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.
Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.
Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.
Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.
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...
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