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.

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...

Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded ssDNA annealing proteins (SSAPs) improve HR 1000-fold above endogenous levels; however, they are not broadly functional. Using Escherichia coli , Lactococcus lactis , Mycobacterium smegmatis , Lactobacillus rhamnosus , and Caulobacter crescentus we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host’s single-stranded DNA-binding protein (SSB), and are portable between species if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with a paired SSB can significantly improve activity, in some species enabling SSAP functionality even without host-compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes inaccessible through sequential nucleotide conversions.

Guanylyl cyclase C (GCC) is a cell-surface protein that is expressed by normal intestinal epithelial cells, more than 95% of metastatic colorectal cancers (mCRC), and the majority of gastric and pancreatic cancers. Due to strict apical localization, systemically delivered GCC-targeting agents should not reach GCC in normal intestinal tissue, while accessing antigen in tumor. We generated an investigational antibody-drug conjugate (TAK-264, formerly MLN0264) comprising a fully human anti-GCC monoclonal antibody conjugated to monomethyl auristatin E via a protease-cleavable peptide linker. TAK-264 specifically bound, was internalized by, and killed GCC-expressing cells in vitro in an antigen-density-dependent manner. In GCC-expressing xenograft models with similar GCC expression levels/patterns observed in human mCRC samples, TAK-264 induced cell death, leading to tumor regressions and long-term tumor growth inhibition. TAK-264 antitumor activity was generally antigen-density-dependent, although some GCC-expressing tumors were refractory to TAK-264-targeted high local concentrations of payload. These data support further evaluation of TAK-264 in the treatment of GCC-expressing tumors.