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Supplementary Figure 2. Effect of BEZ235 on MYC-dependent gene expression in colorectal cancer cell lines.

Supplementary Figure 3. Effect of BEZ235 on MAP-kinase signaling.

Supplementary Figure Legends, Table Legends. Table 1. Reagents used in this study.

Supplementary Figure 4. Characterization of the 4E-BP1(4A) allele and eIF4A inhibitors.

Supplementary Figure 7. Additional characterization of silvestrol and BEZ235 effects in vivo.

Supplementary Figure 1. Characterization of BEZ235 effects on MYC protein stability and cell proliferation.

Supplementary Figure 5. Additional characterization of translation in colon carcinoma.

Supplementary Figure 6. BEZ235 does not alter proliferation or MYC levels in wild type or APC deficient intestinal enterocytes.

Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing "glutamine addiction". Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3'-UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA polymerase II (RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the MYC 3'-UTR is critical for maintaining genome stability when ribonucleotide levels are low.

MYC is an oncogenic transcription factor that binds globally to active promoters and promotes transcriptional elongation by RNA polymerase II (RNAPII) 1,2 . Deregulated expression of the paralogous protein MYCN drives the development of neuronal and neuroendocrine tumours and is often associated with a particularly poor prognosis 3 . Here we show that, similar to MYC, activation of MYCN in human neuroblastoma cells induces escape of RNAPII from promoters. If the release of RNAPII from transcriptional pause sites (pause release) fails, MYCN recruits BRCA1 to promoter-proximal regions. Recruitment of BRCA1 prevents MYCN-dependent accumulation of stalled RNAPII and enhances transcriptional activation by MYCN. Mechanistically, BRCA1 stabilizes mRNA decapping complexes and enables MYCN to suppress R-loop formation in promoter-proximal regions. Recruitment of BRCA1 requires the ubiquitin-specific protease USP11, which binds specifically to MYCN when MYCN is dephosphorylated at Thr58. USP11, BRCA1 and MYCN stabilize each other on chromatin, preventing proteasomal turnover of MYCN. Because BRCA1 is highly expressed in neuronal progenitor cells during early development 4 and MYC is less efficient than MYCN in recruiting BRCA1, our findings indicate that a cell-lineage-specific stress response enables MYCN-driven tumours to cope with deregulated RNAPII function.

Summary MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle., Graphical Abstract, Highlights • N-MYC forms complexes with TFIIIC, RAD21, and TOP2A • TFIIIC recruits RAD21 and is required for N-MYC-dependent pause release of Pol II • Aurora-A displaces TFIIIC, TOP2A, and RAD21 from N-MYC during S phase • Aurora-A inhibits pause release of Pol II during S phase, Büchel et al. demonstrate that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21. Aurora-A competes with TFIIIC and RAD21 for binding to N-MYC, and Aurora-A displaces the three proteins from N-MYC during S phase. As consequence, N-MYC-dependent pause release is inhibited during S phase, preventing activation of the ATR checkpoint kinase.

Deregulated expression of MYC is a driver of colorectal carcinogenesis, suggesting that inhibiting MYC may have significant therapeutic value. The PI3K and mTOR pathways control MYC turnover and translation, respectively, providing a rationale to target both pathways to inhibit MYC. Surprisingly, inhibition of PI3K does not promote MYC turnover in colon carcinoma cells, but enhances MYC expression because it promotes FOXO-dependent expression of growth factor receptors and MAPK-dependent transcription of MYC. Inhibition of mTOR fails to inhibit translation of MYC, because levels of 4EBPs are insufficient to fully sequester eIF4E and because an internal ribosomal entry site element in the 5′-untranslated region of the MYC mRNA permits translation independent of eIF4E. A small-molecule inhibitor of the translation factor eIF4A, silvestrol, bypasses the signaling feedbacks, reduces MYC translation, and inhibits tumor growth in a mouse model of colorectal tumorigenesis. We propose that targeting translation initiation is a promising strategy to limit MYC expression in colorectal tumors. Significance: Inhibiting MYC function is likely to have a significant therapeutic impact in colorectal cancers. Here, we explore several strategies to target translation initiation in order to block MYC expression. We show that a small-molecule inhibitor of eIF4A inhibits MYC expression and suppresses tumor growth in vivo. Cancer Discov; 5(7); 768–81. ©2015 AACR. See related commentary by Castell and Larsson, p. 701. This article is highlighted in the In This Issue feature, p. 681

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.

We demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21(Cip1) and subsequent cell cycle arrest depends upon endogenous Miz-1 in HPV-negative C33A cervical cancer cells containing mutated p53. Transient expression of E7 in C33A inhibits UV-induced expression of p21(Cip1) and overcomes Miz-1-induced G1-phase arrest. The C-terminal E7Δ79LEDLL83-mutant with reduced Miz-1-binding capacity was impaired in its capability to repress p21(Cip1) expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21(Cip1) expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21(Cip1) core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21(Cip1) promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21(Cip1) expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21(Cip1) expression. E7C24G retained E7-wild-type capability to inhibit Miz-1-dependent transactivation. These findings suggest that HPV-16 E7 can repress Miz-1-induced p21(Cip1) gene expression.

Deregulated expression of MYC contributes to the genesis of multiple human tumours. The encoded protein, MYC, functions through the transcriptional regulation of large numbers of target genes. Recent publications show that MYC is closely involved in DNA replication and the checkpoint processes that monitor progress through the S phase, and suggest that limiting replication stress is a key function of this protein. These findings could have considerable implications for our understanding of how MYC transforms cells and which mechanisms protect normal cells from transformation by activated oncogenes.

The Myc-associated zinc-finger protein, Miz1, activates transcription of the p21cip1 gene in response to UV irradiation. Miz1 associates with topoisomerase II binding protein1 (TopBP1), an essential activator of the Atr kinase. We show here that Miz1 is required for the recruitment of a fraction of TopBP1 to chromatin, for the protection of TopBP1 from proteasomal degradation and for Atr-dependent signal transduction. TopBP1 that is not bound to chromatin is degraded by the HectH9 (Mule, ARF-BP1 and HUWE1) ubiquitin ligase. Myc antagonizes the binding of TopBP1 to Miz1; as a result, expression of Myc leads to dissociation of TopBP1 from chromatin, reduces the amount of total TopBP1 and attenuates Atr-dependent signal transduction. Our data show that Miz1 and Myc affect the activity of the Atr checkpoint through their effect on TopBP1 chromatin association and stability.

The cellular levels of the Myc oncoprotein are critical determinants of cell proliferation, cell growth and apoptosis and are tightly regulated by external growth factors. Levels of Myc oncoprotein also decline in response to intracellular stress signals such as DNA damage. We show here that this decline is in part due to proteasomal degradation and that it is mediated by the Fbw7 ubiquitin ligase. We have shown previously that the ubiquitin-specific protease Usp28, binds to the nucleoplasmic isoform of Fbw7, Fbw7alpha, and counteracts its function in mammalian cells. Usp28 dissociates from Fbw7alpha in response to UV irradiation, providing a mechanism how Fbw7-mediated degradation of Myc is enhanced upon DNA damage. Our data extend previous observations that link Myc function to the cellular response to DNA damage.

Myc plays a key role in homeostasis of the skin. We show that Miz1, which mediates Myc repression of gene expression, is expressed in the epidermal basal layer. A large percentage of genes regulated by the Myc–Miz1 complex in keratinocytes encode proteins involved in cell adhesion, and some, including the α6 and β1 integrins, are directly bound by Myc and Miz1 in vivo. Using a Myc mutant deficient in Miz1 binding (MycV394D), we show that Miz1 is required for the effects of Myc on keratinocyte responsiveness to TGF-β. Myc, but not MycV394D, decreases keratinocyte adhesion and spreading. In reconstituted epidermis, Myc induces differentiation and loss of cell polarization in a Miz1-dependent manner. In vivo, overexpression of β1 integrins restores basal layer polarity and prevents Myc-induced premature differentiation. Our data show that regulation of cell adhesion is a major function of the Myc–Miz1 complex and suggest that it may contribute to Myc-induced exit from the epidermal stem cell compartment.

The Myc oncoprotein represses initiator-dependent transcription through the POZ domain transcription factor Miz-1. We now show that transactivation by Miz-1 is negatively regulated by association with topoisomerase II binding protein (TopBP1); UV irradiation downregulates expression of TopBP1 and releases Miz-1. Miz-1 binds to the p21Cip1 core promoter in vivo and is required for upregulation of p21Cip1 upon UV irradiation. Using both c-myc −/− cells and a point mutant of Myc that is deficient in Miz-1 dependent repression, we show that Myc negatively regulates transcription of p21Cip1 upon UV irradiation and facilitates recovery from UV-induced cell cycle arrest through binding to Miz-1. Our data implicate Miz-1 in a pathway that regulates cell proliferation in response to UV irradiation.

We used the Sleeping Beauty (SB) transposable element as a tool to probe transposon–host cell interactions in vertebrates. The Miz-1 transcription factor was identified as an interactor of the SB transposase in a yeast two-hybrid screen. Through its association with Miz-1, the SB transposase down-regulates cyclin D1 expression in human cells, as evidenced by differential gene expression analysis using microarray hybridization. Down-regulation of cyclin D1 results in a prolonged G 1 phase of the cell cycle and retarded growth of transposase-expressing cells. G 1 slowdown is associated with a decrease of cyclin D1/cdk4-specific phosphorylation of the retinoblastoma protein. Both cyclin D1 down-regulation and the G 1 slowdown induced by the transposase require Miz-1. A temporary G 1 arrest enhances transposition, suggesting that SB transposition is favored in the G 1 phase of the cell cycle, where the nonhomologous end-joining pathway of DNA repair is preferentially active. Because nonhomologous end-joining is required for efficient SB transposition, the transposase-induced G 1 slowdown is probably a selfish act on the transposon’s part to maximize the chance for a successful transposition event.

Nature 511, 483–487 (2014); doi:10.1038/nature13473 In this Letter, the ArrayExpress microarray data set accession number was wrongly given as E-MTAB-1524; the correct accession number is E-MTAB-1886. This has been corrected in the online versions of the paper.

The transcription factor Miz1 is required for DNA-damage-induced cell-cycle arrest. We have now identified 14-3-3eta as a gene that inhibits Miz1 function through interaction with its DNA binding domain. Binding of 14-3-3eta to Miz1 depends on phosphorylation by Akt and regulates the recovery of cells from arrest after DNA damage. Miz1 has two functions in response to DNA damage: first, it is required for upregulation of a large group of genes, a function that is regulated by c-Myc, but not by 14-3-3eta; second, Miz1 represses the expression of many genes in response to DNA damage in an Akt- and 14-3-3eta-regulated manner.

The Myc oncoprotein is a transcription factor that can both activate and repress genes. Transcriptional activation by Myc is well understood, but, by contrast, the mechanisms through which Myc represses transcription have remained elusive. Recent evidence suggests that complex formation by Myc with a zinc-finger transcription factor, Miz-1, plays an important role in mediating repression by Myc. The findings might explain how Myc interferes with cell-cycle arrest in response to TGF-beta, APC and DNA damage.

Arf assembles a complex containing Miz1, heterochromatin, and histone H3K3 to block expression of genes involved in cell adhesion and signal transduction. The resulting blockade of cell–cell and cell–matrix interactions facilitates elimination of cells carrying oncogenic mutations., Oncogenic stress induces expression of the alternate reading frame (Arf) tumor suppressor protein. Arf then stabilizes p53, which leads to cell cycle arrest or apoptosis. The mechanisms that distinguish both outcomes are incompletely understood. In this study, we show that Arf interacts with the Myc-associated zinc finger protein Miz1. Binding of Arf disrupts the interaction of Miz1 with its coactivator, nucleophosmin, induces the sumoylation of Miz1, and facilitates the assembly of a heterochromatic complex that contains Myc and trimethylated H3K9 in addition to Miz1. Arf-dependent assembly of this complex leads to the repression of multiple genes involved in cell adhesion and signal transduction and induces apoptosis. Our data point to a tumor-suppressive pathway that weakens cell–cell and cell–matrix interactions in response to expression of Arf and that may thereby facilitate the elimination of cells harboring an oncogenic mutation.