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IMP dehydrogenase-2 drives aberrant nucleolar activity and structure in glioblastoma

ABSTRACT: Ribonucleotides serve important cellular functions as energy carriers, signaling molecules and building blocks of RNA. In eukaryotes, rRNA and tRNA synthesis in the nucleolus accounts together for over 90% of all RNA molecules. In many cancers, high proliferation rates correlated with elevation of rRNA and tRNA levels4 and nucleolar hypertrophy. However, the metabolic changes that lead to the increased nucleolar transcription and in turn foster tumorigenesis are incompletely understood. Here we show that in the highly lethal brain cancer glioblastoma (GBM), inosine monophosphate dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed. This leads to increased rRNA and tRNA synthesis and stabilization of the oncogenic GTP-binding protein nucleostemin, and enlarged and malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 reverses these malignant effects and inhibits GBM cell proliferation, whereas untransformed glia cells are unaffected. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of GBM cells even in the absence of functional p53. Our results have uncovered upregulation of IMPDH2 as a prerequisite for aberrant nucleolar function and translational capacity in GBM. This GTP metabolic reprogramming constitutes a primary event in gliomagenesis and has implications for GBM treatment and possible malignant transformation in other cancers.

ORGANISM(S): Homo sapiens

PROVIDER: GSE124727 | GEO | 2019/07/30

REPOSITORIES: GEO

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IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency

Project description:Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use a mouse model of AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that AMPD deficiency in mice primarily leads to hippocampal dentate gyrus degeneration despite causing a generalized reduction of brain GTP levels. Remarkably, we uncover that neurodegeneration resistant regions of the hippocampus accumulate micron sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis. In contrast, IMPDH2 filaments are barely detectable in the dentate gyrus, which show a progressively neuroinflammation and neurodegeneration. Furthermore, using a human AMPD2 neural cell culture model, we show that blocking IMPDH2 polymerization with a dominant negative IMPDH2 variant impairs AMPD2 deficient neural progenitor growth. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation caused by AMPD2 deficiency, providing resistance to neurodegeneration. This data opens the possibility of exploring the involvement of IMPDH2 assembly as a therapeutic intervention for neurodegeneration.

2024-01-16 | GSE253045 | GEO

Local GTP availability is an essential step in regulation of RAC1 activity and cell invasion

Project description:Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because GTP concentrations determined by chromatography or mass spectrometry were shown to be substantially higher than the RAC1 GTP dissociation constant (RAC1-GTP Kd) determined in vitro. Here, using a combination of genetically engineered intracellular GTP sensors, recently generated by us, and RAC1 activity reporter, we demonstrate that fluctuations in GTP levels within the range of RAC1-GTP Kd, correlate with changes in RAC1 activity in live cells. We further demonstrate that RAC1 co-localize in protrusions of invading cells with the de novo guanylate biosynthesis enzymes including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2) and guanosine monophosphate synthase (GMPS). This colocalization is partly driven by direct interaction of RAC1 with the Bateman domain of IMPDH2. Substitution of endogenous IMPDH2 with the IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Accordingly, targeting of IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased local GTP levels in cell protrusions, downregulated RAC1 activity and decreased cell invasion. These data provide previously unrecognized mechanistic insight into the regulation of RAC1 activity by local GTP pools in live cells.

2021-11-04 | PXD028540 | Pride

Transcriptional effect of an off-target shRNA targeting nucleostemin in human glioblastoma-derived cancer stem cells

Project description:Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples. The significance of its expression was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). We found that an off-target nucleostemin RNAi (shRNA22) abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is severely compromised. Attempts were made to identify the primary target of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways. The use of this shRNA may offer a new therapeutic approach for this incurable type of brain tumors. The transcriptional profile of neurosphere cultures infected with lentiviral particles containing shRNA22 was compared with the profile of neurosphere cultures infected with lentiviral particles containing control shRNA. Experiments were done in triplicate.

2011-10-20 | E-GEOD-30448 | biostudies-arrayexpress

The gene profile of zebrafish guanine binding protein-like 2 (Gnl2) mutant embryos versus wild-type embryos at 22 hours post-fertilization

Project description:Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells and implicated in the control of cell cycle progression, the maintenance of stem cells, and the regulation of cancer cells. The depletion of NS in cell lines and invertebrate animals has been demonstrated to result in defective ribosome biogenesis, while both depletion and overexpression of NS are able to stabilize the tumor suppressor p53 protein. While the existence of p53-independent functions of NS has been previously suggested, these roles to date remain unclear. Here, we report two zebrafish mutants recovered from forward and reverse genetic screens that encode two members of this nucleolar protein family, Guanine nucleotide binding-protein-like 2 (Gnl2) and Gnl3/Nucleostemin (Ns). We show that Gnl2 and Ns are required for pre-rRNA processing and the production of the 60S ribosomal subunit in the developing zebrafish embryo. In addition, we demonstrate that these proteins are required for correct timing of neural differentiation in the brain and retina. In both mutants, cell cycle regulators such as cyclinD1 and the cell cycle inhibitor cdkn1c/p57kip2 are aberrantly expressed. Concomitantly, retinal progenitors fail to exit the cell cycle in a timely fashion and differentiation of retinal cell types is delayed. We show that loss of either Gnl2 or Ns induces the stabilization of p53 and apoptosis in the brain, however the retinal cell differentiation defect in gnl2 mutants is independent of p53 status. This work demonstrates that the loss of either Gnl2 or Ns results in ribosome biogenesis and neural differentiation defects in vertebrates. In addition, the data suggests that the latter phenotype of gnl2 mutants is due to an increase in expression of cell cycle markers cyclinD1 and p57/kip2 and not to the stabilization of p53.

2010-03-31 | GSE21134 | GEO

Transcriptional effect of an off-target shRNA targeting nucleostemin in human glioblastoma-derived cancer stem cells

2011-10-21 | GSE30448 | GEO

Project description:Overexpression of Arabidopsis nucleolar GTP-binding 1

| PRJNA805197 | ENA

De Novo Purine Synthesis Mediates Circadian Control of Cell Cycle in Zebrafish (HTS)

Project description:Inosine 5'-phosphate dehydrogenase (impdh) has been well known as a key enzyme in GTP biosynthesis pathway. We found that three isoforms of impdh in zebrafish, namely impdh1a, impdh1b and impdh2, all show robust circadian expression.To examine the molecular functions of three impdh isoforms in zebrafish on the genome scale, we measured the global expression changes of impdh1a, impdh1b and impdh2 morpholino injected larvae (morphants) respectively using RNA-seq. Wild type (WT), control and three impdh morphants were collected at 32 hpf. In our RNA-seq result, we identified 468, 331 and 1166 significant genes affected by impdh1a, impdh1b and impdh2 morpholino (MO) knock-down respectively. Among them, there are limited overlaps between genes affected by different MOs and only 36 genes in common among all three MOs. This indicates that the three impdh isoforms have distinct molecular functions.

2013-10-01 | GSE51278 | GEO

De Novo Purine Synthesis Mediates Circadian Control of Cell Cycle in Zebrafish (HTS)

2013-10-01 | E-GEOD-51278 | biostudies-arrayexpress

Ahmed_Shireman Brain 2021 ChIPSeq

Project description:Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumor is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of GBM cells to adapt to complex changes within the tumor microenvironment. To elucidate the molecular mechanisms of this adaptation, specifically during chemotherapy, we employed ChIP-Sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ALR13B is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with Liquid Chromatography-Mass Spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme IMPDH2. Further, radioisotope tracing revealed that this interaction function as a negative regulator for purine salvaging. Inhibition of ARL13B-IMPDH2 interaction enhances temozolomide (TMZ)-induced DNA damage by forcing GBM cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved by using an FDA-approved drug, Mycophenolate Mofetil, that can block the IMPDH2 activity and enhance the therapeutic efficacy of TMZ. Our results suggest and support clinical evaluation of MMF in combination with TMZ treatment in glioma patients.

2021-01-07 | GSE164352 | GEO

Association of prognosis of acute myeloid leukemia patients with leukemia stem cell gene signature based on nucleostemin promoter activity

Project description:Acute myeloid leukemia (AML) is a heterogeneous neoplastic disorder, in which only a subset of cells have function as leukemia-initiating cells (LICs). In this study, we prospectively evaluated leukemia-initiating capacity of fractionated subpopulations of AML cells depending on expression of a nucleolar GTP binding protein, Nucleostemin (NS). To monitor NS expression in living AML cells, we generated a mouse AML model using a transgenic mouse, in which green florescence protein (GFP) is expressed under the control of particular region of NS promoter (NS-GFP). In AML cells, NS-GFP levels were correlated with endogenous NS mRNA. AML cells with the highest expression of NS-GFP were very immature blast-like cells, efficiently formed leukemia colonies in vitro, and exhibited high leukemia-initiating capacity in vivo. Gene expression profiling analysis revealed that cell cycle regulators and nucleotide metabolism-related genes were highly enriched in a gene set associated with leukemia-initiating capacity, i.e., leukemia stem cell gene signature. Furthermore, we found that the leukemia stem cell gene signature stratified human AML patients into two distinct clusters, reflecting prognosis. These data demonstrate that the mouse leukemia stem cell gene signature is significantly associated with malignant properties of human AML patients. Further analyses of gene regulation related with leukemia stem cells would provide novel insights for the progress in diagnostic and therapeutic approach of AML patients. A mouse acute myeloid leukemia model was generated by introduction of HoxA9/Meis1 to bone marrow cells from a transgenic mouse, in which green florescence protein (GFP) is expressed under the control of particular region of NS promoter (NS-GFP).The leukemia cells were fractionated into 4 subpopulations based on NS-GFP level and c-Kit expression, (a) c-Kit-NS-GFPlow, (b) c-Kit-NS-GFPmiddle, (c) c-Kit+NS-GFPmiddle and (d) c-Kit+NS-GFPhigh cells.

2014-07-30 | E-GEOD-58032 | biostudies-arrayexpress

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