The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

IntroductionMLN4924 is a potent and selective small-molecule inhibitor of NEDD8-activating enzyme (NAE) that is currently in phase 1 clinical trials. 1-3 NAE plays an essential role in regulating the activity of a subset of ubiquitin E3 ligases, the cullin-RING ligases (CRLs), which are responsible for regulating destruction of many intracellular proteins. 4 NAE activates the small ubiquitin-like molecule NEDD8 as the first step in the neddylation cascade. 5 NAE hydrolyzes adenosine triphosphate (ATP) to adenylate NEDD8 at its C-terminus and transfers NEDD8 from the adenyl group to a specific cysteine within NAE. The activated NEDD8 is then transferred to the active-site cysteine of Ubc12 or UBE2F the E2s specific for the NEDD8 pathway. Finally, NEDD8 is conjugated on a conserved lysine near the C-terminal end of a cullin protein; this covalent modification is required for the cullin complex to recruit a ubiquitin-charged E2 protein facilitating polyubiquitination of proteins, targeting them for proteasomal degradation. Thus, NAE plays a key role in regulating the levels (and therefore the function) of a subset of proteins.Many of the proteins that are substrates for CRL-mediated polyubiquitination have key roles in cell-cycle progression and signal transduction, making NAE inhibition an attractive target for anticancer therapy. MLN4924 potently inhibits NAE in vitro, resulting in inhibition of CRL neddylation and an increase in levels of CRL substrate proteins (eg, Cdt-1, Nrf-2). 3 The primary mechanism of action of NAE inhibition in many cell types is induction of DNA rereplication because of blocking degradation of Cdt-1, a critical factor required for licensing origins of DNA replication. 3 Dysregulation of Cdt-1 activity leads to DNA rereplication. 6,7 For example, overexpression of Cdt-1 and Cdc6 induces DNA rereplication, activates DNA damage repair pathways, and induces cell death. 7 Induction of DNA rereplication by MLN4924 results in S-phase accumulation, DNA-damage responses, and cell death 3 (M.A.M., U.N., T.A.S., P. Veiby, P.G.S., B. Amidon, manuscript in preparation). Similar effects were observed in human tumor xenografts where MLN4924 inhibited NAE in vivo leading to tumor growth inhibition. 3 The nuclear factor-B (NF-B) signaling pathway plays a key role in many aspects of cancer initiation and progression through transcriptional control of genes involved in growth, angiogenesis, antiapoptosis, invasiveness, and metastasis. 8 Regulation of NF-B signaling occurs at many levels, one of which is through the regulation of protein turnover by the action of CRLs. Under normal conditions, NF-B transcription factors are maintained in an inactive state by binding to IB proteins. In canonical NF-B signaling, IB␣ binds to p50-p65, sequesters the transcription factors in the cytoplasm rendering them inactive. On stimulation of the IKK complex, IB␣ is phosphorylated at Ser32 and Ser36, resulting in its polyubiquitination and degradation, 9-11 thus resulting in nuclear accumulation of the complex and transcri...

MLN4924 is a first-in-class experimental cancer drug that inhibits the NEDD8-activating enzyme, thereby inhibiting cullin-RING E3 ubiquitin ligases and stabilizing many cullin substrates. The mechanism by which MLN4924 inhibits cancer cell proliferation has not been defined, although it is accompanied by DNA rereplication and attendant DNA damage. Here we show that stabilization of the DNA replication factor Cdt1, a substrate of cullins 1 and 4, is critical for MLN4924 to trigger DNA rereplication and inhibit cell proliferation. Even only 1 hour of exposure to MLN4924, which was sufficient to elevate Cdt1 for 4-5 hours, was found to be sufficient to induce DNA rereplication and to activate apoptosis and senescence pathways. Cells in S phase were most susceptible, suggesting that MLN4924 will be most toxic on highly proliferating cancers. Although MLN4924-induced cell senescence seems to be dependent on induction of p53 and its downstream effector p21Waf1 , we found that p53 À/À and p21 À/À cells were even more susceptible than wild-type cells to MLN4924. Our results suggested that apoptosis, not senescence, might be more important for the antiproliferative effect of MLN4924. Furthermore, our findings show that transient exposure to this new investigational drug should be useful for controlling p53-negative cancer cells, which often pose significant clinical challenge.

Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (Ͻ1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activitydependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A.

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for proteinsynthesis-dependent synaptic plasticity.Fragile X syndrome is the most common form of inherited mental retardation and is caused by a functional absence of the RNA-binding protein, fragile X mental retardation protein, FMRP.3 The protein, FMRP, is known to associate with ϳ3% of the mammalian brain mRNAs, repressing their translation; many of these target mRNAs indeed appear overtranslated in the absence of FMRP (1). Electrophysiology of the fragile X mouse model has revealed a synaptic deficit in the hippocampus with elevated group I metabotropic glutamate receptor (mGluR)-mediated long term depression. Accordingly, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization is enhanced in the absence of FMRP, all thought due to constitutive overtranslation of an long-term depression (LTD)-required FMRP target mRNA(s) (2-4). Group I mGluR activity is also known to influence FMRP transport and synthesis (5, 6); however, little is known about regulation of FMRP itself.Post-translational modifications are known regulators of activity-dependent protein synthesis (7), and FMRP is known to be phosphorylated at a highly conserved serine at position 499. The effects of FMRP phosphorylation on translation by ribosomal run-off assays suggested that non-phosphorylated FMRP associates with actively translating ribosomes, whereas phosphorylated FMRP is found in the context of potentially stalled ribosomes (8). We recently determined that FMRP is dephosphorylated by protein phosphatase 2A (PP2A) and, immediately following mGluR stimulation, PP2A dephosphorylates FMRP, corresponding with the translation of SAPAP3, an FMRP target mRNA (9). SAPAP3 is a post-synaptic scaffolding protein associated with PSD95, whose message was previously identified as an FMRP target mRNA in the mouse brain following FMRP immunoprecipitation and microchip analyses (10,35,36). Less than 2 min following mGluR activation, FMRP is rephosphorylated in a PP2A-and mamm...

Fragile X syndrome (FXS), a common inherited form of mental retardation, is caused by the functional absence of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates the translation of specific mRNAs at synapses. Altered synaptic plasticity has been described in a mouse FXS model. However, the mechanism by which the loss of FMRP alters synaptic function, and subsequently causes the mental impairment, is unknown. Here, in cultured hippocampal neurons, we used siRNAs against Fmr1 to demonstrate that a reduction of FMRP in dendrites leads to an increase in internalization of the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit, GluR1, in dendrites. This abnormal AMPAR trafficking was caused by spontaneous action potential-driven network activity without synaptic stimulation by an exogenous agonist and was rescued by 2-methyl-6-phenylethynyl-pyridine (MPEP), an mGluR5-specific inverse agonist. Because AMPAR internalization depends on local protein synthesis after mGluR5 stimulation, FMRP, a negative regulator of translation, may be viewed as a counterbalancing signal, wherein the absence of FMRP leads to an apparent excess of mGluR5 signaling in dendrites. Because AMPAR trafficking is a driving process for synaptic plasticity underlying learning and memory, our data suggest that hypersensitive AMPAR internalization in response to excess mGluR signaling may represent a principal cellular defect in FXS, which may be corrected by using mGluR antagonists.fragile X syndrome ͉ autism

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