Mir-21-Sox2 Axis Delineates Glioblastoma Subtypes with Prognostic Impact.

Glioblastoma (GBM) is the most aggressive human brain tumor. Although several molecular subtypes of GBM are recognized, a robust molecular prognostic marker has yet to be identified. Here, we report that the stemness regulator Sox2 is a new, clinically important target of microRNA-21 (miR-21) in GBM, with implications for prognosis. Using the MiR-21-Sox2 regulatory axis, approximately half of all GBM tumors present in the Cancer Genome Atlas (TCGA) and in-house patient databases can be mathematically classified into high miR-21/low Sox2 (Class A) or low miR-21/high Sox2 (Class B) subtypes. This classification reflects phenotypically and molecularly distinct characteristics and is not captured by existing classifications. Supporting the distinct nature of the subtypes, gene set enrichment analysis of the TCGA dataset predicted that Class A and Class B tumors were significantly involved in immune/inflammatory response and in chromosome organization and nervous system development, respectively. Patients with Class B tumors had longer overall survival than those with Class A tumors. Analysis of both databases indicated that the Class A/Class B classification is a better predictor of patient survival than currently used parameters. Further, manipulation of MiR-21-Sox2 levels in orthotopic mouse models supported the longer survival of the Class B subtype. The MiR-21-Sox2 association was also found in mouse neural stem cells and in the mouse brain at different developmental stages, suggesting a role in normal development. Therefore, this mechanism-based classification suggests the presence of two distinct populations of GBM patients with distinguishable phenotypic characteristics and clinical outcomes. SIGNIFICANCE STATEMENT: Molecular profiling-based classification of glioblastoma (GBM) into four subtypes has substantially increased our understanding of the biology of the disease and has pointed to the heterogeneous nature of GBM. However, this classification is not mechanism based and its prognostic value is limited. Here, we identify a new mechanism in GBM (the miR-21-Sox2 axis) that can classify ∼50% of patients into two subtypes with distinct molecular, radiological, and pathological characteristics. Importantly, this classification can predict patient survival better than the currently used parameters. Further, analysis of the miR-21-Sox2 relationship in mouse neural stem cells and in the mouse brain at different developmental stages indicates that miR-21 and Sox2 are predominantly expressed in mutually exclusive patterns, suggesting a role in normal neural development.

Transcriptional Regulation of Protein Synthesis by Mediator Kinase in MYC-driven Medulloblastoma.

MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening across MB cell lines, we identified the Mediator-associated kinase CDK8 as the top dependence for MYC-driven MB. Loss of CDK8 markedly reduces MYC expression and impedes MB growth. Mechanistically, we demonstrate that CDK8 depletion suppresses ribosome biogenesis and mRNA translation. CDK8 regulates occupancy of phospho-Polymerase II at specific chromatin loci facilitating an epigenetic alteration that promotes transcriptional regulation of ribosome biogenesis. Additionally, CDK8-mediated phosphorylation of 4EBP1 plays a crucial role in initiating eIF4E-dependent translation. Targeting CDK8 effectively suppresses cancer stem and progenitor cells, characterized by increased ribosome biogenesis activity. We also report the synergistic inhibition of CDK8 and mTOR in vivo and in vitro . Overall, our findings establish a connection between transcription and translation regulation, suggesting a promising therapeutic approach targets multiple points in the protein synthesis network for MYC-driven MB.

EYA2 tyrosine phosphatase inhibition reduces MYC and prevents medulloblastoma progression.

BACKGROUND: Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. METHODS: Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. RESULTS: EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. CONCLUSIONS: Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.

Secondary RNA structure and nucleotide specificity contribute to internal initiation mediated by the human tau 5' leader.

Mechanisms by which eukaryotic internal ribosomal entry sites (IRESs) initiate translation have not been well described. Viral IRESs utilize a combination of secondary/tertiary structure concomitant with sequence specific elements to initiate translation. Eukaryotic IRESs are proposed to utilize the same components, although it appears that short sequence specific elements are more common. In this report we perform an extensive analysis of the IRES in the human tau mRNA. We demonstrate that the tau IRES exhibits characteristics similar to viral IRESs. It contains two main structural domains that exhibit secondary interactions, which are essential for internal initiation. Moreover, the tau IRES is extremely sensitive to small nucleotide substitutions. Our data also indicates that the 40S ribosome is recruited to the middle of the IRES, but whether it scans to the initiation codon in a linear fashion is questioned. Overall, these results identify structural and sequence elements critical for tau IRES activity and consequently, provide a novel target to regulate tau protein expression in disease states including Alzheimer disease and other tauopathies.

SMYD3 Promotes Cell Cycle Progression by Inducing Cyclin D3 Transcription and Stabilizing the Cyclin D1 Protein in Medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Maximum safe resection, postoperative craniospinal irradiation, and chemotherapy are the standard of care for MB patients. MB is classified into four subgroups: Shh, Wnt, Group 3, and Group 4. Of these subgroups, patients with Myc+ Group 3 MB have the worst prognosis, necessitating alternative therapies. There is increasing interest in targeting epigenetic modifiers for treating pediatric cancers, including MB. Using an RNAi functional genomic screen, we identified the lysine methyltransferase SMYD3, as a crucial epigenetic regulator that drives the growth of Group 3 Myc+ MB cells. We demonstrated that SMYD3 directly binds to the cyclin D3 promoter to activate its transcription. Further, SMYD3 depletion significantly reduced MB cell proliferation and led to the downregulation of cyclin D3, cyclin D1, pRBSer795, with concomitant upregulations in RB in vitro. Similar results were obtained following pharmacological inhibition of SMYD3 using BCI-121 ex vivo. SMYD3 knockdown also promoted cyclin D1 ubiquitination, indicating that SMYD3 plays a vital role in stabilizing the cyclin D1 protein. Collectively, our studies demonstrate that SMYD3 drives cell cycle progression in Group 3 Myc+ MB cells and that targeting SMYD3 has the potential to improve clinical outcomes for high-risk patients.

A novel PLK1 inhibitor onvansertib effectively sensitizes MYC-driven medulloblastoma to radiotherapy.

BACKGROUND: Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. METHODS: Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay, and RNA-Seq. ALDH activity, cell-cycle distribution, and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. RESULTS: PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compared with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. CONCLUSIONS: These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.

A Regulatory Loop of FBXW7-MYC-PLK1 Controls Tumorigenesis of MYC-Driven Medulloblastoma.

Polo-like kinase 1 (PLK1) is highly expressed in group 3 medulloblastoma (MB), and it has been preclinically validated as a cancer therapeutic target in medulloblastoma. Here, we demonstrate that PLK1 inhibition with PCM-075 or BI6727 significantly reduces the growth of MB cells and causes a decrease of c-MYC mRNA and protein levels. We show that MYC activates PLK1 transcription, while the inhibition of PLK1 suppresses MB tumor development and causes a decrease in c-MYC protein level by suppressing FBXW7 auto poly-ubiquitination. FBXW7 physically interacts with PLK1 and c-MYC, facilitating their protein degradation by promoting ubiquitination. These results demonstrate a PLK1-FBXW7-MYC regulatory loop in MYC-driven medulloblastoma. Moreover, FBXW7 is significantly downregulated in group 3 patient samples. The overexpression of FBXW7 induced apoptosis and suppressed proliferation in vitro and in vivo, while constitutive phosphorylation mutation attenuated its tumor suppressor function. Altogether, these findings demonstrated that PLK1 inhibition stabilizes FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing medulloblastoma progression.

Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in MYC-driven medulloblastoma.

MYC-driven medulloblastoma is a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. MYC gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in MYC-amplified medulloblastoma, we employ a CRISPR-Cas9 essentiality screen targeting 1,140 genes. We identify CDK7 as a mediator of medulloblastoma tumorigenesis. Using chemical inhibitors and genetic depletion, we observe cessation of tumor growth in xenograft mouse models and increases in apoptosis. The results are attributed to repression of a core set of MYC-driven transcriptional programs mediating DNA repair. CDK7 inhibition alters RNA polymerase II (RNA Pol II) and MYC association at DNA repair genes. Blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage, extending survival and tumor latency in xenograft mouse models. Our studies establish the selective inhibition of MYC-driven medulloblastoma by CDK7 inhibition combined with radiation as a viable therapeutic strategy.

Translation initiation of the human tau mRNA through an internal ribosomal entry site.

Neurofibrillary tangles are a pathological phenotype in Alzheimer's disease (AD) and are caused by the hyperphosphorylation of the microtubule associated protein tau. In mouse models of AD, decreasing tau protein expression limits the severity of symptoms and inhibits progression of AD. We now report that the 5' leader in the human tau mRNA contains an internal ribosomal entry site (IRES) and that IRES-dependent translation plays a role in the synthesis of tau protein. Consequently, targeting the tau IRES provides a novel target for regulating tau expression in AD and other tauopathies.

Combined functional genomic and chemical screens identify SETD8 as a therapeutic target in MYC-driven medulloblastoma.

Medulloblastoma (MB) is the most prevalent malignant brain tumor in children, accounting for 20% of all childhood brain tumors. The molecular profiling of MB into 4 major subgroups (WNT, SHH, Grp3, and Grp4) emphasizes the heterogeneity of MB and opens paths in which treatments may be targeted to molecularly aggressive and distinct tumors. Current therapeutic strategies for Group 3 MB are challenging and can be accompanied by long-term side effects from treatment. The involvement of altered epigenetic machinery in neoplastic transformation in MB has become more evident. Thus, we performed an epigenomic RNAi and chemical screen and identified SETD8/PRE-SET7/KMT5a as a critical player in maintaining proliferation and cell survival of MB cells. We have found that inhibition of SETD8 effects the migration/invasive ability of MB cells. SETD8 alters H4K20me chromatin occupancy at key genes involved in tumor invasiveness and pluripotency. Interestingly, these results link the aggressive and metastatic behavior of MYC-driven MB with SETD8 activity. Based on our results, we suggest that SETD8 has a critical role mediating Group 3 MB tumorigenesis. Establishing a role for SETD8 as a factor in MYC-driven MB has potential to lead to more effective therapies needed to improve outcomes in high-risk patients.

REST-DRD2 mechanism impacts glioblastoma stem cell-mediated tumorigenesis.

BACKGROUND: Glioblastoma (GBM) is a lethal, heterogeneous human brain tumor, with regulatory mechanisms that have yet to be fully characterized. Previous studies have indicated that the transcriptional repressor REST (repressor element-1 silencing transcription factor) regulates the oncogenic potential of GBM stem cells (GSCs) based on level of expression. However, how REST performs its regulatory role is not well understood. METHODS: We examined 2 independent high REST (HR) GSC lines using genome-wide assays, biochemical validations, gene knockdown analysis, and mouse tumor models. We analyzed in-house patient tumors and patient data present in The Cancer Genome Atlas (TCGA). RESULTS: Genome-wide transcriptome and DNA-binding analyses suggested the dopamine receptor D2 (DRD2) gene, a dominant regulator of neurotransmitter signaling, as a direct target of REST. Biochemical analyses and mouse intracranial tumor models using knockdown of REST and double knockdown of REST and DRD2 validated this target and suggested that DRD2 is a downstream target of REST regulating tumorigenesis, at least in part, through controlling invasion and apoptosis. Further, TCGA GBM data support the presence of the REST-DRD2 axis and reveal that high REST/low DRD2 (HRLD) and low REST/high DRD2 (LRHD) tumors are specific subtypes, are molecularly different from the known GBM subtypes, and represent functional groups with distinctive patterns of enrichment of gene sets and biological pathways. The inverse HRLD/LRHD expression pattern is also seen in in-house GBM tumors. CONCLUSIONS: These findings suggest that REST regulates neurotransmitter signaling pathways through DRD2 in HR-GSCs to impact tumorigenesis. They further suggest that the REST-DRD2 mechanism forms distinct subtypes of GBM.

REST overexpression in mice causes deficits in spontaneous locomotion.

Overexpression of REST has been implicated in brain tumors, ischemic insults, epilepsy, and movement disorders such as Huntington's disease. However, owing to the lack of a conditional REST overexpression animal model, the mechanism of action of REST overexpression in these disorders has not been established in vivo. We created a REST overexpression mouse model using the human REST (hREST) gene. Our results using these mice confirm that hREST expression parallels endogenous REST expression in embryonic mouse brains. Further analyses indicate that REST represses the dopamine receptor 2 (Drd2) gene, which encodes a critical nigrostriatal receptor involved in regulating movement, in vivo. Overexpression of REST using Drd2-Cre in adult mice results in increased REST and decreased DRD2 expression in the striatum, a major site of DRD2 expression, and phenocopies the spontaneous locomotion deficits seen upon global DRD2 deletion or specific DRD2 deletion from indirect-pathway medium spiny neurons. Thus, our studies using this mouse model not only reveal a new function of REST in regulating spontaneous locomotion but also suggest that REST overexpression in DRD2-expressing cells results in spontaneous locomotion deficits.

FOXG1 expression shows correlation with neuronal differentiation in cerebellar development, aggressive phenotype in medulloblastomas, and survival in a xenograft model of medulloblastoma.

FOXG1 is a transcription factor that interacts with multiple signaling pathways and modulates neuronal differentiation in the telencephalon. Dysregulation of FOXG1 expression has been previously reported in medulloblastoma. In this study, we demonstrate a regional specific expression of FOXG1 and its colocalization with Nestin expression in the premigratory mitotically active (outer) layer of the external granular layer of the cerebellum. An inverse expression of the granular precursor cell markers, Math1 and Musashi1, in the inner nonmitotic migratory layer of the external granular layer and in the internal granular layer was observed. Furthermore, modulation of FOXG1 in the medulloblastoma cell line, DAOY, was associated with the induction of neuronal differentiation markers and significant changes in multiple signaling pathways regulating cell proliferation, differentiation, survival, and apoptosis. Additionally, we observed enhanced survival in intracerebellar mice xenografts injected with DAOY cells bearing shFOXG1 constructs versus shLuciferase construct. Overall, these findings suggest that down-modulation of FOXG1 is a prerequisite for the onset of neuronal differentiation during cerebellar development and that a decrease of FOXG1 in medulloblastoma cells offers a survival advantage in mice. We propose that the disruption of signaling pathways that promote mature neuronal differentiation by overexpressed FOXG1 is a contributing event in the neoplastic transformation of cerebellar stem cells.

Implicating SCF complexes in organogenesis in Caenorhabditis elegans.

Development of the Caenorhabditis elegans foregut (pharynx) is regulated by a network of proteins that includes the Retinoblastoma protein (pRb) ortholog LIN-35; the ubiquitin pathway components UBC-18 and ARI-1; and PHA-1, a cytoplasmic protein. Loss of pha-1 activity impairs pharyngeal development and body morphogenesis, leading to embryonic arrest. We have used a genetic suppressor approach to dissect this complex pathway. The lethality of pha-1 mutants is suppressed by loss-of-function mutations in sup-35/ztf-21 and sup-37/ztf-12, which encode Zn-finger proteins, and by mutations in sup-36. Here we show that sup-36 encodes a divergent Skp1 family member that binds to several F-box proteins and the microtubule-associated protein PLT-1/τ. Like SUP-35, SUP-36 levels were negatively regulated by UBC-18-ARI-1. We also found that SUP-35 and SUP-37 physically associated and that SUP-35 could bind microtubules. Thus, SUP-35, SUP-36, and SUP-37 may function within a pathway or complex that includes cytoskeletal components. Additionally, SUP-36 may regulate the subcellular localization of SUP-35 during embryogenesis. We carried out a genome-wide RNAi screen to identify additional regulators of this network and identified 39 genes, most of which are associated with transcriptional regulation. Twenty-three of these genes acted via the LIN-35 pathway. In addition, several S-phase kinase-associated protein (Skp)1-Cullin-F-Box (SCF) components were identified, further implicating SCF complexes as part of the greater network controlling pharyngeal development.

Dynamic status of REST in the mouse ESC pluripotency network.

BACKGROUND: REST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network. METHODS: Employing biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature. RESULTS: We show that Rest+/- and Rest-/- AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/- and Rest-/- AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a REST-dependent pluripotency when cultured under feeder-free conditions, as well as Rest-/- AB-1 ESCs, showed no REST-dependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST's role in ESC pluripotency. CONCLUSIONS: REST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner.

A regulatory module controlling pharyngeal development and function in Caenorhabditis elegans.

In Caenorhabditis elegans, the differentiation and morphogenesis of the foregut are controlled by several transcriptional regulators and cell signaling events, and by PHA-1, an essential cytoplasmic protein of unknown function. Previously we have shown that LIN-35 and UBC-18-ARI-1 contribute to the regulation of pha-1 and pharyngeal development through the Zn-finger protein SUP-35/ZTF-21. Here we characterize SUP-37/ZTF-12 as an additional component of the PHA-1 network regulating pharyngeal development. SUP-37 is encoded by four distinct splice isoforms, which contain up to seven C2H2 Zn-finger domains, and is localized to the nucleus, suggesting a role in transcription. Similar to sup-35, sup-37 loss-of-function mutations can suppress both LOF mutations in pha-1 as well as synthetic-lethal double mutants, including lin-35; ubc-18, which are defective in pharyngeal development. Genetic, molecular, and expression data further indicate that SUP-37 and SUP-35 may act at a common step to control pharyngeal morphogenesis, in part through the transcriptional regulation of pha-1. Moreover, we find that SUP-35 and SUP-37 effect pharyngeal development through a mechanism that can genetically bypass the requirement for pha-1 activity. Unlike SUP-35, SUP-37 expression is not regulated by either the LIN-35 or UBC-18-ARI-1 pathways. In addition, SUP-37 carries out two essential functions that are distinct from its role in regulating pharyngeal development with SUP-35. SUP-37 is required within a subset of pharyngeal muscle cells to facilitate coordinated rhythmic pumping and in the somatic gonad to promote ovulation. These latter observations suggest that SUP-37 may be required for the orchestrated contraction of muscle cells within several tissues.

The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.