In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.

Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules 1,2 . Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.To identify kinases that are specifically required for the viability of cells expressing deregulated MYC, we used U2OS cells expressing c-MYC fused to the oestrogen receptor ligand binding domain (MYC-ER) (Fig. 1a). Activation of MYC-ER by 4-hydroxytamoxifen (OHT) had little effect on apoptosis when cells were grown at low density in the presence of growth factors. Under these conditions, we performed a short interfering (si)RNA screen of the human kinome, using automated microscopy to identify siRNAs that induced poly-ADP-ribose-polymerase cleavage specifically in the presence of OHT. This screen yielded two hits, ARK5 and AMPK (Supplementary Table 1).Depletion of ARK5 induced the accumulation of MYC-expressing cells that stained positive for annexin V and propidium iodide (Fig. 1a and Supplementary Fig. 1a). Similarly, expressing different short hairpin (sh)RNAs targeting ARK5 induced levels of MYC-dependent death that correlated with the degree of knockdown (Fig. 1b). Titration of OHT revealed that levels of MYC that cause a dependence on ARK5 are higher than those required to promote proliferation ( Supplementary Fig. 1b). Depletion of ARK5 induced death in U2OS cells constitutively expressing MYC and suppressed propagation of MRC5 fibroblasts in a MYCdependent manner (Fig. 1c and Supplementary Fig. 1c). Expression of murine ARK5, which is not targeted by the shRNAs used, prevented death upon depletion of human ARK5 (Fig. 1d). This rescue required LKB1-dependent phosphorylation of T212, but not AKT-dependent phosphorylation of S601 (refs 3, 4). Mutation of K85 within the ATPbinding domain blocked the ability of murine ARK5 to prevent death, demonstrating that rescue requires ARK5 catalytic activity. Accordingly, a small-molecule inhibitor of ARK5, BX795, mimicked the effects o...

Amplification of MYCN is a driver mutation in a subset of human neuroendocrine tumors, including neuroblastoma. No small molecules that target N-Myc, the protein encoded by MYCN, are clinically available. N-Myc forms a complex with the Aurora-A kinase, which protects N-Myc from proteasomal degradation. Although stabilization of N-Myc does not require the catalytic activity of Aurora-A, we show here that two Aurora-A inhibitors, MLN8054 and MLN8237, disrupt the Aurora-A/N-Myc complex and promote degradation of N-Myc mediated by the Fbxw7 ubiquitin ligase. Disruption of the Aurora-A/N-Myc complex inhibits N-Myc-dependent transcription, correlating with tumor regression and prolonged survival in a mouse model of MYCN-driven neuroblastoma. We conclude that Aurora-A is an accessible target that makes destabilization of N-Myc a viable therapeutic strategy.

Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3′ untranslated region (3′UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3′UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3′ end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3′ end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3′UTR lengths, especially for chicken, and APADB provides necessary information to close this gap.Database URL: http://tools.genxpro.net/apadb/

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