4.4mM, which partially corresponded to the growth rates of these cells. Our study will advance our understanding of molecular pathways driving meningioma and identify potential targeted therapies to bridge the current gap in treatment of the disease.

Despite radiation therapy (RT) being an integral part of the treatment of most pediatric cancers and the recent discovery of novel molecular-targeted agents (MTAs) in this era of precision medicine with the potential to improve the therapeutic ratio of modern chemoradiotherapy regimens, there are only a few preclinical trials being conducted to discover novel radiosensitizers and radioprotectors. This has resulted in a paucity of translational clinical trials combining RT and novel MTAs. This report describes the opportunities and challenges of investigating RT together with MTAs in preclinical testing for immunotherapy, brain tumors, and sarcomas in pediatric oncology. We discuss the need for improving the collaboration between radiation oncologists, biologists, and physicists to improve the reliability, reproducibility, and translational potential of RT-based preclinical research. Current translational clinical trials using RT and MTAs for immunotherapy, brain tumors, and sarcomas are described. The technologic advances in experimental RT, availability of novel experimental tumor models, advances in immunology and tumor biology, and the discovery of novel MTAs together hold considerable promise for good quality preclinical and clinical multimodality research to improve the current rates of survival and toxicity in children afflicted with cancer.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults. There is no treatment that provides durable relief for the vast majority of GBM patients. In this study, we’ve tested a bispecific antibody comprised of single-chain variable regions (scFvs) against T cell CD3ε and GBM cell interleukin 13 receptor alpha 2 (IL13Rα2). We demonstrate that this BiTE (BiTELLON) engages peripheral and tumor-infiltrating lymphocytes harvested from patient’s tumors, and in so doing exerts anti-GBM activityex vivo. The interaction of BiTELLONwith T cells and engagement of IL13Rα2-expressing GBM cells stimulates T cell proliferation as well as production of pro-inflammatory cytokines INFγ and TNFα. We have modified neural stem cells (NSCs) to produce and secrete the BiTE (NSCsLLON). When injected intracranially in mice with brain tumor, NSCsLLONshow tropism for tumor, secrete BiTELLON, and remain viable for several days. When injected directly into tumor, NSCLLONprovide significant survival benefit to mice bearing IL13Rα2+ GBM. Our results support further investigation and development of this therapeutic for clinical translation.

Malignant gliomas are heterogeneous neoplasms. Glioma stem-like cells (GSCs) are undifferentiated and self-renewing cells that develop and maintain these tumors. These cells are the main population that resist current therapies. Genomic and epigenomic analyses has identified various molecular subtypes. Bone morphogenetic protein 4 (BMP4) reduces the number of GSCs through differentiation and induction of apoptosis, thus increasing therapeutic sensitivity. However, the short half-life of BMP4 impedes its clinical application. We have previously reviewed BMP4 signaling in central nervous system development and glioma tumorigenesis and its’ potential as a treatment target in human gliomas. Recent advances in understanding both adult and pediatric malignant gliomas highlight critical roles of BMP4 signaling pathways in the regulation of tumor biology, and indicate its’ potential as a therapeutic molecule. Furthermore, significant progress has been made on synthesizing BMP4 biocompatible delivery materials, which can bind to and markedly extend BMP4 half-life. Here, we review current research associated with BMP4 in brain tumors, especially in pediatric malignant gliomas. We also summarize BMP4 delivery strategies, with a focus on biocompatible BMP4 binding peptide amphiphile nanostructures as promising novel delivery platforms for treatment of these devastating tumors.

Purpose: Indoleamine 2,3 dioxygenase 1 (IDO1) mediates potent immunosuppression in multiple preclinical models of cancer. However, the basis for elevated IDO1 expression in human cancer, including the most common primary malignant brain tumor in adults, glioblastoma (GBM), is poorly understood. The major objective of this study is to address this gap in our understanding of how IDO1 expression contributes to the biology of GBM, and whether its level of expression is a determinant of GBM patient outcome. Experimental Design: Patient-resected GBM, The Cancer Genome Atlas, human T-cell:GBM cocultures, as well as nu/nu, NOD-scid, and humanized (NSG-SGM3-BLT) mice-engrafted human GBM form the basis of our investigation. Results: In situ hybridization for IDO1 revealed transcript expression throughout patient-resected GBM, whereas immunohistochemical IDO1 positivity was highly variable. Multivariate statistical analysis revealed that higher levels of IDO1 transcript predict a poor patient prognosis (P = 0.0076). GBM IDO1 mRNA levels positively correlated with increased gene expression for markers of cytolytic and regulatory T cells, in addition to decreased patient survival. Humanized mice intracranially engrafted human GBM revealed an IFNγ-associated T-cell–mediated increase of intratumoral IDO1. Conclusions: Our data demonstrate that high intratumoral IDO1 mRNA levels correlate with a poor GBM patient prognosis. It also confirms the positive correlation between increased GBM IDO1 levels and human-infiltrating T cells. Collectively, this study suggests that future efforts aimed at increasing T-cell–mediated effects against GBM should consider combinatorial approaches that coinhibit potential T-cell–mediated IDO1 enhancement during therapy. Clin Cancer Res; 23(21); 6650–60. ©2017 AACR.

Human glioma, in particular, malignant forms such as glioblastoma exhibit dismal survival rates despite advances in treatment strategies. A population of glioma cells with stem-like features, glioma cancer stem-like cells (GCSCs), contribute to renewal and maintenance of the tumor cell population and appear responsible for chemotherapeutic and radiation resistance. Bone morphogenetic protein 4 (BMP4), drives differentiation of GCSCs and thus improves therapeutic efficacy. Based on this observation it is imperative that the clinical merits of BMP4 in treating human gliomas should be addressed. This article reviews BMP4 signaling in central nervous system development and in glioma tumorigenesis, and the potential of this molecule as a treatment target in human gliomas. Further work needs to be done to determine if distinct lineages of GCSCs, associated with different glioma sub-classifications, proneural, neural, classical and mesenchymal, differ in responsiveness to BMP4 treatment. Additionally, interaction among BMP4 and cell matrix, tumor-vascular molecules and microglial immune cells also needs to be investigated, as this will enhance our knowledge about the role of BMP4 in human glioma and lead to the identification and/or development of novel therapeutic approaches that improve treatment outcomes of these devastating tumors.

Aichi virus 1 (AiV-1) is a human pathogen from the Kobuvirus genus of the Picornaviridae family. Worldwide, 80 to 95% of adults have antibodies against the virus. AiV-1 infections are associated with nausea, gastroenteritis, and fever. Unlike most picornaviruses, kobuvirus capsids are composed of only three types of subunits: VP0, VP1, and VP3. We present here the structure of the AiV-1 virion determined to a resolution of 2.1 Å using X-ray crystallography. The surface loop puff of VP0 and knob of VP3 in AiV-1 are shorter than those in other picornaviruses. Instead, the 42-residue BC loop of VP0 forms the most prominent surface feature of the AiV-1 virion. We determined the structure of AiV-1 empty particle to a resolution of 4.2 Å using cryo-electron microscopy. The empty capsids are expanded relative to the native virus. The N-terminal arms of capsid proteins VP0, which mediate contacts between the pentamers of capsid protein protomers in the native AiV-1 virion, are disordered in the empty capsid. Nevertheless, the empty particles are stable, at least in vitro , and do not contain pores that might serve as channels for genome release. Therefore, extensive and probably reversible local reorganization of AiV-1 capsid is required for its genome release. IMPORTANCE Aichi virus 1 (AiV-1) is a human pathogen that can cause diarrhea, abdominal pain, nausea, vomiting, and fever. AiV-1 is identified in environmental screening studies with higher frequency and greater abundance than other human enteric viruses. Accordingly, 80 to 95% of adults worldwide have suffered from AiV-1 infections. We determined the structure of the AiV-1 virion. Based on the structure, we show that antiviral compounds that were developed against related enteroviruses are unlikely to be effective against AiV-1. The surface of the AiV-1 virion has a unique topology distinct from other related viruses from the Picornaviridae family. We also determined that AiV-1 capsids form compact shells even after genome release. Therefore, AiV-1 genome release requires large localized and probably reversible reorganization of the capsid.

BACKGROUND Anaplastic meningiomas pose a serious health concern due to their propensity for recurrence and brain invasion. Five-year overall survival of patients with anaplastic meningiomas is less than 10%, similar to that of glioblastoma patients. Standard treatment of recurrent anaplastic meningioma is radiotherapy (RT), but radioresistance is common. New therapeutic strategies, used singularly or in combination with RT, are urgently needed. Since deletion of the gene encoding the p16 cell cycle checkpoint protein is common in anaplastic meningioma, we hypothesized that such tumors would be sensitive to Palbociclib, a cdk4/6 inhibitor, and that Palbociclib could enhance RT efficacy. METHODS CH157 anaplastic meningioma cells were used in vitro for investigating tumor cell proliferation response to RT and Palbociclib, either alone or in combination, and as indicated by MTT assay results. CH157 cells were also used to establish intracranial xenografts in mice treated with RT or Palbociclib. Accuracy of RT dose was verified with calibrated MOSFET\OSLD dosimeters. Mice were monitored for intracranial tumor growth and response to treatment, as indicated by bioluminescence imaging. Length of survival data was collected and analyzed by log-rank test. RESULTS At two days post RT, the in vitro anti-proliferative effect of 2, 6, and 10 Gy radiation was significantly enhanced with concurrent 1mM Palbociclib (p < 0.001). Daily administration of Palbociclib (150 mg/kg) for 1 week significantly extended the survival of mice with intracranial CH157 xenografts (group mean value of 18.4 vs. 13.6 days with vehicle control, p < 0.001). A single dose of 10 Gy RT was superior to 6 Gy administered 1x and 2Gy/day administered on 5 consecutive days (group mean values of 37.4 vs. 24.0 vs. 24.6, respectively: p < 0.001). CONCLUSIONS These data support further investigation of RT + Palbociclib for treating p16-deficient anaplastic meningiomas.

We stress that these are long-term processes and that current expectations of donors and others for short-term solutions have been unrealistic. We see such work as preventing violence and encouraging integration.

Diffuse intrinsic pontine glioma (DIPG), an infiltrative, high grade glioma (HGG) affecting young children, has the highest mortality rate of all pediatric cancers. Despite treatment, average survival is less than twelve months, and five-year survival under 5%. We previously detected increased expression of Tenascin-C (TNC) protein in DIPG cerebrospinal fluid and tumor tissue relative to normal specimens. TNC is an extracellular matrix (ECM) glycoprotein that mediates cell-matrix interactions, guides migrating neurons during normal brain development and is thought to maintain the periventricular stem cell niche in the developing brain. Tumor TNC expression is reported in adult glioma and other cancers. However, the pattern and effects of TNC expression in DIPG has not been previously explored. Here, we characterize TNC expression in patient derived pediatric supratentorial HGG (n = 3) and DIPG (n = 6) cell lines, as well as pediatric glioma tumor (n = 50) and normal brain tissue specimens (n = 3). We found tumor specific TNC gene and protein overexpression that directly correlated with higher tumor grade (WHO III and IV, p = 0.05), H3K27 M mutation (p = 0.012), shorter progression free survival (p = 0.034), and poorer overall survival (0.041) in association with these factors. TNC knockdown via lentiviral shRNA transfection of HGG (n = 1) and DIPG (n = 3) cell lines resulted in decreased cell proliferation, migration, and invasion in vitro (p

Expression of the constitutively active BRAF(V600E)-mutant kinase occurs in pediatric and predominantly young adult high-grade glioma patients (Cancer Res. 2010, 70:512; Acta Neuropathol. 2011, 121:397). BRAF(V600E) expression frequently coincides with CDKN2A deletion and marks a subgroup of predominantly hemispheric tumors with epigenetic similarities to pleomorphic xanthastrocytoma (PXA) and histologic features of grade IV astrocytoma (Cancer Cell 2018, 33:829). The origin of BRAF(V600E) mutant astrocytoma is under investigation. Here, we are using transgenic mice to assess neuroanatomical aspects and effects on differentiation of BRAF(V600E) expression. In previous studies, expression of BRAF(V600E) in astroglial cells and neural stem cells of the developing brain led to hyperplasia in transgenic mice. To circumvent the early postnatal lethality associated with BRAF(V600E) expression during development, my lab injected adenovirus expressing Cre recombinase into the corpus callosum of BRAFCA Ink4a/Arf floxed young adult mice. In these mice, Cre expression induces BRAF(V600E) expression and deletion of Ink4a/Arf, the mouse locus homologous to human CDKN2A. This approach showed that BRAF(V600E) expression and CDKN2A (Ink4a/Arf) deficient corpus callosum cells form high-grade astrocytoma-like tumors in young adult mice (Proc Natl Acad Sci. 2012, 109:8710). To further investigate the neuroanatomical aspects of tumor formation we directed cre expression to distinct neuroanatomical areas, including the cortex, of the young adult mouse brain. Histo-pathologic and survival analyses showed that each injected area was susceptible to tumor formation albeit with different latency. Induction of BRAF(V600E) expression in Ink4a/Arf-deleted neurospheres in vitro decreased the capacity for neuronal and glial differentiation and led to expansion of oligodendrocyte progenitor-like cells (OPC). Ongoing studies investigate directly the extent to which BRAF(V600E) expression in Ink4a/Arf-deleted OPC in transgenic mice cause tumor formation. Results from cell of origin studies are expected to inform which mouse model most faithfully recapitulates the human diseases.

Tumors in patients with recurrent GBM are resistant to standard therapies. Available models for testing experimental therapies to treat GBM are primarily derived from or representative of primary, untreated GBM. We have developed a clinically relevant model of recurrent, therapy-resistant glioblastoma (GBM6R) from a patient-derived xenograft (PDX) of primary glioblastoma (GBM6) that underwent multiple rounds of temozolomide (TMZ) treatment following intracranial engraftment in a host mouse. Initial characterizations of GBM6R revealed a significant decrease in sensitivity to TMZ both in vitro (p< 0.008) and in vivo (p=0.0079). An unbiased screen to identify proteins with altered expression in GBM6R, relative to treatment naïve GBM6, revealed nuclear lamina protein LMNA as highly expressed in GBM6R. From in silico analysis we found that high LMNA expression is associated with poor survival for GBM patients (p=0.0001, Gravendeel; p=0.0078, TCGA_GBM; p< 0.0001, CCGA; GlioVis). GBM5 and GBM43 PDX explant cultures treated with multiple doses of TMZ increased their expression of LMNA. We found that in TMZ treated and TMZ resistant tumors, that LMNA preferentially localizes to the nuclear lamina (p< 0.001), which is not observed in primary, untreated tumors (p< 0.005). Analysis of LMNA by immunoprecipitation revealed that LMNA interacts with DNA-PK and γH2AX. From these results, we hypothesize that LMNA may be acting to stabilize DNA damage foci at the nuclear periphery, which results in enhanced DNA repair. Our observations support the testing of selective DNA-PK inhibitors for treating GBM, which would inhibit TMZ-induced DNA damage repair, and enhance the cytotoxic effects of TMZ.

PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

Pediatric high-grade gliomas (pHGGs) account for ~20% of childhood brain tumors, are incurable in nearly all instances, and have an average patient survival of less than 15 months. Histone posttranslational modifications (PTMs) are deregulated in pHGGs. There are many gaps in our knowledge regarding the mechanisms by which “normal” PTM is subverted during pHGG development. Preliminary results from our lab show pHGGs are enriched in H3K4me3 and that the extent of this specific PTM is predictive of patient survival length. H3K4me3 levels are controlled by methyltransferase and demethylase activity. Gene expression analysis indicates SETD1A and WDR82, enzymatic subunits of human SETD1A containing methyltransferase complex (referred to as hSETD1A-COMPASS), are responsible for H3K4 methylation. SETD1A and WDR82 are significantly elevated in pHGGs, relative to pediatric low-grade gliomas (pLGGs), and both show a direct association with glioma malignancy. Interestingly, these associations do not exist in adult gliomas. The results suggest that hSETD1A-COMPASS specifically impacts pHGG development and is a potential therapeutic target for these tumors.

Immune dysfunction is a hallmark of chronic HCV infection and viral clearance with direct antivirals recover some of these immune defects. TCRVγ9Vδ2 T cell dysfunction in treated HCV patients however is not well studied and was the subject of this investigation. Peripheral blood cells from patients who had achieved sustained virologic response (SVR) or those who had relapsed after interferon-free therapy were phenotyped using flow cytometry. Functional potential of Vγ9Vδ2-T cells was tested by measuring proliferation in response to aminobisphosphonate Zoledronic acid, and cytotoxicity against HepG2 hepatoma cell line. TCR sequencing was performed to analyze impact of HCV infection on Vδ2-T cell repertoire. Vγ9Vδ2 cells from patients were activated and therapy resulted in reduction of CD38 expression on these cells in SVR group. Relapsed patients had Vδ2 cells with persistently activated and terminally differentiated cytotoxic phenotype (CD38(+)CD45RA(+)CD27(−)CD107a(+)). Irrespective of outcome with therapy, majority of patients had persistently poor Vδ2-T cell proliferative response to Zoledronate along with lower expression of CD56, which identifies anti-tumor cytotoxic subset, relative to healthy controls. There was no association between the number of antigen reactive Vγ2-Jγ1.2 TCR rearrangements at baseline and levels of proliferation indicating non-response to Zoledronate is not due to depletion of phosphoantigen responding chains. Thus, HCV infection results in circulating Vγ9Vδ2-T cells with a phenotype equipped for immediate effector function but poor cytokine response and expansion in response to antigen, a functional defect that may have implications for susceptibility for carcinogenesis despite HCV cure.

Pediatric diffuse midline glioma is a highly morbid glial neoplasm that may arise in the thalamus or brainstem (also known as diffuse intrinsic pontine glioma or DIPG). Because tumor anatomic location precludes surgical resection, diagnosis and treatment is based on MR imaging and analysis of biopsy specimens. Up to 80% of pediatric diffuse midline gliomas harbor a histone H3 mutation resulting in the replacement of lysine 27 with methionine (K27M) in genes encoding histone H3 variant H3.3 (H3F3A) or H3.1 (HIST1H3B). H3K27M mutant glioma responds more poorly to treatment and is associated with worse clinical outcome than wild-type tumors, so mutation detection is now diagnostic for a new clinical entity, diffuse midline glioma H3K27M mutant, as defined in the most recent WHO classification system. We previously reported patterns of histone H3 trimethylation (H3K27me3) and acetylation (H3K27Ac) associated with H3K27M mutation that impact transcription regulation and contribute to tumorigenesis. Given the clinical implications of the H3K27M mutation and these associated H3 post-translational modifications (PTMs), we set to determine whether they can be characterized via immunohistochemistry (IHC) in a cohort of pediatric glioma (n = 69) and normal brain tissue (n = 4) specimens. We observed 100% concordance between tissue IHC and molecular sequencing for detecting H3K27M mutation. In turn, H3K37M and H3K27me3 results, but not H3K27Ac staining patterns, were predictive of clinical outcomes. Our results demonstrate H3K27M and H3K27me3 staining of pediatric glioma tissue may be useful for diagnosis, stratification to epigenetic targeted therapies, and longitudinal monitoring of treatment response.

Meningiomas are the most common primary intracranial tumor. Important advances are occurring in meningioma research. These are expected to accelerate, potentially leading to impactful changes on the management of meningiomas in the near and medium term. This review will cover the histo- and molecular pathology of meningiomas, including recent 2016 updates to the WHO classification of CNS tumors. We will discuss clinical and radiographic presentation and therapeutic management. Surgery and radiotherapy, the two longstanding primary therapeutic modalities, will be discussed at length. In addition, data from prior and ongoing investigations of other treatment modalities, including systemic and targeted therapies, will be covered. This review will quickly update the reader on the contemporary management and future directions in meningiomas. [Formula: see text]

Ependymomas are the third most common form of brain tumors in children. These tumors are resistant to chemotherapy and despite genomic sequencing, there is a lack of effective molecular treatment targets. Efficient treatment targets need to be identified. Increasing evidence shows epigenetic alterations including posttranslational histone modifications (PTMs), associated with malignancy, chemotherapeutic resistance and prognosis in pediatric ependymomas. In this study we examined histone PTMs in ependymomas and identified potential targets to improve chemotherapeutic efficacy. Global levels of trimethylation at lysine 4 on histone H3 (H3K4me3), detected with immunohistochemistry and western blots, positively correlated with malignancy in pediatric primary ependymomas. Micro-array analysis of 22 pediatric ependymomoas identified upregulated candidate drug resistance genes. Promoter H3K4me3 occupancy was examined for two of these genes, CCND1 and ERBB2. Chromatin-immunoprecipitation with H3K4me3 coupled with real-time PCR, showed higher levels of H3K4me3 at these genes in grade III tumors, in comparison to grade II ones. When H3K4me3 levels were reduced in primary cultured ependymoma cells in vitro, cell response to chemotherapy increased. In summary, these results indicate that H3K4me3 levels are high at promoters of genes which confer chemotherapeutic resistance. Consequently, a novel treatment regimen which targets H3K4me3 in combination with traditional protocols may increase chemotherapeutic efficacy and improve prognosis for children who present with ependymoma.

INTRODUCTION: Glioblastoma (GBM) is the most frequent and aggressive primary tumor of the central nervous system (CNS), accounting for over 50% of all primary malignant gliomas arising in the adult brain. Even after surgical resection, adjuvant radiotherapy (RT) and temozolomide (TMZ) chemotherapy, as well as tumor-treating fields, the median survival is only 15–20 months. We have identified a pathogenic mechanism that contributes to the tumor-induced immunosuppression in the form of increased indoleamine 2,3 dioxygenase 1 (IDO1) expression; an enzyme that metabolizes the essential amino acid, tryptophan (Trp), into kynurenine (Kyn). However, real-time measurements of IDO1 activity has yet to become mainstream in clinical protocols for assessing IDO1 activity in GBM patients. METHODS: Pre-treatment and on-treatment α-[(11)C]-methyl-L-Trp (AMT) positron emission tomography (PET) with co-registered MRI was performed on patients with recurrent GBM treated with the IDO1 pathway inhibitor indoximod (D1-MT) and temozolomide. RESULTS: Regional intratumoral variability of AMT within enhancing and non-enhancing tumor was noted at baseline. On treatment imaging revealed decreased regional uptake suggesting IDO1 pathway modulation with treatment. CONCLUSIONS: Here, we have validated the ability to use PET of the Trp probe, AMT, for use in visualizing and quantifying intratumoral Trp uptake in GBM patients treated with an IDO1 pathway inhibitor. These data serve as rationale to utilize AMT-PET imaging in the future evaluation of GBM patients treated with IDO1 enzyme inhibitors.

In this study, laboratory scale experiments were performed to degrade highly concentrated organic matter in the form of color in the distillery spent wash through batch oxidative methods such as electrocoagulation (EC), electrofenton (EF) and Fenton process. The effect of corresponding operating parameters, namely initial pH: 2–10; current intensity: 1–5 A; electrolysis time: 0.5–4 h; agitation speed: 100–500 rpm; inter-electrode distance: 0.5–4 cm and Fenton's reagent dosage: 5–40 mg/L was employed for optimizing the process of spent wash color removal. The performance of all the three processes was compared and assessed in terms of percentage color removal. For EC, 79% color removal was achieved using iron electrodes arranged with 0.5 cm of inter-electrode space and at optimum conditions of pH 7, 5 A current intensity, 300 rpm agitation speed and in 2 h of electrolysis time. In EF, 44% spent wash decolorization was observed using carbon (graphite) electrodes with an optimum conditions of 0.5 cm inter-electrode distance, pH 3, 4 A current intensity, 20 mg/L FeSO4 and agitation speed of 400 rpm for 3 h of electrolysis time. By Fenton process, 66% decolorization was attained by Fenton process at optimized conditions of pH 3, 40 mg/L of Fenton's reagent and at 500 rpm of agitation speed for 4 h of treatment time.

Signaling through the Fas/Apo-1/CD95 death receptor is known to affect virus-specific cell-mediated immune (CMI) responses. We tested whether modulating the Fas-apoptotic pathway can enhance immune responses to DNA vaccination or lymphocytic choriomeningitis virus (LCMV) infection. Mice were electroporated with plasmids expressing a variety of pro- or anti-apoptotic molecules related to Fas signaling and then either LCMV-infected or injected with plasmid DNA expressing SIV or HIV antigens. Whereas Fas or FasL knockout mice had improved CMI, down-regulation of Fas or FasL by shRNA or antibody failed to improve CMI and was accompanied by increases in regulatory T cells (Treg). Two “adjuvant” plasmids were discovered that significantly enhanced plasmid immunizations. The adjuvant effects of Fas-associated death domain (FADD) and of cellular FLICE-inhibitory protein (cFLIP) were consistently accompanied by increased effector memory T lymphocytes and increased T cell proliferation. This adjuvant effect was also observed when comparing murine infections with LCMV-Armstrong and its persisting variant LCMV-Clone 13. LCMV-Armstrong was cleared in 100% of mice nine days after infection, while LCMV-Clone 13 persisted in all mice. However, half of the mice pre-electroporated with FADD or cFLIP plasmids were able to clear LCMV-Clone 13 by day nine, and, in the case of cFLIP, increased viral clearance was accompanied by higher CMI. Our studies imply that molecules in the Fas pathway are likely to affect a number of events in addition to the apoptosis of cells involved in immunity.

This study focuses on coagulation–flocculation method of degradation of pollutants from the distillery spent wash. The optimisation of major operating parameter, that is the coagulant dosage for the degradation of distillery effluent in terms of colour, chemical oxygen demand (COD) and residual turbidity removal, was studied. In addition to conventional coagulants namely aluminium sulphate, ferric sulphate and calcium sulphate, a novel initiative of using Moringa oleifera seed extract (MOSE) as a coagulant aid to enhance the degradation efficiency was investigated. The objective of this study aims at the utilisation of plant-based organic coagulant resources, hence minimising the dosage of inorganic chemical coagulants. The effectiveness of the optimised dosage of chemical coagulants with and without the addition of MOSE was assessed in terms of percentage colour, COD and turbidity removal. The results showed that, in addition to conventional chemical coagulants, M. oleifera seed extract (MOSE) po...

Purpose There is strong biological plausibility for a causal role of reactive oxygen species in vascular pathology but no direct epidemiological evidence linking elevated reactive oxygen species levels to hypertension development. We examined cross-sectional and prospective associations between oxidative status (urinary F 2 -isoprostanes) and hypertension in the Insulin Resistance Atherosclerosis Study cohort ( n = 831). Methods The cohort included non-Hispanic white, Hispanic, and non-Hispanic black individuals, with 252 (30%) having prevalent hypertension and 579 participants normotensive at baseline, 122 (21%) of whom developed hypertension during the 5-year follow-up. Four urinary F 2 -isoprostane isomers were quantified in baseline specimens using LC/MS–MS and were summarized as a composite index. Examined outcomes included hypertension status (yes/no), systolic (SBP) and diastolic blood pressure (DBP), pulse pressure (PP), and mean arterial pressure (MAP). Results Prevalent and incident hypertension were associated with greater age, Black race, impaired glucose tolerance, and greater BMI. F 2 -IsoP levels were lower among men and among non-Hispanic Blacks, were inversely associated with age, and were directly associated with BMI. No cross-sectional association was found between F 2 -isoprostanes and hypertension status (OR = 0.93, 0.77–0.12). Among the continuous measures of blood pressure only PP was associated with F 2 -isoprostanes at baseline (beta-coefficient = 0.99, 0.11–1.86). No prospective association was found between F 2 -isoprostanes and incident hypertension: OR = 0.98, 0.77–1.25. No prospective associations were found for systolic blood pressure and diastolic blood pressure, and pulse pressure. Mean arterial pressure showed an inverse association (beta-coefficient = −0.16, −0.31 to −0.01). Conclusions Elevated F 2 -isoprostane levels do not increase the risk of hypertension.

RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood-brain barrier-/blood-tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.

O6-methylguanine-DNA methyltransferase (MGMT) expression has been recently proposed as a useful prognostic and/or predictive marker in glioblastoma patients receiving adjuvant therapy after the surgery. We studied samples from 50 patients with histologically confirmed GBM to evaluate MGMT expression by immunohistochemistry and its relation to promoter methylation status. Genomic DNA was extracted from scrapings of formalin-fixed, paraffin-embedded tissue corresponding to hematoxylin and eosin sections. Using the mouse monoclonal antibody MT3.1, MGMT expression was assessed and scored in tumor cells: (1=negative or limited to10% positive tumor cells, 2=10% to 50%, 3=50%). Methylation-specific polymerase chain reaction was performed after bisulfite treatment. Assessment of MGMT expression in neoplastic tissue required careful scrutiny because of its expression in a variety of non-neoplastic cells. MGMT expression was present in tumor cells with a score of 1, 2, and 3, respectively in 36 (72%), 13 (26%), and 1 (2%) cases. Methylation-specific polymerase chain reaction yielded interpretable results in 39 cases (78%). MGMT promoter methylation was detected in 15 cases (38.5%), whereas 24 (61.5%) were unmethylated. Among the methylated samples, 14 (of 15) had a score of 1, and 1 had a score of 3 by immunohistochemistry. Of the 24 unmethylated samples, 18 had a score of 1, and 6 of 2. There was no significant correlation between MGMT expression and methylation, and no significant survival difference was observed between patients whose tumors were negative versus positive for MGMT protein by immunohistochemistry. This study underscores some of the difficulties in applying immunohistochemistry to assess MGMT expression.

Pediatric gliomas, particularly high-grade gliomas, which include diffuse intrinsic pontine gliomas (DIPGs), are among the most formidable and devastating cancers in children. These tumors remain incurable, despite many treatment approaches. We recently identified a small population of glioma cells with stem-like features in pediatric gliomas (glioma stem cells: GSCs), that may be responsible, for therapeutic resistance. Bone morphogenetic protein 4 (BMP4), essential for CNS development, increases GSC therapeutic sensitivity and is a promising adjuvant for glioma treatment. Mechanisms through which BMP4 increases therapeutic sensitivity need to be elucidated, as this can lead to identification of additional treatment targets and delivery systems for BMP4 administration in a clinical setting. Additionally, extension of BMP4 short half-life would enhance its’ clinical application. Here we show that BMP4 increases chemosensitivity by decreasing H3K4me3 at the promoter of multidrug resistant gene 1 (MDR1), resulting in decreased MDR1 expression. BMP4 appears to bring about this effect by decreasing hSETD1A, an H3K4me3 methyltransferase. Our work also demonstrates the first use of a novel sulfated glycopeptide (glyco-PA) nanostructure as a vector for BMP4 delivery. Glyco-PA markedly extended and enhanced BMP4 function, and increased chemotherapeutic anti-tumor activity against pediatric malignant glioma cells in culture. Overall, this work illuminates BMP4 effects on pediatric glioma therapeutic sensitivity through epigenetic mechanisms, and demonstrates the potential of bioactive glyco-PA nanostructures as a delivery mechanism for treating pediatric malignant gliomas and other tumors.

BACKGROUND Temozolomide (TMZ) is routinely used in the post-surgical treatment of patients with newly diagnosed glioblastoma (GBM). Nearly all GBM recur following TMZ treatment, and there is an increasing body of data that detail TMZ-associated mutations in recurrent GBM. In vivo models of recurrent GBM are scarce in comparison to models of treatment-naïve GBM. MATERIAL AND METHODS Mice with intracranial treatment naïve GBM patient derived xenograft (PDX) were treated with radiotherapy (RT) only, TMZ only, or RT + TMZ. Tumors were monitored for recovery from treatment by bioluminescence imaging (BLI), and upon animal subject decline due to growing tumor, subjects were euthanized, with viable intracranial tumor dissected and used to establish subcutaneous tumor in new animal hosts that received no treatment. Subcutaneous tumors were sequentially passaged through two additional animal hosts. DNA was extracted from third passage subcutaneous tumors and subjected to full exome sequencing. PDX exome sequence data were examined for genes with nonsynonymous frameshift and nonsense variants (mutations). RESULTS Exome sequence analysis showed striking differences between PDX receiving RT only vs. TMZ with or without RT. Most notable is the substantially greater incidence of G-C to A-T transition mutations in TMZ-treated PDX, which are a signature of TMZ treatment. All TMZ-treated PDX also showed substantially more genes being mutated than PDX receiving RT only: one cycle RT only = 88; one cycle TMZ only = 339; one cycle of concurrent RT + TMZ = 531; one cycle of concurrent RT + TMZ followed by two additional cycles of TMZ only = 1502. The PDX subjected to multiple cycles of TMZ treatment prior to establishment as a subcutaneous tumor has shown stable resistance to TMZ treatment in subsequent experiments. Importantly, TMZ-treated PDX had mutations in LTBP4, MSH6, and PREX1 genes that are enriched in patient GBM following recurrence after initial TMZ treatment (Nat Genet 2016;48:768–76); these genes were not mutated in PDX receiving RT only. CONCLUSION Our study results support the following: 1) Intracranial GBM PDX treated with TMZ develop a global genomic treatment signature, as well as specific mutations that are observed in recurrent patient GBMs; 2) Mutations detected in TMZ-treated intracranial tumors are stable, persisting after repeated subcutaneous passages without further treatment; 3) Paired naïve and TMZ-treated GBM PDX will facilitate studies aimed at increasing our understanding of TMZ resistance, and help identify new therapies for more effective treatment of recurrent GBM.

Background Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. Methods Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 occupancy was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. Results H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells, increased cell response to chemotherapy. Conclusions Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors. Key words: promoter, H3K4me3, human SETD1A, chemotherapy, pediatric ependymoma Citation Format: Guifa Xi, Nitin Wadhwani, Rintaro Hashizume, Barbara Mania-Farnell, Marcelo Bento Soares, Charles D. James, Tadanori Tomita. Global reduction of H3K4me3 improves chemotherapeutic efficacy for pediatric ependymomas [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 2871.

Pediatric diffuse midline glioma is a highly morbid glial neoplasm that may arise in the thalamus or brainstem (also known as diffuse intrinsic pontine glioma or DIPG). Because tumor anatomic location precludes surgical resection, diagnosis and treatment is based on MR imaging and analysis of biopsy specimens. Up to 80% of pediatric diffuse midline gliomas harbor a histone H3 mutation resulting in the replacement of lysine 27 with methionine (K27M) in genes encoding histone H3 variant H3.3 (H3F3A) or H3.1 (HIST1H3B). H3K27M mutant glioma responds more poorly to treatment and is associated with worse clinical outcome than wild-type tumors, so mutation detection is now diagnostic for a new clinical entity, diffuse midline glioma H3K27M mutant, as defined in the most recent WHO classification system. We previously reported patterns of histone H3 trimethylation (H3K27me3) and acetylation (H3K27Ac) associated with H3K27M mutation that impact transcription regulation and contribute to tumorigenesis. Given the clinical implications of the H3K27M mutation and these associated H3 post-translational modifications (PTMs), we set to determine whether they can be characterized via immunohistochemistry (IHC) in a cohort of pediatric glioma (n = 69) and normal brain tissue (n = 4) specimens. We observed 100% concordance between tissue IHC and molecular sequencing for detecting H3K27M mutation. In turn, H3K37M and H3K27me3 results, but not H3K27Ac staining patterns, were predictive of clinical outcomes. Our results demonstrate H3K27M and H3K27me3 staining of pediatric glioma tissue may be useful for diagnosis, stratification to epigenetic targeted therapies, and longitudinal monitoring of treatment response.

Background: Breast cancer (BC) is a heterogeneous disease with most common metastatic sites of liver, lung, brain, and bone. Endocrine resistance in hormone receptor-positive (HR+) advanced BC (ABC) cancer is a clinical challenge. ESR1 mutations are a key mechanism in acquired resistance, primarily occurs after exposure to endocrine therapy such as aromatase inhibitors but also selective estrogen modulators and degraders (i.e. Tamoxifen and Fulvestrant). Circulating tumor cells (CTCs) enumeration is a prognostic biomarker in ABC but the relation between the onset of ESR1 mutations and CTCs status is still unclear. Aim of this project is to define the clinical behavior of ESR1 mutated ABC in terms of metastasizing potential, through CTC enumeration and pattern; and to establish ESR1 mutated HR+ ABC PDX models able to recapitulate these characteristics. Methods: CTCs and circulating tumor DNA (ctDNA) were characterized in 55 HR+ ABC patients. ESR1 mutations status from 55 patient plasma cell-free DNA were generated using Guardant Next Generation Sequencing. Samples were also examined for numbers of CTCs by CellSearch. Association of ESR1 mutations with sites of distant organ metastasis and with CTC enumeration was analyzed by Chi square test and Kruskal–Wallis test, respectively. In preclinical model development, six samples of pleural effusion-derived tumor cells from Stage IV HR+ ABC patients were collected to establish HR+ ABC with ESR1 mutation PDX tumor model and its derived 3D organoid/spheroid cultures Results: ESR1 mutations were identified in 10 out of 55 patients (4 Y537S variant and 3 D538G variant, 4 other variants, 1 patient with both variants). In 55 patients, 72 visceral vs 27 bone metastatic incidences were observed; the data indicated 9 observed vs 4.5 expected in ESR1 mutated and 16 observed vs 20.5 expected in wild type (WT) (P=0.003) for liver metastasis; 10 observed vs 7.1 expected in ESR1 mutated and 29 observed vs 31.9 expected in WT (P=0.026) for bone metastasis. Further liver metastasis analysis of individual hot spot mutation site indicated 4 observed vs 1.8 expected in Y537S and 21 observed vs 23.2 expected in WT (P=0.037); and 3 observed vs 1.4 expected in D538G and 22 observed vs 23.6 expected in wild type (P=0.088). The analysis of correlation/distribution between CTCs numbers and ESR1 mutated suggested CTCs median of 13 (IQR 7-49) in ESR1 mutated and 0 (IQR 0-4) in WT HR+ patients (P=0.0044). Four ABC PDX tumor models were developed in immunodeficient NSG female mice demonstrated by pathology to have highly heterogeneous characteristics and metastatic features of the origin patient tumor, in particular, breast fat pad xenografted PDX tumor can result in metastasis to liver and lung tissue. In addition, two patient 3D tumor organoid/spheroid cultures were successfully established. Conclusions: ESR1 mutated ABC is associated with more aggressive (Stage IV) clinical behavior demonstrated by association with visceral metastases and CTCs detection. ESR1-mutated PDX models recapitulate aggressive features of the disease and can be used for preclinical testing of novel agents in endocrine resistant disease. Citation Format: Qiang W, Zhong Z, Gerratana L, Zhang Y, Zhang Q, Gursel D, Wei J-J, Bleher R, James C, O'Halloran T, Cristofanilli M. Development of patient-derived xenograft tumor model with organ-specific metastatic potential for evaluation of new therapeutics for hormone receptor-positive advanced breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-03-01.

Aim: The aim of this work is to evaluate combining targeting strategy and convection-enhanced delivery in brain tumor models by imaging quantum dot–immunoliposome hybrid nanoparticles. Materials & methods: An EGF receptor-targeted, quantum dot–immunoliposome hybrid nanoparticle (QD-IL) was synthesized. In vitro uptake was measured by flow cytometry and intracellular localization was imaged by confocal microscopy. In the in vivo study, QD-ILs were delivered to intracranial xenografts via convection-enhanced delivery and fluorescence was monitored noninvasively in real-time. Results: QD-ILs exhibited specific and efficient uptake in vitro and exhibited approximately 1.3- to 5.0-fold higher total fluorescence compared with nontargeted counterpart in intracranial brain tumor xenografts in vivo. Conclusion: QD-ILs serve as an effective imaging agent in vitro and in vivo, and the data suggest that ligand-directed liposomal nanoparticles in conjunction with convection-enhanced delivery may offer therapeutic benefits for glioblastoma treatment as a result of specific and efficient uptake by malignant cells. Original submitted 15 June 2012; Revised submitted 12 November 2012; Published online 30 April 2013

WOS:000369207000023; International audience; Consumption of sugar-sweetened beverages is associated with overweight and obesity. In this study, we hypothesized that obesity-prone (OP) mice fed a high-fat high-sucrose diet (HFHS) are more sensitive to consumption of sucrose-sweetened water (SSW) than obesity-resistant (OR) mice. After 3 weeks of ad libitum access to the HFHS diet (7.5 h/day), 180 male mice were classified as either OP (upper quartile of body weight gain, 5.2 +/- 0.1 g, n = 45) or OR (lower quartile, 3.2 +/- 0.1 g, n = 45). OP and OR mice were subsequently divided into 3 subgroups that had access to HFHS (7.5 h/day) for 16 weeks, supplemented with: i) water (OP/water and OR/water); ii) water and SSW (12.6% w/v), available for 2 h/day randomly when access to HFHS was available and for 5 randomly-chosen days/week (OP/SSW and OR/SSW); or iii) water and SSW for 8 weeks, then only water for 8 weeks (OP/SSW-water and OR/SSW-water). OR/SSW mice decreased their food intake compared to OR/water mice, while OP/SSW mice exhibited an increase in food and total energy intake compared to OP/water mice. OP/SSW mice also gained more body weight and fat mass than OP/water mice, showed an increase in liver triglycerides and developed insulin resistance. These effects were fully reversed in OP/SSW-water mice. In the gut, OR/SSW mice, but not OP/SSW mice, had an increase GLP-1 and CCK response to a liquid meal compared to mice drinking only water. OP/SSW mice had a decreased expression of melanocortin receptor 4 in the hypothalamus and increased expression of delta opioid receptor in the nucleus accumbens compared to OP/water mice when fasted that could explain the hyperphagia in these mice. When access to the sucrose solution was removed for 8 weeks, OP mice had increased dopaminergic and opioidergic response to a sucrose solution. Thus, intermittent access to a sucrose solution in mice fed a HFHS diet induces changes in the gut and brain signaling, leading to increased energy intake and adverse metabolic consequences only in mice prone to HFHSinduced obesity. (C) 2015 Elsevier Inc. All rights reserved.

Diffuse intrinsic pontine gliomas arise almost exclusively in children, and despite advances in treatment, the majority of patients die within 2 years after initial diagnosis. Because of their infiltrative nature and anatomic location in an eloquent area of the brain, most pontine gliomas are treated without a surgical biopsy. The corresponding lack of tissue samples has resulted in a limited understanding of the underlying genetic and molecular biologic abnormalities associated with pontine gliomas, and is a substantial obstacle for the preclinical testing of targeted therapeutic agents for these tumors. We have established a human glioma cell line that originated from surgical biopsy performed on a patient with a pontine glioma. To insure sustainable in vitro propagation, tumor cells were modified with hTERT (human telomerase ribonucleoprotein reverse transcriptase), and with a luciferase reporter to enable non-invasive bioluminescence imaging. The hTERT modified cells are tumorigenic in athymic rodents, and produce brainstem tumors that recapitulate the infiltrative growth of brainstem gliomas in patients. © Springer Science+Business Media, LLC. 2012.