MLL4 Is Required to Maintain Broad H3K4me3 Peaks and Super-Enhancers at Tumor Suppressor Genes.

Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes.

Overview of CD70 as a Potential Therapeutic Target for Osteosarcoma.

Osteosarcoma is a primary malignant bone tumor. Effective chemotherapy regimens for refractory disease are scarce, accounting for no improvement in survival. Immune-based cell therapies have emerged as novel alternatives. However, advancements with these therapies have been seen mostly when immune cells are armed to target specific tumor Ags. Recent studies identified cluster of differentiation 70 (CD70) as a promising target to osteosarcoma particularly because CD70 is highly expressed in osteosarcoma lung metastases (Pahl et al. 2015. Cancer Cell Int. 15: 31), and its overexpression by tumors has been correlated with immune evasion and tumor proliferation (Yang et al. 2007. Blood 110: 2537-2544). However, the limited knowledge of the overall CD70 expression within normal tissues and the potential for off-target effect pose several challenges (Flieswasser et al. 2022. J. Exp. Clin. Cancer Res. 41: 12). Nonetheless, CD70-based clinical trials are currently ongoing and are preliminarily showing promising results for patients with osteosarcoma. The present review sheds light on the recent literature on CD70 as it relates to osteosarcoma and highlights the benefits and challenges of targeting this pathway.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

Evaluation of potential role of R-loop and G-quadruplex DNA in the fragility of c-MYC during chromosomal translocation associated with Burkitt's lymphoma.

t(8;14) translocation is the hallmark of Burkitt's lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt's lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt's lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100 bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID in vitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation.

Dissociation protocols used for sarcoma tissues bias the transcriptome observed in single-cell and single-nucleus RNA sequencing.

BACKGROUND: Single-cell RNA-seq has emerged as an innovative technology used to study complex tissues and characterize cell types, states, and lineages at a single-cell level. Classification of bulk tumors by their individual cellular constituents has also created new opportunities to generate single-cell atlases for many organs, cancers, and developmental models. Despite the tremendous promise of this technology, recent evidence studying epithelial tissues and diverse carcinomas suggests the methods used for tissue processing, cell disaggregation, and preservation can significantly bias gene expression and alter the observed cell types. To determine whether sarcomas - tumors of mesenchymal origin - are subject to the same technical artifacts, we profiled patient-derived tumor explants (PDXs) propagated from three aggressive subtypes: osteosarcoma (OS), Ewing sarcoma (ES), desmoplastic small round cell tumor (DSRCT). Given the rarity of these sarcoma subtypes, we explored whether single-nuclei RNA-seq from more widely available archival frozen specimens could accurately be identified by gene expression signatures linked to tissue phenotype or pathognomonic fusion proteins. RESULTS: We systematically assessed dissociation methods across different sarcoma subtypes. We compared gene expression from single-cell and single-nucleus RNA-sequencing of 125,831 whole-cells and nuclei from ES, DSRCT, and OS PDXs. We detected warm dissociation artifacts in single-cell samples and gene length bias in single-nucleus samples. Classic sarcoma gene signatures were observed regardless of the dissociation method. In addition, we showed that dissociation method biases could be computationally corrected. CONCLUSIONS: We highlighted transcriptional biases, including warm dissociation and gene-length biases, introduced by the dissociation method for various sarcoma subtypes. This work is the first to characterize how the dissociation methods used for sc/snRNA-seq may affect the interpretation of the molecular features in sarcoma PDXs.

Bempegaldesleukin (BEMPEG; NKTR-214) efficacy as a single agent and in combination with checkpoint-inhibitor therapy in mouse models of osteosarcoma.

Survival of patients with relapsed/refractory osteosarcoma has not improved in the last 30 years. Several immunotherapeutic approaches have shown benefit in murine osteosarcoma models, including the anti-programmed death-1 (anti-PD-1) and anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) immune checkpoint inhibitors. Treatment with the T-cell growth factor interleukin-2 (IL-2) has shown some clinical benefit but has limitations due to poor tolerability. Therefore, we evaluated the efficacy of bempegaldesleukin (BEMPEG; NKTR-214), a first-in-class CD122-preferential IL-2 pathway agonist, alone and in combination with anti-PD-1 or anti-CTLA-4 immune checkpoint inhibitors in metastatic and orthotopic murine models of osteosarcoma. Treatment with BEMPEG delayed tumor growth and increased overall survival of mice with K7M2-WT osteosarcoma pulmonary metastases. BEMPEG also inhibited primary tumor growth and metastatic relapse in lungs and bone in the K7M3 orthotopic osteosarcoma mouse model. In addition, it enhanced therapeutic activity of anti-CTLA-4 and anti-PD-1 checkpoint blockade in the DLM8 subcutaneous murine osteosarcoma model. Finally, BEMPEG strongly increased accumulation of intratumoral effector T cells and natural killer cells, but not T-regulatory cells, resulting in improved effector:inhibitory cell ratios. Collectively, these data in multiple murine models of osteosarcoma provide a path toward clinical evaluation of BEMPEG-based regimens in human osteosarcoma.

SCR7, an inhibitor of NHEJ can sensitize tumor cells to ionization radiation.

Nonhomologous end joining (NHEJ), one of the major DNA double-strand break repair pathways, plays a significant role in cancer cell proliferation and resistance to radio and chemotherapeutic agents. Previously, we had described a small molecule inhibitor, SCR7, which inhibited NHEJ in a DNA Ligase IV dependent manner. Here, we report that SCR7 potentiates the effect of γ-radiation (IR) that induces DNA breaks as intermediates to eradicate cancer cells. Dose fractionation studies revealed that coadministration of SCR7 and IR (0.5 Gy) in mice Dalton's lymphoma (DLA) model led to a significant reduction in mice tumor cell proliferation, which was equivalent to that observed for 2 Gy dose when both solid and liquid tumor models were used. Besides, co-treatment with SCR7 and 1 Gy of IR further improved the efficacy. Notably, there was no significant change in blood parameters, kidney and liver functions upon combinatorial treatment of SCR7 and IR. Further, the co-treatment of SCR7 and IR resulted in a significant increase in unrepaired DSBs within cancer cells compared to either of the agent alone. Anatomy, histology, and other studies in tumor models confirmed the cumulative effects of both agents in activating apoptotic pathways to induce cytotoxicity by modulating DNA damage response and repair pathways. Thus, we report that SCR7 has the potential to reduce the side effects of radiotherapy by lowering its effective dose ex vivo and in mice tumor models, with implications in cancer therapy.

EpiSAFARI: sensitive detection of valleys in epigenetic signals for enhancing annotations of functional elements.

MOTIVATION: Functional genomics experiments generate genomewide signal profiles that are dense information sources for annotating the regulatory elements. These profiles measure epigenetic activity at the nucleotide resolution and they exhibit distinctive patterns as they fluctuate along the genome. Most notable of these patterns are the valley patterns that are prevalently observed in assays such as ChIP Sequencing and bisulfite sequencing. The genomic positions of valleys pinpoint locations of cis-regulatory elements such as enhancers and insulators. Systematic identification of the valleys provides novel information for delineating the annotation of regulatory elements. Nevertheless, the valleys are not reported by majority of the analysis pipelines. RESULTS: We describe EpiSAFARI, a computational method for sensitive detection of valleys from diverse types of epigenetic profiles. EpiSAFARI employs a novel smoothing method for decreasing noise in signal profiles and accounts for technical factors such as sparse signals, mappability and nucleotide content. In performance comparisons, EpiSAFARI performs favorably in terms of accuracy. The histone modification valleys detected by EpiSAFARI exhibit high conservation, transcription factor binding and they are enriched in nascent transcription. In addition, the large clusters of histone valleys are found to be enriched at the promoters of the developmentally associated genes. Differential histone valleys exhibit concordance with differential DNase signal at cell line specific valleys. DNA methylation valleys exhibit elevated conservation and high transcription factor binding. Specifically, we observed enriched binding of transcription factors associated with chromatin structure around methyl-valleys. AVAILABILITY AND IMPLEMENTATION: EpiSAFARI is publicly available at https://github.com/harmancilab/EpiSAFARI. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Licochalcone A is a Natural Selective Inhibitor of Arginine Methyltransferase 6.

Arginine methylation is a post-translational modification that is implicated in multiple biological functions including transcriptional regulation. The expression of protein arginine methyltransferases (PRMT) has been shown to be upregulated in various cancers. PRMTs have emerged as attractive targets for the development of new cancer therapies. Here, we describe the identification of a natural compound, licochalcone A, as a novel, reversible and selective inhibitor of PRMT6. Since expression of PRMT6 is upregulated in human breast cancers and is associated with oncogenesis, we used the human breast cancer cell line system to study the effect of licochalcone A treatment on PRMT6 activity, cell viability, cell cycle, and apoptosis. We demonstrated that licochalcone A is a non-S-adenosyl L-methionine (SAM) binding site competitive inhibitor of PRMT6. In MCF-7 cells, it inhibited PRMT6-dependent methylation of histone H3 at arginine 2 (H3R2), which resulted in a significant repression of estrogen receptor activity. Licochalcone A exhibited cytotoxicity towards human MCF-7 breast cancer cells, but not MCF-10A human breast epithelial cells, by upregulating p53 expression and blocking cell cycle progression at G2/M, followed by apoptosis. Thus, licochalcone A has potential for further development as a therapeutic agent against breast cancer.

Medulloblastoma epigenetics and the path to clinical innovation.

INTRODUCTION: In the last decade, a number of genomic and pharmacological studies have demonstrated the importance of epigenetic dysregulation in medulloblastoma initiation and progression. High throughput approaches including gene expression array, next-generation sequencing (NGS), and methylation profiling have now clearly identified at least four molecular subgroups within medulloblastoma, each with distinct clinical and prognostic characteristics. These studies have clearly shown that despite the overall paucity of mutations, clinically relevant events do occur within the cellular epigenetic machinery. Thus, this review aims to provide an overview of our current understanding of the spectrum of epi-oncogenetic perturbations in medulloblastoma. METHODS: Comprehensive review of epigenetic profiles of different subgroups of medulloblastoma in the context of molecular features. Epigenetic regulation is mediated mainly by DNA methylation, histone modifications and microRNAs (miRNA). Importantly, epigenetic mis-events are reversible and have immense therapeutic potential. CONCLUSION: The widespread epigenetic alterations present in these tumors has generated intense interest in their use as therapeutic targets. We provide an assessment of the progress that has been made towards the development of molecular subtypes-targeted therapies and the current status of clinical trials that have leveraged these recent advances.

ST09, A Novel Curcumin Derivative, Blocks Cell Migration by Inhibiting Matrix Metalloproteases in Breast Cancer Cells and Inhibits Tumor Progression in EAC Mouse Tumor Models.

PURPOSE: Curcumin is known for its anticancer and migrastatic activity in various cancers, including breast cancer. Newer curcumin derivatives are being explored to overcome limitations of curcumin like low bioavailability, stability, and side effects due to its higher dose. In this study, the synthesis of ST09, a novel curcumin derivative, and its antiproliferative, cytotoxic, and migrastatic properties have been explored both in vitro and in vivo. METHODS: After ST09 synthesis, anticancer activity was studied by performing standard cytotoxicity assays namely, lactate dehydrogenase (LDH) release assay, 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyletrazolium bromide (MTT), and trypan blue exclusion assay. Annexin-FITC, cell cycle analysis using flow cytometry, and Western blotting were performed to elucidate cell death mechanisms. The effect on the inhibition of cell migration was studied by transwell migration assay. An EAC (Ehrlich Ascites carcinoma) induced mouse tumor model was used to study the effect of ST09 on tumor regression. Drug toxicity was measured using aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and flow-cytometry based lymphocyte count. Histological analysis was performed for assessment of any tissue injury post ST09 treatment. RESULTS: ST09 shows an approximate 100-fold higher potency than curcumin, its parent compound, on breast tumor cell lines MCF-7 and MDA-MB231. ST09 arrests the cell cycle in a cell type-specific manner and induces an intrinsic apoptotic pathway both in vitro and in vivo. ST09 inhibits migration by downregulating matrix metalloprotease 1,2 (MMP1,2) and Vimentin. In vivo, ST09 administration led to decreased tumor volume in a mouse allograft model by boosting immunity with no significant drug toxicity. CONCLUSION: ST09 exhibits antiproliferative and cytotoxic activity at nanomolar concentrations. It induces cell death by activation of the intrinsic pathway of apoptosis both in vitro and in vivo. It also inhibits migration and invasion. This study provides evidence that ST09 can potentially be developed as a novel antitumor drug candidate for highly metastatic and aggressive breast cancer.

Characterization of DNA double-strand break repair pathways in diffuse large B cell lymphoma.

Efficient DNA repair is indispensable for maintaining genomic integrity in humans. Cancer associated deletions and mutations are mainly due to misrepaired DNA double-strand breaks (DSBs). Classical nonhomologous end joining (c-NHEJ) and homologous recombination (HR) are two major DSB repair pathways in humans. An error prone, alternative NHEJ pathway that utilizes microhomology was also reported in cancer cells and to a lesser extent in normal cells. In the present study, we evaluated the efficiency of various DSB repair pathways in the most common lymphoma, the diffuse large B cell lymphoma (DLBCL). Here we show that DNA repair through c-NHEJ pathway is limited in SUDHL8, a cell line derived from a DLBCL patient. Unlike c-NHEJ, microhomology mediated end joining (MMEJ) was predominant at physiological temperature. Consistent with the observation, expression level of repair proteins such as LIGASE I, LIGASE III, PARP1, CtIP, and MRE11 was higher in DLBCL cells when compared to c-NHEJ proteins. Further, inhibition of LIGASE I or MRE11, led to reduction in the efficiency of MMEJ in DLBCL cells. Besides, HR-mediated DSB repair occurring through gene conversion was observed. Thus, our results reveal the predominance of MMEJ over c-NHEJ in repairing DSBs in DLBCL cells, while error-free repair through HR was also evident.

Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

Investigation of anti-cancer and migrastatic properties of novel curcumin derivatives on breast and ovarian cancer cell lines.

BACKGROUND: Curcumin is known for its multitude of medicinal properties, including anti-cancer and migrastatic activity. Efforts to overcome poor bioavailability, stability, and side effects associated with the higher dose of curcumin has led to the development of newer derivatives of curcumin. Thus, the focus of this study is to screen novel curcumin derivatives, namely ST03 and ST08, which have not been reported before, for their cytotoxicity and migrastatic property on cancer cells. METHODS: Anti-cancer activity of ST03 and ST08 was carried out using standard cytotoxicity assays viz., LDH, MTT, and Trypan blue on both solid and liquid cancer types. Flow cytometric assays and western blotting was used to investigate the cell death mechanisms. Transwell migration assay was carried out to check for migrastatic properties of the compounds. RESULTS: Both the compounds, ST03 and ST08, showed ~ 100 fold higher potency on liquid and solid tumour cell lines compared to its parent compound curcumin. They induced cytotoxicity by activating the intrinsic pathway of apoptosis in the breast (MDA-MB-231) and ovarian cancer cell lines (PA-1) bearing metastatic and stem cell properties, respectively. Moreover, ST08 also showed inhibition on breast cancer cell migration by inhibiting MMP1 (matrix metalloproteinase 1). CONCLUSION: Both ST03 and ST08 exhibit anti-cancer activity at nanomolar concentration. They induce cell death by activating the intrinsic pathway of apoptosis. Also, they inhibit migration of the cancer cells by inhibiting MMP1 in breast cancer cells.

The ubiquitin-proteasome pathway in adult and pediatric brain tumors: biological insights and therapeutic opportunities.

Nearly 20 years ago, the concept of targeting the proteasome for cancer therapy began gaining momentum. This concept was driven by increased understanding of the biology/structure and function of the 26S proteasome, insight into the role of the proteasome in transformed cells, and the synthesis of pharmacological inhibitors with clinically favorable features. Subsequent in vitro, in vivo, and clinical testing culminated in the FDA approval of three proteasome inhibitors-bortezomib, carfilzomib, and ixazomib -for specific hematological malignancies. However, despite in vitro and in vivo studies pointing towards efficacy in solid tumors, clinical responses broadly have been evasive. For brain tumors, a malignancy in dire need of new approaches both in adult and pediatric patients, this has also been the case. Elucidation of proteasome-dependent processes in specific types of brain tumors, the evolution of newer proteasome targeting strategies, and the use of proteasome inhibitors in combination strategies will clarify how these agents can be leveraged more effectively to treat central nervous system malignancies. Since brain tumors represent a heterogeneous subset of solid tumors, and in particular, pediatric brain tumors possess distinct biology from adult brain tumors, tailoring of proteasome inhibitor-based strategies to specific subtypes of these tumors will be critical for advancing care for affected patients, and will be discussed in this review.

Pharmacological inhibition of LSD1 activity blocks REST-dependent medulloblastoma cell migration.

BACKGROUND: Medulloblastoma (MB) is the most common malignant brain tumor in children. Current problems in the clinic include metastasis, recurrence, and treatment-related sequelae that highlight the need for targeted therapies. Epigenetic perturbations are an established hallmark of human MB and expression of Lysine Specific Demethylase 1 (LSD1) is elevated in MBs compared to normal tissue, suggesting that LSD1 inhibitors may have efficacy against human MB tumors. METHODS: Expression of LSD1 was examined across a publicly-available database and correlated with patient outcomes. Sonic Hedgehog (SHH) MB samples were clustered based on expression of LSD1 and LSD1-associated RE-1 silencing transcription factor (REST) target genes as well as genes involved in metastasis. Resulting clusters were examined for patient outcomes associated with LSD1 and REST expression. Human SHH MB cell lines were transduced with a REST-transgene to create isogenic cell pairs. In vitro viability and cell migration assays were used to examine the effect of LSD1 knockdown or inhibition on these parameters. RESULTS: We demonstrate that subsets of SHH MB tumors have elevated LSD1 expression coincident with increased expression of its deubiquitylase, USP7, and REST. Patients with co-elevation of USP7, REST, and LSD1 have poorer outcomes compared to those with lower expression of these genes. In SHH MB cell lines, REST elevation increased cell growth and LSD1 protein levels. Surprisingly, while genetic loss of LSD1 reduced cell viability, pharmacological targeting of its activity using LSD1 inhibitors did not affect cell viability. However, a reduction in REST-dependent cell migration was seen in wound healing, suggesting that REST-LSD1 interaction regulates cell migration. Ingenuity pathway analyses validated these findings and identified Hypoxia Inducible Factor 1 alpha (HIF1A) as a potential target. In line with this, ectopic expression of HIF1A rescued the loss of migration seen following LSD1 inhibition. CONCLUSIONS: A subset of SHH patients display increased levels of LSD1 and REST, which is associated with poor outcomes. REST elevation in MB in conjunction with elevated LSD1 promotes MB cell migration. LSD1 inhibition blocks REST-dependent cell migration of MB cells in a HIF1A-dependent manner.

Methotrexate administration directly into the fourth ventricle in children with malignant fourth ventricular brain tumors: a pilot clinical trial.

We hypothesize that chemotherapy can be safely administered directly into the fourth ventricle to treat recurrent malignant brain tumors in children. For the first time in humans, methotrexate was infused into the fourth ventricle in children with recurrent, malignant brain tumors. A catheter was surgically placed into the fourth ventricle and attached to a ventricular access device. Cerebrospinal fluid (CSF) flow was confirmed by CINE MRI postoperatively. Each cycle consisted of 4 consecutive daily methotrexate infusions (2 milligrams). Disease response was monitored with serial MRI scans and CSF cytologic analysis. Trough CSF methotrexate levels were sampled. Five patients (3 with medulloblastoma and 2 with ependymoma) received 18, 18, 12, 9, and 3 cycles, respectively. There were no serious adverse events or new neurological deficits attributed to methotrexate. Two additional enrolled patients were withdrawn prior to planned infusions due to rapid disease progression. Median serum methotrexate level 4 h after infusion was 0.04 µmol/L. Range was 0.02-0.13 µmol/L. Median trough CSF methotrexate level 24 h after infusion was 3.18 µmol/L (range 0.53-212.36 µmol/L). All three patients with medulloblastoma had partial response or stable disease until one patient had progressive disease after cycle 18. Both patients with ependymoma had progressive disease after 9 and 3 cycles, respectively. Low-dose methotrexate can be infused into the fourth ventricle without causing neurological toxicity. Some patients with recurrent medulloblastoma experience a beneficial anti-tumor effect both within the fourth ventricle and at distant sites.

Synthesis and antiproliferative activity of imidazo[2,1-b][1,3,4]thiadiazole derivatives.

A series of 2,5,6-substituted imidazo[2,1-b][1,3,4]thiadiazole derivatives have been prepared and were tested for antiproliferative activity on cancer cells at the National Cancer Institute. Results showed that molecules with a benzyl group at position 2, exhibited an increase in activity for the introduction of a formyl group at the 5 position. The compound 2-benzyl-5-formyl-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazole 22 has been chosen for understanding the mechanism of action by various molecular and cellular biology studies. Results obtained from cell cycle evaluation analysis, analysis of mitochondrial membrane potential and Annexin V-FITC by flow cytometric analysis, ROS production and expression of apoptotic and DNA-repair proteins suggested that compound 22 induced cytotoxicity by activating extrinsic pathway of apoptosis, however, without affecting cell cycle progression.

REST-dependent downregulation of von Hippel-Lindau tumor suppressor promotes autophagy in SHH-medulloblastoma.

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)-a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein - a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.