R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m6A/MYC/CEBPA Signaling.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.

Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors.

Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or ß (PI5P4Kα and ß) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and ß in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K ß and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A(-/-), PIP4K2B(+/-), and TP53(-/-) mice were viable and had a dramatic reduction in tumor formation compared to TP53(-/-) littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

Systemic elevation of PTEN induces a tumor-suppressive metabolic state.

Decremental loss of PTEN results in cancer susceptibility and tumor progression. PTEN elevation might therefore be an attractive option for cancer prevention and therapy. We have generated several transgenic mouse lines with PTEN expression elevated to varying levels by taking advantage of bacterial artificial chromosome (BAC)-mediated transgenesis. The "Super-PTEN" mutants are viable and show reduced body size due to decreased cell number, with no effect on cell size. Unexpectedly, PTEN elevation at the organism level results in healthy metabolism characterized by increased energy expenditure and reduced body fat accumulation. Cells derived from these mice show reduced glucose and glutamine uptake and increased mitochondrial oxidative phosphorylation and are resistant to oncogenic transformation. Mechanistically we find that PTEN elevation orchestrates this metabolic switch by regulating PI3K-dependent and -independent pathways and negatively impacting two of the most pronounced metabolic features of tumor cells: glutaminolysis and the Warburg effect.

A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

The Lipid Kinase PI5P4Kß Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kß, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kß preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kß is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kß is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kß. The critical role of the GTP-sensing activity of PI5P4Kß in cancer signifies this lipid kinase as a cancer therapeutic target.

Multimodal action of KRP203 on phosphoinositide kinases in vitro and in cells.

Increased phosphoinositide signaling is commonly associated with cancers. While "one-drug one-target" has been a major drug discovery strategy for cancer therapy, a "one-drug multi-targets" approach for phosphoinositide enzymes has the potential to offer a new therapeutic approach. In this study, we sought a new way to target phosphoinositides metabolism. Using a high-throughput phosphatidylinositol 5-phosphate 4-kinase-alpha (PI5P4Kα) assay, we have identified that the immunosuppressor KRP203/Mocravimod induces a significant perturbation in phosphoinositide metabolism in U87MG glioblastoma cells. Despite high sequence similarity of PI5P4K and PI4K isozymes, in vitro kinase assays showed that KRP203 activates some (e.g., PI5P4Kα, PI4KIIß) while inhibiting other phosphoinositide kinases (e.g., PI5P4Kß, γ, PI4KIIα, class I PI3K-p110α, δ, γ). Furthermore, KRP203 enhances PI3P5K/PIKFYVE's substrate selectivity for phosphatidylinositol (PI) while preserving its selectivity for PI(3)P. At cellular levels, 3 h of KRP203 treatment induces a prominent increase of PI(3)P and moderate increase of PI(5)P, PI(3,5)P2, and PI(3,4,5)P3 levels in U87MG cells. Collectively, the finding of multimodal activity of KRP203 towards multi-phosphoinositide kinases may open a novel basis to modulate cellular processes, potentially leading to more effective treatments for diseases associated with phosphoinositide signaling pathways.

Homogeneous luminescent quantitation of cellular guanosine and adenosine triphosphates (GTP and ATP) using QT-LucGTP&ATP assay.

Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are essential nucleic acid building blocks and serve as energy molecules for a wide range of cellular reactions. Cellular GTP concentration fluctuates independently of ATP and is significantly elevated in numerous cancers, contributing to malignancy. Quantitative measurement of ATP and GTP has become increasingly important to elucidate how concentration changes regulate cell function. Liquid chromatography-coupled mass spectrometry (LC-MS) and capillary electrophoresis-coupled MS (CE-MS) are powerful methods widely used for the identification and quantification of biological metabolites. However, these methods have limitations related to specialized instrumentation and expertise, low throughput, and high costs. Here, we introduce a novel quantitative method for GTP concentration monitoring (GTP-quenching resonance energy transfer (QRET)) in homogenous cellular extracts. CE-MS analysis along with pharmacological control of cellular GTP levels shows that GTP-QRET possesses high dynamic range and accuracy. Furthermore, we combined GTP-QRET with luciferase-based ATP detection, leading to a new technology, termed QT-LucGTP&ATP, enabling high-throughput compatible dual monitoring of cellular GTP and ATP in a homogenous fashion. Collectively, GTP-QRET and QT-LucGTP&ATP offer a unique, high-throughput opportunity to explore cellular energy metabolism, serving as a powerful platform for the development of novel therapeutics and extending its usability across a range of disciplines.

Schlafen 11 predicts response to platinum-based chemotherapy in gastric cancers.

BACKGROUND: Although unresectable or recurrent gastric cancers (GC) are frequently treated with platinum-based chemotherapy, response to treatment remains unpredictable. Because Schlafen 11 (SLFN11) is recently identified as a critical determinant of platinum sensitivity, we investigated the potential clinical utility of SLFN11 in the treatment of GC. METHODS: We analysed the correlation between SLFN11 expression and overall survival in 169 GC patients by our established immunohistochemical approach. The impact of SLFN11 expression on the response to platinum and transition of SLFN11 expression upon long-term treatment with platinum were examined using GC cell lines and organoids. RESULTS: GC patients with high-SLFN11 expression exhibited significantly better survival than those with low-SLFN11 expression, and the significance increased when we selected patients treated with platinum-based chemotherapy. Knockout of SLFN11 and reactivation of SLFN11 in GC cells conferred resistance and sensitivity to platinum, respectively. In GC cells and organoids, long-term treatment with oxaliplatin suppressed SLFN11 expression while imparting drug resistance. The acquired resistance to oxaliplatin was reversed by reactivation of SLFN11 with epigenetic modifying drugs. CONCLUSIONS: This is the first report revealing definitive clinical implications of SLFN11 in the treatment of GC patients and providing novel strategies for the drug selection based on SLFN11 expression.

Depletion of phosphatidylinositol 4-phosphate at the Golgi translocates K-Ras to mitochondria.

Ras proteins are small GTPases localized to the plasma membrane (PM), which regulate cellular proliferation, apoptosis and differentiation. After a series of post-translational modifications, H-Ras and N-Ras traffic to the PM from the Golgi via the classical exocytic pathway, but the exact mechanism of K-Ras trafficking to the PM from the ER is not fully characterized. ATP5G1 (also known as ATP5MC1) is one of the three proteins that comprise subunit c of the F0 complex of the mitochondrial ATP synthase. In this study, we show that overexpression of the mitochondrial targeting sequence of ATP5G1 perturbs glucose metabolism, inhibits oncogenic K-Ras signaling, and redistributes phosphatidylserine (PtdSer) to mitochondria and other endomembranes, resulting in K-Ras translocation to mitochondria. Also, it depletes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. Glucose supplementation restores PtdSer and K-Ras PM localization and PI4P at the Golgi. We further show that inhibition of the Golgi-localized PI4-kinases (PI4Ks) translocates K-Ras, and PtdSer to mitochondria and endomembranes, respectively. We conclude that PI4P at the Golgi regulates the PM localization of PtdSer and K-Ras.This article has an associated First Person interview with the first author of the paper.

Mycophenolate Mofetil Hepatotoxicity Associated With Mitochondrial Abnormality in Liver Transplant Recipients and Mice.

OBJECTIVES: Mycophenolate mofetil (MMF) is a widely used immunosuppressive agent. MMF hepatotoxicity has been reported in non-transplant and renal transplant patients with minimal histologic description. This is the first study describing detailed histology and ultrastructure of MMF hepatotoxicity. METHODS: Four liver-transplant recipients (Cases 1-4) were suspected to have MMF hepatotoxicity. Cases 1-3 (two females and one male; 4-17 years) had multiple biopsies for liver function test (LFT) abnormalities. Case 4 (female; 16 years) had a surveillance biopsy. Electron-microscopic examination (EM) was requested on Cases 1-3 for unexplained, persistent LFT elevation and histologic abnormalities despite therapy and Case 4 for unexplained histologic abnormalities despite a stable clinical course. To confirm the pathologic changes in the human allografts, livers from MMF-treated and untreated mice were also reviewed. RESULTS: While the allograft biopsies showed nonspecific histologic changes, EM revealed unequivocal mitochondrial abnormalities similar to those seen in primary and secondary mitochondrial disorders. In Cases 1 and 2, LFTs improved after stopping and reducing MMF, respectively. In Case 3, pre- and post-MMF treatment biopsies were performed and only the post-MMF biopsy demonstrated mitochondrial abnormalities. Mitochondrial abnormality in Case 4 was subclinical. The mouse study confirmed that MMF caused various stress changes in the mitochondria; number of mitochondria/cell (mean ± standard deviation; untreated group: 58.25 ±â€Š8.426; MMF-treated group: 76.37 ±â€Š18.66), number of lipid droplets/cell (untreated: 0.9691 ±â€Š1.150; MMF-treated: 3.649 ±â€Š4.143) and sizes of mitochondria (µm, untreated: 0.8550 ±â€Š0.3409; MMF-treated: 0.9598 ±â€Š0.5312) were significantly increased in hepatocytes in the MMF-treated mice compared with the untreated mice (P < 0.0001). CONCLUSIONS: Although MMF is safe for the majority of patients, MMF can cause mitochondrial stress, which may trigger more severe mitochondrial abnormalities in a small subset. MMF hepatotoxicity should be considered for MMF-treated patients with unexplained, persistent LFT abnormalities and nonspecific histologic findings. EM should be requested for these cases.

Dynamic compartmentalization of purine nucleotide metabolic enzymes at leading edge in highly motile renal cell carcinoma.

Compartmentalization is vital for biological systems at multiple levels, including biochemical reactions in metabolism. Organelle-based compartments such as mitochondria and peroxisomes sequester the responsible enzymes and increase the efficiency of metabolism while simultaneously protecting the cell from dangerous intermediates, such as radical oxygen species. Recent studies show intracellular nucleotides, such as ATP and GTP, are heterogeneously distributed in cells with high concentrations at the lamellipodial and filopodial projections, or leading edge. However, the intracellular distribution of purine nucleotide enzymes remains unclear. Here, we report the enhanced localization of GTP-biosynthetic enzymes, including inosine monophosphate dehydrogenase (IMPDH isotype 1 and 2), GMP synthase (GMPS), guanylate kinase (GUK1) and nucleoside diphosphate kinase-A (NDPK-A) at the leading edge in renal cell carcinoma cells. They show significant co-localization at the membrane subdomain, and their co-localization pattern at the membrane is distinct from that of the cell body. While other purine nucleotide biosynthetic enzymes also show significant localization at the leading edge, their co-localization pattern with IMPDH is divergent. In contrast, a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), predominantly localized in the cytoplasm. Mechanistically, we found that plasma membrane localization of IMPDH isozymes requires active actin polymerization. Our results demonstrate the formation of a discrete metabolic compartment for localized purine biosynthesis at the leading edge, which may promote localized nucleotide metabolism for cell migration and metastasis in cancers.

Degradation of activated K-Ras orthologue via K-Ras-specific lysine residues is required for cytokinesis.

Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.

Differences in the regulation of K-Ras and H-Ras isoforms by monoubiquitination.

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation.

Selenite targets eIF4E-binding protein-1 to inhibit translation initiation and induce the assembly of non-canonical stress granules.

Stress granules (SGs) are large cytoplasmic ribonucleoprotein complexes that are assembled when cells are exposed to stress. SGs promote the survival of stressed cells by contributing to the reprogramming of protein expression as well as by blocking pro-apoptotic signaling cascades. These cytoprotective effects implicated SGs in the resistance of cancer cells to radiation and chemotherapy. We have found that sodium selenite, a selenium compound with chemotherapeutic potential, is a potent inducer of SG assembly. Selenite-induced SGs differ from canonical mammalian SGs in their morphology, composition and mechanism of assembly. Their assembly is induced primarily by eIF4E-binding protein1 (4EBP1)-mediated inhibition of translation initiation, which is reinforced by concurrent phosphorylation of eIF2α. Selenite-induced SGs lack several classical SG components, including proteins that contribute to pro-survival functions of canonical SGs. Our results reveal a new mechanism of mammalian SG assembly and provide insights into how selenite cytotoxicity may be exploited as an anti-neoplastic therapy.

Functional molecular evolution of a GTP sensing kinase: PI5P4Kß.

Over 4 billion years of evolution, multiple mutations, including nucleotide substitutions, gene and genome duplications and recombination, have established de novo genes that translate into proteins with novel properties essential for high-order cellular functions. However, molecular processes through which a protein evolutionarily acquires a novel function are mostly speculative. Recently, we have provided evidence for a potential evolutionary mechanism underlying how, in mammalian cells, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) evolved into a GTP sensor from ATP-utilizing kinase. Mechanistically, PI5P4Kß has acquired the guanine efficient association (GEA) motif by mutating its nucleotide base recognition sequence, enabling the evolutionary transition from an ATP-dependent kinase to a distinct GTP/ATP dual kinase with its KM for GTP falling into physiological GTP concentrations-the genesis of GTP sensing activity. Importantly, the GTP sensing activity of PI5P4Kß is critical for the manifestation of cellular metabolism and tumourigenic activity in the multicellular organism. The combination of structural, biochemical and biophysical analyses used in our study provides a novel framework for analysing how a protein can evolutionarily acquire a novel activity, which potentially introduces a critical function to the cell.

IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

Epigenetic upregulation of Schlafen11 renders WNT- and SHH-activated medulloblastomas sensitive to cisplatin.

BACKGROUND: Intensive chemotherapeutic regimens with craniospinal irradiation have greatly improved survival in medulloblastoma patients. However, survival markedly differs among molecular subgroups and their biomarkers are unknown. Through unbiased screening, we found Schlafen family member 11 (SLFN11), which is known to improve response to DNA damaging agents in various cancers, to be one of the top prognostic markers in medulloblastomas. Hence, we explored the expression and functions of SLFN11 in medulloblastoma. METHODS: SLFN11 expression for each subgroup was assessed by immunohistochemistry in 98 medulloblastoma patient samples and by analyzing transcriptomic databases. We genetically or epigenetically modulated SLFN11 expression in medulloblastoma cell lines and determined cytotoxic response to the DNA damaging agents cisplatin and topoisomerase I inhibitor SN-38 in vitro and in vivo. RESULTS: High SLFN11 expressing cases exhibited significantly longer survival than low expressing cases. SLFN11 was highly expressed in the WNT-activated subgroup and in a proportion of the SHH-activated subgroup. While WNT activation was not a direct cause of the high expression of SLFN11, a specific hypomethylation locus on the SLFN11 promoter was significantly correlated with high SLFN11 expression. Overexpression or deletion of SLFN11 made medulloblastoma cells sensitive and resistant to cisplatin and SN-38, respectively. Pharmacological upregulation of SLFN11 by the brain-penetrant histone deacetylase-inhibitor RG2833 markedly increased sensitivity to cisplatin and SN-38 in SLFN11-negative medulloblastoma cells. Intracranial xenograft studies also showed marked sensitivity to cisplatin by SLFN11-overexpression in medulloblastoma cells. CONCLUSIONS: High SLFN11 expression is one factor which renders favorable outcomes in WNT-activated and a subset of SHH-activated medulloblastoma possibly through enhancing response to cisplatin.

Inhibition of lung cancer growth: ATP citrate lyase knockdown and statin treatment leads to dual blockade of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/AKT pathways.

ATP citrate lyase (ACL) catalyzes the conversion of cytosolic citrate to acetyl-CoA and oxaloacetate. A definitive role for ACL in tumorigenesis has emerged from ACL RNAi and chemical inhibitor studies, showing that ACL inhibition limits tumor cell proliferation and survival and induces differentiation in vitro. In vivo, it reduces tumor growth leading to a cytostatic effect and induces differentiation. However, the underlying molecular mechanisms are poorly understood and agents that could enhance the efficacy of ACL inhibition have not been identified. Our studies focus on non-small cell lung cancer (NSCLC) lines, which show phosphatidylinositol 3-kinase (PI3K)/AKT activation secondary to a mutation in the K-Ras gene or the EGFR gene. Here we show that ACL knockdown promotes apoptosis and differentiation, leading to the inhibition of tumor growth in vivo. Moreover, in contrast to most studies, which elucidate how activation/suppression of signaling pathways can modify metabolism, we show that inhibition of a metabolic pathway "reverse signals" and attenuates PI3K/AKT signaling. Additionally, we find that statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which act downstream of ACL in the cholesterol synthesis pathway, dramatically enhance the anti-tumor effects of ACL inhibition, even regressing established tumors. With statin treatment, both PI3K/AKT and the MAPK pathways are affected. Moreover, this combined treatment is able to reduce the growth of EGF receptor resistant tumor cell types. Given the essential role of lipid synthesis in numerous cancers, this work may impact therapy in a broad range of tumors.

G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility.

Phosphoinositide 3-kinase (PI3K)gamma and Dictyostelium PI3K are activated via G protein-coupled receptors through binding to the Gbetagamma subunit and Ras. However, the mechanistic role(s) of Gbetagamma and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gbeta null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P(3). Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras-guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.

SI-MOIRAI: a new method to identify and quantify the metabolic fate of nucleotides.

Since the discovery of nucleotides over 100 years ago, extensive studies have revealed the importance of nucleotides for homeostasis, health and disease. However, there remains no established method to investigate quantitatively and accurately intact nucleotide incorporation into RNA and DNA. Herein, we report a new method, Stable-Isotope Measure Of Influxed Ribonucleic Acid Index (SI-MOIRAI), for the identification and quantification of the metabolic fate of ribonucleotides and their precursors. SI-MOIRAI, named after Greek goddesses of fate, combines a stable isotope-labelling flux assay with mass spectrometry to enable quantification of the newly synthesized ribonucleotides into r/m/tRNA under a metabolic stationary state. Using glioblastoma (GBM) U87MG cells and a patient-derived xenograft (PDX) GBM mouse model, SI-MOIRAI analyses showed that newly synthesized GTP was particularly and disproportionally highly utilized for rRNA and tRNA synthesis but not for mRNA synthesis in GBM in vitro and in vivo. Furthermore, newly synthesized pyrimidine nucleotides exhibited a significantly lower utilization rate for RNA synthesis than newly synthesized purine nucleotides. The results reveal the existence of discrete pathways and compartmentalization of purine and pyrimidine metabolism designated for RNA synthesis, demonstrating the capacity of SI-MOIRAI to reveal previously unknown aspects of nucleotide biology.