SUMMARY MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved “MYC box IIIb” motif that engages a shallow, hydrophobic, cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC to ~80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anti-cancer therapies against MYC-driven tumors.

SUMMARY The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the “WIN site” of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.

The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacologically inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MYC–WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt's lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC–WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC–WDR5 inhibitors could be developed as anticancer agents.

Author Contributions JT, KBT, JRA, JJM, KMM, RDG, CH, and JDM designed and synthesized compounds. ERA and LRT conducted mechanism of action studies featured in Figure 4,5 and 7. JS and JGS obtained the biochemical and cell-based data in Table 1-3. JLS conducted western blot and caspase assay in Figure 6. BZ, TAR, and WGP performed X-ray crystallography studies of complexes. JK, MI, andRJC conducted CTOSs assay in Figure 8. WJM GMS helped design experiments. WPT, SRS, TL, and SWF design and directed experiments and helped write the paper. All authors have given approval to the final version of the manuscript. Supporting Information. X-ray refinement statistics, MLL1 HMT assay details and titration curves of compound 16. This material is available free of charge via the internet at http://pubs.acs.org. Accession Codes. Atom coordinates and structure factors for WDR5-ligand complexes can be accessed in the PDB via the following accession codes: Compound 13/WDR5 complex (6UFX), Compound 16/WDR5 complex (6UCS). Authors will release the atomic coordinates upon article publication.

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Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors. Members of the tumor necrosis factor (TNF)1 superfamily of receptors induce a variety of cellular responses including apoptosis, cellular differentiation, and proliferation. A subfamily of these receptors contains a death domain (DD) that is essential for transducing the apoptotic signal. Fas/CD95 is the best characterized member of this family. Binding of Fas ligand (FasL) to a preformed Fas/CD95 trimer (1) results in dimerization of two trimers (2) and higher levels of oligomerization (3). These activated receptors signal the apoptotic response by recruiting FADD to the cytoplasmic DD of the receptor to form the deathinducing signaling complex (DISC). FADD consists of two distinct domains, a DD that binds to the DD of Fas/CD95 and a death effector domain (DED) that binds to DEDs on caspase-8 and caspase-10 (4). The DD and DED have similar structural folds consisting of six anti-parallel ␣-helices, and both form globular protein structures whose only known function is to interact with other proteins (5). Thus, it is thought that binding of ligand to Fas/CD95 results in the recruitment of FADD through DD interactions followed by caspases through DED interactions. The induced proximity of two or more initiator caspases in a complex with FADD and the receptor results in their dimerization and activation (6, 7). Proteolytic processing leads to a fully processed, active form of the caspase that can dissociate from the receptor complex. Once activated, these caspases can then cleave and activate effector caspases such as caspase-3 and other substrates to induce the characteristic phenotypes associated with apoptosis.Activation of TNF receptor 1 requires an additional adaptor protein, TRADD. Binding of TNF␣ to TNF receptor 1 results in...

Although evasion of apoptosis is thought to be required for the development of cancer, it is unclear which cell death pathways are evaded. We previously identified a novel epithelial cell death pathway that works in normal cells but is inactivated in tumor cells, implying that it may be targeted during tumor development. The pathway can be activated by the Fas-associated death domain (FADD) of the adaptor protein but is distinct from the known mechanism of FADDinduced apoptosis through caspase-8. Here, we show that a physiological signal (tumor necrosis factor-related apoptosisinducing ligand) can kill normal epithelial cells through the endogenous FADD protein by using the novel FADD death domain pathway, which activates both apoptosis and autophagy. We also show that selective resistance to this pathway occurs when primary epithelial cells are immortalized and that this occurs through a mechanism that is independent of known events (telomerase activity, and loss of function of p53, Rb, INK4a, and ARF) that are associated with immortalization. These data identify a novel cell death pathway that combines apoptosis and autophagy and that is selectively inactivated at the earliest stages of epithelial cancer development. INTRODUCTIONBecause apoptosis can suppress tumor development, it is sometimes thought that cancer cells are generally resistant to apoptosis, whereas normal cells are sensitive. In fact, cancer cells are closer to their apoptotic threshold than their normal counterparts and often die more easily than normal cells in response to apoptotic stimuli (Evan and Vousden, 2001;Lowe et al., 2004). Apoptosis sensitization in cancer cells occurs because growth-promoting oncogenic events such as Myc expression (Evan and Littlewood, 1998;Evan and Vousden, 2001;Pelengaris et al., 2002), Rb inactivation (Chau and Wang, 2003), E2F activation (Nahle et al., 2002), and cyclin D3 expression (Mendelsohn et al., 2002) raise the levels of apoptotic proteins or make it easier to activate these molecules and thus reduce the threshold at which apoptosis is activated. Activated oncogenes can also sensitize cells to apoptosis by promoting loss of inhibitors of apoptosis that exist in primary cells (Duelli and Lazebnik, 2000). Immortalization and transformation also sensitize cells to nonapoptotic death (Fehrenbacher et al., 2004).If cancer cells die more easily than their normal counterparts, which cell death pathways are evaded during tumor development? One answer is that cancer cells must remain below the lowered apoptotic threshold for undergoing stress-induced apoptosis that is caused by the oncogenes that drive cell growth. Indeed, it has been suggested that this may be sufficient to cause cancer without any other cellular defects (Green and Evan, 2002). However, this model does not exclude the possibility that there may also be specific cell death pathways that inhibit cancer development in normal cells that are specifically inactivated during tumor development. Such a pathway would be expected to have the unusual cha...

Genetic variations in the PSA promoter are associated with serum PSA levels in men without prostatic disease. PSA promoter genotype information may help to refine models of PSA cutoff values.