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.

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Increased Aurora A expression occurs in a variety of human cancers and induces chromosomal abnormalities during mitosis associated with tumor initiation and progression. MLN8054 is a selective smallmolecule Aurora A kinase inhibitor that has entered Phase I clinical trials for advanced solid tumors. MLN8054 inhibits recombinant Aurora A kinase activity in vitro and is selective for Aurora A over the family member Aurora B in cultured cells. MLN8054 treatment results in G2/M accumulation and spindle defects and inhibits proliferation in multiple cultured human tumor cells lines. Growth of human tumor xenografts in nude mice was dramatically inhibited after oral administration of MLN8054 at well tolerated doses. Moreover, the tumor growth inhibition was sustained after discontinuing MLN8054 treatment. In human tumor xenografts, MLN8054 induced mitotic accumulation and apoptosis, phenotypes consistent with inhibition of Aurora A. MLN8054 is a selective inhibitor of Aurora A kinase that robustly inhibits growth of human tumor xenografts and represents an attractive modality for therapeutic intervention of human cancers.cancer ͉ mitosis ͉ apoptosis

The Syk cytoplasmic protein-tyrosine kinase has two amino-terminal SH2 domains and a carboxy-terminal catalytic domain. Syk, and its close relative ZAP-70, are apparently pivotal in coupling antigen- and Fc-receptors to downstream signalling events. Syk associates with activated Fc receptors, the T cell receptor complex and the B-cell antigen-receptor complex (BCR) in immature and mature B lymphocytes. On receptor activation, the tandem SH2 domains of Syk bind dual phosphotyrosine sites in the conserved ITAM motifs of receptor signalling chains, such as the immunoglobulin alpha and beta-chains of the BCR, leading to Syk activation. Here we have investigated Syk function in vivo by generating a mouse strain with a targeted mutation in the syk gene. Homozygous syk mutants suffered severe haemorrhaging as embryos and died perinatally, indicating that Syk has a critical role in maintaining vascular integrity or in wound healing during embryogenesis. Analysis of syk-/- lymphoid cells showed that the syk mutation impaired the differentiation of B-lineage cells, apparently by disrupting signalling from the pre-BCR complex and thereby preventing the clonal expansion, and further maturation, of pre-B cells.

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

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...

The CD4 T-cell surface antigen is an integral membrane glycoprotein of relative molecular mass 55,000 which binds class II major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). It is thought to stabilize physical interactions between T cells and APCs (for a review, see ref. 1). Evidence is accumulating that suggests that CD4 can transduce an independent signal during T-cell activation. It has recently been shown that CD4 expressed on human and murine T cells is physically associated with the Src-related tyrosine protein kinase p56lck (refs 7, 8). These results indicate that CD4 can function as a signal transducer and suggest that tyrosine phosphorylation events may be important in CD4-mediated signalling. Here, we present evidence that cross-linking of the CD4 receptor induces a rapid increase in the tyrosine-specific protein kinase activity of p56lck and is associated with the rapid phosphorylation of one of the subunits (zeta) of the T-cell receptor complex on tyrosine residues. These data provide direct evidence for a specific CD4 signal transduction pathway that is mediated through p56lck and suggest that some of the tyrosine phosphorylation events detected during antigen-mediated T-cell activation may result from signalling through this surface molecule.

The high-affinity IgE receptor (Fc epsilon RI), which is expressed on the surface of mast cells and basophils, has a central role in immediate allergic responses. In the rat basophilic leukaemia cell line RBL-2H3, which is a model system for the analysis of Fc epsilon RI-mediated signal transduction, surface engagement of Fc epsilon RI induces histamine release and the tyrosine phosphorylation of several distinct proteins. Although the alpha, beta, and gamma subunits of Fc epsilon RI lack intrinsic tyrosine protein kinase (TPK) activity, a kinase that copurifies with Fc epsilon RI phosphorylates the beta and gamma subunits of the receptor on tyrosine residues. We report here that in RBL-2H3 cells, p56lyn and pp60c-src are activated after Fc epsilon RI crosslinking, and p56lyn coimmunoprecipitates with Fc epsilon RI. In the mouse mast-cell line PT-18, another cell type used to study FC epsilon RI-mediated signalling, tyrosine phosphorylation of proteins is also an immediate consequence of receptor crosslinking. Notably, the only detectable src protein-related TPK in PT-18 cells is p62c-yes, and it is this TPK that is activated on Fc epsilon RI engagement and coimmunoprecipitates with the receptor. Therefore, it seems that different src protein-related TPKs can associate with the same receptor and become activated after receptor engagement.