Modulation of Radiation Biomarkers in a Randomized Phase II Study of 131I-MIBG With or Without Radiation Sensitizers for Relapsed or Refractory Neuroblastoma.

PURPOSE: 131I-metaiodobenzylguanidine (131I-MIBG) has demonstrated efficacy as a single agent in neuroblastoma. Recent trials have focused on 131I-MIBG combination strategies, though little is known about the effect of putative radiosensitizers on biological markers of radiation exposure. METHODS AND MATERIALS: NANT2011-01 evaluated 131I-MIBG therapy alone (arm A) or in combination with vincristine/irinotecan (arm B) or vorinostat (arm C) for patients with relapsed or refractory neuroblastoma. Blood samples were collected before and after 131I-MIBG infusion to determine levels of radiation-associated biomarkers (transcript and protein). The association of biomarker with treatment arm, clinical response, and treatment toxicity was analyzed. RESULTS: The cohort included 99 patients who had at least 1 biomarker available for analysis. Significant modulation in most biomarkers between baseline, 72, and 96 hours following 131I-MIBG was observed. Patients in arm C had the lowest degree of modulation in FLT3 ligand protein. Lower baseline BCL2 transcript levels were associated with higher overall response. Patients with greater increases in FLT3 ligand at 96 hours after 131I-MIBG therapy were significantly more likely to have grade 4 thrombocytopenia. Peripheral blood gene expression of the BCL2 family of apoptotic markers (BCL2L1 and BAX transcripts) was significantly associated with grade 4 hematologic toxicity. RNA sequencing demonstrated little overlap in the top modulated peripheral blood transcripts between randomized arms. CONCLUSIONS: Peripheral blood biomarkers relevant to radiation exposure demonstrate significant modulation after 131I-MIBG and concomitant radiation sensitizers affect extent of modulation. Biomarkers related to hematopoietic damage and apoptosis were associated with hematologic toxicity.

A druggable addiction to de novo pyrimidine biosynthesis in diffuse midline glioma.

Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify druggable targets for DMG, we conducted a genome-wide CRISPR screen that reveals a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. This metabolic vulnerability reflects an elevated rate of uridine/uracil degradation that depletes DMG cells of substrates for the alternate salvage pyrimidine biosynthesis pathway. A clinical stage inhibitor of DHODH (rate-limiting enzyme in the de novo pathway) diminishes uridine-5'-phosphate (UMP) pools, generates DNA damage, and induces apoptosis through suppression of replication forks-an "on-target" effect, as shown by uridine rescue. Matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy imaging demonstrates that this DHODH inhibitor (BAY2402234) accumulates in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts, highlighting BAY2402234 as a promising therapy against DMGs.

Wee1 kinase inhibitor adavosertib with radiation in newly diagnosed diffuse intrinsic pontine glioma: A Children's Oncology Group phase I consortium study.

Background: Children with diffuse intrinsic pontine gliomas (DIPG) have a dismal prognosis. Adavosertib (AZD1775) is an orally available, blood-brain barrier penetrant, Wee1 kinase inhibitor. Preclinical efficacy against DIPG is heightened by radiation induced replication stress. Methods: Using a rolling six design, 7 adavosertib dose levels (DLs) (50 mg/m2 alternating weeks, 50 mg/m2 alternating with weeks of every other day, 50 mg/m2, then 95, 130, 160, 200 mg/m2) were assessed. Adavosertib was only given on days of cranial radiation therapy (CRT).The duration of CRT (54 Gy over 30 fractions; 6 weeks) constituted the dose limiting toxicity (DLT) period. Endpoints included tolerability, pharmacokinetics, overall survival (OS) and peripheral blood γH2AX levels as a marker of DNA damage. Results: A total of 46 eligible patients with newly diagnosed DIPG [median (range) age 6 (3-21) years; 52% female] were enrolled. The recommend phase 2 dose (RP2D) of adavosertib was 200 mg/m2/d during days of CRT. Dose limiting toxicity included ALT elevation (n = 1, DL4) and neutropenia (n = 1, DL7). The mean Tmax, T1/2 and Clp on Day 1 were 2 h, 4.4 h, and 45.2 L/hr/m2, respectively. Modest accumulation of adavosertib was observed comparing day 5 versus day 1 AUC0-8h (accumulation ratio = 1.6). OS was 11.1 months (95% CI: 9.4, 12.5) and did not differ from historical control. Conclusion: Adavosertib in combination with CRT is well tolerated in children with newly diagnosed DIPG, however, compared to historical controls, did not improve OS. These results can inform future trial design in children with high-risk cancer.

Harnessing Lactate Metabolism for Radiosensitization.

Cancer cells rewire their metabolism to promote cell proliferation, invasion, and metastasis. Alterations in the lactate pathway have been characterized in diverse cancers, correlate with outcomes, and lead to many downstream effects, including decreasing oxidative stress, promoting an immunosuppressive tumor microenvironment, lipid synthesis, and building chemo- or radio-resistance. Radiotherapy is a key modality of treatment for many cancers and approximately 50% of patients with cancer will receive radiation for cure or palliation; thus, overcoming radio-resistance is important for improving outcomes. Growing research suggests that important molecular controls of the lactate pathway may serve as novel therapeutic targets and in particular, radiosensitizers. In this mini-review, we will provide an overview of lactate metabolism in cancer, discuss three important contributors to lactate metabolism (lactate dehydrogenase, monocarboxylate transporters, and mitochondrial pyruvate carrier), and present data that inhibition of these three pathways can lead to radiosensitization. Future research is needed to further understand critical regulators of lactate metabolism and explore clinical safety and efficacy of inhibitors of lactate dehydrogenase, monocarboxylate transporters, and mitochondrial pyruvate carrier alone and in combination with radiation.

RP58 Represses Transcriptional Programs Linked to Nonneuronal Cell Identity and Glioblastoma Subtypes in Developing Neurons.

How mammalian neuronal identity is progressively acquired and reinforced during development is not understood. We have previously shown that loss of RP58 (ZNF238 or ZBTB18), a BTB/POZ-zinc finger-containing transcription factor, in the mouse brain leads to microcephaly, corpus callosum agenesis, and cerebellum hypoplasia and that it is required for normal neuronal differentiation. The transcriptional programs regulated by RP58 during this process are not known. Here, we report for the first time that in embryonic mouse neocortical neurons a complex set of genes normally expressed in other cell types, such as those from mesoderm derivatives, must be actively repressed in vivo and that RP58 is a critical regulator of these repressed transcriptional programs. Importantly, gene set enrichment analysis (GSEA) analyses of these transcriptional programs indicate that repressed genes include distinct sets of genes significantly associated with glioma progression and/or pluripotency. We also demonstrate that reintroducing RP58 in glioma stem cells leads not only to aspects of neuronal differentiation but also to loss of stem cell characteristics, including loss of stem cell markers and decrease in stem cell self-renewal capacities. Thus, RP58 acts as an in vivo master guardian of the neuronal identity transcriptome, and its function may be required to prevent brain disease development, including glioma progression.

Lipidome-based Targeting of STAT3-driven Breast Cancer Cells Using Poly-l-glutamic Acid-coated Layer-by-Layer Nanoparticles.

The oncogenic transcription factor STAT3 is aberrantly activated in 70% of breast cancers, including nearly all triple-negative breast cancers (TNBCs). Because STAT3 is difficult to target directly, we considered whether metabolic changes driven by activated STAT3 could provide a therapeutic opportunity. We found that STAT3 prominently modulated several lipid classes, with most profound effects on N-acyl taurine and arachidonic acid, both of which are involved in plasma membrane remodeling. To exploit these metabolic changes therapeutically, we screened a library of layer-by-layer (LbL) nanoparticles (NPs) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-l-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to nontransformed cells, and the heightened PLE-NP binding to TNBC cells was attenuated by STAT3 inhibition. This effect was also observed in densely packed three-dimensional breast cancer organoids. As STAT3-transformed cells show greater resistance to cytotoxic agents, we evaluated whether enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven cells at lower doses compared with both unencapsulated cisplatin and cisplatin-loaded nontargeted NPs. In addition, because radiation is commonly used in breast cancer treatment, and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using unique LbL NPs.

Phase I Clinical Trial of the Wee1 Inhibitor Adavosertib (AZD1775) with Irinotecan in Children with Relapsed Solid Tumors: A COG Phase I Consortium Report (ADVL1312).

PURPOSE: Adavosertib (AZD1775), an inhibitor of WEE1 kinase, potentiates replicative stress induced by oncogenes or chemotherapy. Antitumor activity of adavosertib has been demonstrated in preclinical models of pediatric cancer. This phase I trial was performed to define dose-limiting toxicities (DLT), recommended phase II dose (RP2D), and pharmacokinetics of adavosertib in combination with irinotecan in children and adolescents with relapsed or refractory solid tumors or primary central nervous system tumors. PATIENTS AND METHODS: Using a 3+3 escalation design, five dose cohorts of the combination of adavosertib and irinotecan (50/70; 65/70; 65/90; 85/90; 110/90 mg/m2/day) delivered on days 1-5 of a 21-day cycle were studied. Pharmacokinetics and analysis of peripheral blood γH2AX was performed. RESULTS: Thirty-seven patients were enrolled; 27 were evaluable. The median (range) age was 14 (2-20) years. Twenty-five (93%) received prior chemotherapy (median, three regimens) and 21 (78%) received prior radiotherapy. Eleven patients had a primary central nervous system (CNS) malignancy. Common toxicities were hematologic and gastrointestinal. Two patients receiving adavosertib (110 mg/m2) in combination with irinotecan (90 mg/m2) experienced dose-limiting grade 3 dehydration. A patient with Ewing sarcoma had a confirmed partial response and 2 patients (ependymoma and neuroblastoma) had prolonged stable disease (≥ 6 cycles). Pharmacokinetics of adavosertib were variable but generally dose proportional and clearance was lower in younger patients. CONCLUSIONS: Adavosertib (85 mg/m2) in combination with irinotecan (90 mg/m2) administered orally for 5 days was the MTD in children and adolescents with solid and CNS tumors.

Immune landscapes associated with different glioblastoma molecular subtypes.

Recent work has highlighted the tumor microenvironment as a central player in cancer. In particular, interactions between tumor and immune cells may help drive the development of brain tumors such as glioblastoma multiforme (GBM). Despite significant research into the molecular classification of glioblastoma, few studies have characterized in a comprehensive manner the immune infiltrate in situ and within different GBM subtypes.In this study, we use an unbiased, automated immunohistochemistry-based approach to determine the immune phenotype of the four GBM subtypes (classical, mesenchymal, neural and proneural) in a cohort of 98 patients. Tissue Micro Arrays (TMA) were stained for CD20 (B lymphocytes), CD5, CD3, CD4, CD8 (T lymphocytes), CD68 (microglia), and CD163 (bone marrow derived macrophages) antibodies. Using automated image analysis, the percentage of each immune population was calculated with respect to the total tumor cells. Mesenchymal GBMs displayed the highest percentage of microglia, macrophage, and lymphocyte infiltration. CD68+ and CD163+ cells were the most abundant cell populations in all four GBM subtypes, and a higher percentage of CD163+ cells was associated with a worse prognosis. We also compared our results to the relative composition of immune cell type infiltration (using RNA-seq data) across TCGA GBM tumors and validated our results obtained with immunohistochemistry with an external cohort and a different method. The results of this study offer a comprehensive analysis of the distribution and the infiltration of the immune components across the four commonly described GBM subgroups, setting the basis for a more detailed patient classification and new insights that may be used to better apply or design immunotherapies for GBM.

Dual HDAC and PI3K Inhibition Abrogates NFκB- and FOXM1-Mediated DNA Damage Response to Radiosensitize Pediatric High-Grade Gliomas.

Aberrant chromatin remodeling and activation of the PI3K pathway have been identified as important mediators of pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) pathogenesis. As inhibition of these pathways are promising therapeutic avenues and radiation is the only modality to prolong survival of patients with DIPG, we sought to explore radiosensitizing functions of such inhibition and to explore mechanisms of action of such agents. Here, we demonstrate that combined treatment with radiotherapy and CUDC-907, a novel first-in-class dual inhibitor of histone deacetylases (HDAC) and PI3K, evokes a potent cytotoxic response in pHGG and DIPG models. CUDC-907 modulated DNA damage response by inhibiting radiation-induced DNA repair pathways including homologous recombination and nonhomologous end joining. The radiosensitizing effects of CUDC-907 were mediated by decreased NFκB/Forkhead box M1 (FOXM1) recruitment to promoters of genes involved in the DNA damage response; exogenous expression of NFκB/FOXM1 protected from CUDC-907-induced cytotoxicity. Together, these findings reveal CUDC-907 as a novel radiosensitizer with potent antitumor activity in pHGG and DIPG and provide a preclinical rationale for the combination of CUDC-907 with radiotherapy as a novel therapeutic strategy against pHGG and DIPG. More globally, we have identified NFκB and FOXM1 and their downstream transcriptional elements as critical targets for new treatments for pHGG and DIPG.Significance: These findings describe the radiosensitizing effect of a novel agent in pediatric high-grade gliomas, addressing a critical unmet need of increasing the radiation sensitivity of these highly aggressive tumors. Cancer Res; 78(14); 4007-21. ©2018 AACR.

Imaging patterns predict patient survival and molecular subtype in glioblastoma via machine learning techniques.

BACKGROUND: MRI characteristics of brain gliomas have been used to predict clinical outcome and molecular tumor characteristics. However, previously reported imaging biomarkers have not been sufficiently accurate or reproducible to enter routine clinical practice and often rely on relatively simple MRI measures. The current study leverages advanced image analysis and machine learning algorithms to identify complex and reproducible imaging patterns predictive of overall survival and molecular subtype in glioblastoma (GB). METHODS: One hundred five patients with GB were first used to extract approximately 60 diverse features from preoperative multiparametric MRIs. These imaging features were used by a machine learning algorithm to derive imaging predictors of patient survival and molecular subtype. Cross-validation ensured generalizability of these predictors to new patients. Subsequently, the predictors were evaluated in a prospective cohort of 29 new patients. RESULTS: Survival curves yielded a hazard ratio of 10.64 for predicted long versus short survivors. The overall, 3-way (long/medium/short survival) accuracy in the prospective cohort approached 80%. Classification of patients into the 4 molecular subtypes of GB achieved 76% accuracy. CONCLUSIONS: By employing machine learning techniques, we were able to demonstrate that imaging patterns are highly predictive of patient survival. Additionally, we found that GB subtypes have distinctive imaging phenotypes. These results reveal that when imaging markers related to infiltration, cell density, microvascularity, and blood-brain barrier compromise are integrated via advanced pattern analysis methods, they form very accurate predictive biomarkers. These predictive markers used solely preoperative images, hence they can significantly augment diagnosis and treatment of GB patients.

Shift in monocyte apoptosis with increasing viral load and change in apoptosis-related ISG/Bcl2 family gene expression in chronically HIV-1-infected subjects.

UNLABELLED: Although monocytes and macrophages are targets of HIV-1-mediated immunopathology, the impact of high viremia on activation-induced monocyte apoptosis relative to monocyte and macrophage activation changes remains undetermined. In this study, we determined constitutive and oxidative stress-induced monocyte apoptosis in uninfected and HIV(+) individuals across a spectrum of viral loads (n = 35; range, 2,243 to 1,355,998 HIV-1 RNA copies/ml) and CD4 counts (range, 26 to 801 cells/mm(3)). Both constitutive apoptosis and oxidative stress-induced apoptosis were positively associated with viral load and negatively associated with CD4, with an elevation in apoptosis occurring in patients with more than 40,000 (4.6 log) copies/ml. As expected, expression of Rb1 and interferon-stimulated genes (ISGs), plasma soluble CD163 (sCD163) concentration, and the proportion of CD14(++) CD16(+) intermediate monocytes were elevated in viremic patients compared to those in uninfected controls. Although CD14(++) CD16(+) frequencies, sCD14, sCD163, and most ISG expression were not directly associated with a change in apoptosis, sCD14 and ISG expression showed an association with increasing viral load. Multivariable analysis of clinical values and monocyte gene expression identified changes in IFI27, IFITM2, Rb1, and Bcl2 expression as determinants of constitutive apoptosis (P = 3.77 × 10(-5); adjusted R(2) = 0.5983), while changes in viral load, IFITM2, Rb1, and Bax expression were determinants of oxidative stress-induced apoptosis (P = 5.59 × 10(-5); adjusted R(2) = 0.5996). Our data demonstrate differential activation states in monocytes between levels of viremia in association with differences in apoptosis that may contribute to greater monocyte turnover with high viremia. IMPORTANCE: This study characterized differential monocyte activation, apoptosis, and apoptosis-related gene expression in low- versus high-level viremic HIV-1 patients, suggesting a shift in apoptosis regulation that may be associated with disease state. Using single and multivariable analysis of monocyte activation parameters and gene expression, we supported the hypothesis that monocyte apoptosis in HIV disease is a reflection of viremia and activation state with contributions from gene expression changes within the ISG and Bcl2 gene families. Understanding monocyte apoptosis response may inform HIV immunopathogenesis, retention of infected macrophages, and monocyte turnover in low- or high-viral-load states.

Isoform-level gene signature improves prognostic stratification and accurately classifies glioblastoma subtypes.

Molecular stratification of tumors is essential for developing personalized therapies. Although patient stratification strategies have been successful; computational methods to accurately translate the gene-signature from high-throughput platform to a clinically adaptable low-dimensional platform are currently lacking. Here, we describe PIGExClass (platform-independent isoform-level gene-expression based classification-system), a novel computational approach to derive and then transfer gene-signatures from one analytical platform to another. We applied PIGExClass to design a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) based molecular-subtyping assay for glioblastoma multiforme (GBM), the most aggressive primary brain tumors. Unsupervised clustering of TCGA (the Cancer Genome Altas Consortium) GBM samples, based on isoform-level gene-expression profiles, recaptured the four known molecular subgroups but switched the subtype for 19% of the samples, resulting in significant (P = 0.0103) survival differences among the refined subgroups. PIGExClass derived four-class classifier, which requires only 121 transcript-variants, assigns GBM patients' molecular subtype with 92% accuracy. This classifier was translated to an RT-qPCR assay and validated in an independent cohort of 206 GBM samples. Our results demonstrate the efficacy of PIGExClass in the design of clinically adaptable molecular subtyping assay and have implications for developing robust diagnostic assays for cancer patient stratification.

Isoform level expression profiles provide better cancer signatures than gene level expression profiles.

BACKGROUND: The majority of mammalian genes generate multiple transcript variants and protein isoforms through alternative transcription and/or alternative splicing, and the dynamic changes at the transcript/isoform level between non-oncogenic and cancer cells remain largely unexplored. We hypothesized that isoform level expression profiles would be better than gene level expression profiles at discriminating between non-oncogenic and cancer cellsgene level. METHODS: We analyzed 160 Affymetrix exon-array datasets, comprising cell lines of non-oncogenic or oncogenic tissue origins. We obtained the transcript-level and gene level expression estimates, and used unsupervised and supervised clustering algorithms to study the profile similarity between the samples at both gene and isoform levels. RESULTS: Hierarchical clustering, based on isoform level expressions, effectively grouped the non-oncogenic and oncogenic cell lines with a virtually perfect homogeneity-grouping rate (97.5%), regardless of the tissue origin of the cell lines. However, gene levelthis rate was much lower, being 75% at best based on the gene level expressions. Statistical analyses of the difference between cancer and non-oncogenic samples identified the existence of numerous genes with differentially expressed isoforms, which otherwise were not significant at the gene level. We also found that canonical pathways of protein ubiquitination, purine metabolism, and breast-cancer regulation by stathmin1 were significantly enriched among genes thatshow differential expression at isoform level but not at gene level. CONCLUSIONS: In summary, cancer cell lines, regardless of their tissue of origin, can be effectively discriminated from non-cancer cell lines at isoform level, but not at gene level. This study suggests the existence of an isoform signature, rather than a gene signature, which could be used to distinguish cancer cells from normal cells.

Alternative transcription and alternative splicing in cancer.

In recent years, the notion of "one gene makes one protein that functions in one signaling pathway" in mammalian cells has been shown to be overly simplistic. Recent genome-wide studies suggest that at least half of the human genes, including many therapeutic target genes, produce multiple protein isoforms through alternative splicing and alternative usage of transcription initiation and/or termination. For example, alternative splicing of the vascular endothelial growth factor gene (VEGFA) produces multiple protein isoforms, which display either pro-angiogenic or anti-angiogenic activities. Similarly, for the majority of human genes, the inclusion or exclusion of exonic sequences enhances the generation of transcript variants and/or protein isoforms that can vary in structure and functional properties. Many of the isoforms produced in this manner are tightly regulated during normal development but are misregulated in cancer cells. Altered expression of transcript variants and protein isoforms for numerous genes is linked with disease and its prognosis, and cancer cells manipulate regulatory mechanisms to express specific isoforms that confer drug resistance and survival advantages. Emerging insights indicate that modulating the expression of transcript and protein isoforms of a gene may hold the key to impeding tumor growth and act as a model for efficient targeting of disease-associated genes at the isoform level. This review highlights the role and regulation of alternative transcription and splicing mechanisms in generating the transcriptome, and the misuse and diagnostic/prognostic potential of alternative transcription and splicing in cancer.

IsoformEx: isoform level gene expression estimation using weighted non-negative least squares from mRNA-Seq data.

BACKGROUND: mRNA-Seq technology has revolutionized the field of transcriptomics for identification and quantification of gene transcripts not only at gene level but also at isoform level. Estimating the expression levels of transcript isoforms from mRNA-Seq data is a challenging problem due to the presence of constitutive exons. RESULTS: We propose a novel algorithm (IsoformEx) that employs weighted non-negative least squares estimation method to estimate the expression levels of transcript isoforms. Validations based on in silico simulation of mRNA-Seq and qRT-PCR experiments with real mRNA-Seq data showed that IsoformEx could accurately estimate transcript expression levels. In comparisons with published methods, the transcript expression levels estimated by IsoformEx showed higher correlation with known transcript expression levels from simulated mRNA-Seq data, and higher agreement with qRT-PCR measurements of specific transcripts for real mRNA-Seq data. CONCLUSIONS: IsoformEx is a fast and accurate algorithm to estimate transcript expression levels and gene expression levels, which takes into account short exons and alternative exons with a weighting scheme. The software is available at http://bioinformatics.wistar.upenn.edu/isoformex.

Alternative transcription exceeds alternative splicing in generating the transcriptome diversity of cerebellar development.

Despite our growing knowledge that many mammalian genes generate multiple transcript variants that may encode functionally distinct protein isoforms, the transcriptomes of various tissues and their developmental stages are poorly defined. Identifying the transcriptome and its regulation in a cell/tissue is the key to deciphering the cell/tissue-specific functions of a gene. We built a genome-wide inventory of noncoding and protein-coding transcripts (transcriptomes), their promoters (promoteromes) and histone modification states (epigenomes) for developing, and adult cerebella using integrative massive-parallel sequencing and bioinformatics approach. The data consists of 61,525 (12,796 novel) distinct mRNAs transcribed by 29,589 (4792 novel) promoters corresponding to 15,669 protein-coding and 7624 noncoding genes. Importantly, our results show that the transcript variants from a gene are predominantly generated using alternative transcriptional rather than splicing mechanisms, highlighting alternative promoters and transcriptional terminations as major sources of transcriptome diversity. Moreover, H3K4me3, and not H3K27me3, defined the use of alternative promoters, and we identified a combinatorial role of H3K4me3 and H3K27me3 in regulating the expression of transcripts, including transcript variants of a gene during development. We observed a strong bias of both H3K4me3 and H3K27me3 for CpG-rich promoters and an exponential relationship between their enrichment and corresponding transcript expression. Furthermore, the majority of genes associated with neurological diseases expressed multiple transcripts through alternative promoters, and we demonstrated aberrant use of alternative promoters in medulloblastoma, cancer arising in the cerebellum. The transcriptomes of developing and adult cerebella presented in this study emphasize the importance of analyzing gene regulation and function at the isoform level.

Protein arginine methyltransferase 5 suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells.

The proper epigenetic modification of chromatin by protein arginine methyltransferases (PRMTs) is crucial for normal cell growth and health. The human SWI/SNF-associated PRMT5 is involved in the transcriptional repression of target genes by directly methylating H3R8 and H4R3. To further understand the impact of PRMT5-mediated histone methylation on cancer, we analyzed its expression in normal and transformed human B lymphocytes. Our findings reveal that PRMT5 protein levels are enhanced in various human lymphoid cancer cells, including transformed chronic lymphocytic leukemia (B-CLL) cell lines. PRMT5 overexpression is caused by the altered expression of the PRMT5-specific microRNAs 19a, 25, 32, 92, 92b, and 96 and results in the increased global symmetric methylation of H3R8 and H4R3. An evaluation of both epigenetic marks at PRMT5 target genes such as RB1 (p105), RBL1 (p107), and RBL2 (p130) showed that promoters H3R8 and H4R3 are hypermethylated, which in turn triggers pocket protein transcriptional repression. Furthermore, reducing PRMT5 expression in WaC3CD5 B-CLL cells abolishes H3R8 and H4R3 hypermethylation, restores RBL2 expression, and inhibits cancer cell proliferation. These results indicate that PRMT5 overexpression epigenetically alters the transcription of key tumor suppressor genes and suggest a causal role of the elevated symmetric methylation of H3R8 and H4R3 at the RBL2 promoter in transformed B-lymphocyte pathology.

Interplay between chromatin remodelers and protein arginine methyltransferases.

Chromatin modifying enzymes have emerged as key regulators of all DNA based processes, which control cell growth, development, and differentiation. Recently, it has become clear that different chromatin remodeling and histone-modifying activities are involved in transcriptional activation and repression. Among the enzymes involved in regulating chromatin structure is the family of protein arginine methyltransferases (PRMTs) that specializes in methylating both histones as well as key cellular proteins. There are eleven different PRMT genes (PRMT1-11) whose biological function remains under explored. PRMTs regulate various cellular processes such as DNA repair and transcription, RNA processing, signal transduction, and nucleo-cytoplasmic localization. Like histone lysine methylation, methylation of histone arginine residues can either induce or inhibit transcription depending on the residue being modified and the type of methylation being introduced. In this review, we will focus on the latest findings and biological roles of ATP-dependent chromatin remodeling complexes and PRMT enzymes, and how their aberrant expression is linked to cancer.

Low levels of miR-92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma.

Protein arginine methyltransferase PRMT5 interacts with human SWI/SNF complexes and methylates histones H3R8 and H4R3. To elucidate the role of PRMT5 in human cancer, we analyzed PRMT5 expression in normal human B lymphocytes and a panel of lymphoid cancer cell lines as well as mantle cell lymphoma (MCL) clinical samples. We show that PRMT5 protein levels are elevated in all cancer cells, including clinical samples examined despite its low rate of transcription and messenger RNA stability. Remarkably, polysome profiling revealed that PRMT5 mRNA is translated more efficiently in Mino and JeKo MCL cells than in normal B cells, and that decreased miR-92b and miR-96 expression augments PRMT5 translation. Consequently, global methylation of H3R8 and H4R3 is increased and is accompanied by repression of suppressor of tumorigenecity 7 (ST7) in lymphoid cancer cells. Furthermore, knockdown of PRMT5 expression reduces proliferation of transformed JeKo and Raji cells. Thus, our studies indicate that aberrant expression of PRMT5 leads to altered epigenetic modification of chromatin, which in turn impacts transcriptional performance of anti-cancer genes and growth of transformed lymphoid cells.

The protein arginine methyltransferase Prmt5 is required for myogenesis because it facilitates ATP-dependent chromatin remodeling.

Skeletal muscle differentiation requires the coordinated activity of transcription factors, histone modifying enzymes, and ATP-dependent chromatin remodeling enzymes. The type II protein arginine methyltransferase Prmt5 symmetrically dimethylates histones H3 and H4 and numerous nonchromatin proteins, and prior work has implicated Prmt5 in transcriptional repression. Here we demonstrate that MyoD-induced muscle differentiation requires Prmt5. One of the first genes activated during differentiation encodes the myogenic regulator myogenin. Prmt5 and dimethylated H3R8 (histone 3 arginine 8) are localized at the myogenin promoter in differentiating cells. Modification of H3R8 required Prmt5, and reduction of Prmt5 resulted in the abrogation of promoter binding by the Brg1 ATPase-associated with the SWI/SNF chromatin remodeling enzymes and all subsequent events associated with gene activation, including increases in chromatin accessibility and stable binding by MyoD. Prmt5 and dimethylated H3R8 were also associated with the myogenin promoter in activated satellite cells isolated from muscle tissue, further demonstrating the physiological relevance of these observations. The data indicate that Prmt5 facilitates myogenesis because it is required for Brg1-dependent chromatin remodeling and gene activation at a locus essential for differentiation. We therefore conclude that a histone modifying enzyme is necessary to permit an ATP-dependent chromatin remodeling enzyme to function.