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The MYC oncogenes encode a family of transcription factors that feature prominently in cancer. MYC proteins are overexpressed or deregulated in a majority of malignancies and drive tumorigenesis by inducing widespread transcriptional reprogramming that promotes cell proliferation, metabolism, and genomic instability. The ability of MYC to regulate transcription depends on its dimerization with MAX, which creates a DNA-binding domain that recognizes specific sequences in the regulatory elements of MYC target genes. Recently, we discovered that recognition of target genes by MYC also depends on its interaction with WDR5, a WD40-repeat protein that exists as part of several chromatin-regulatory complexes. Here, we discuss how interaction of MYC with WDR5 could create an avidity-based chromatin recognition mechanism that allows MYC to select its target genes in response to both genetic and epigenetic determinants. We rationalize how the MYC-WDR5 interaction provides plasticity in target gene selection by MYC and speculate on the biochemical and genomic contexts in which this interaction occurs. Finally, we discuss how properties of the MYC-WDR5 interface make it an attractive point for discovery of small-molecule inhibitors of MYC function in cancer cells.
©2015 American Association for Cancer Research.

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 at ∼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 anticancer therapies against MYC-driven tumors.
Copyright © 2015 Elsevier Inc. All rights reserved.

Caveolin-1 (Cav1) is the primary scaffolding protein of caveolae, flask-shaped invaginations of the plasma membrane thought to function in endocytosis, mechanotransduction, signaling and lipid homeostasis. A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins. However, how different tags affect the behavior of ectopically expressed Cav1 is still largely unknown. To address this question, we performed a comparative analysis of the subcellular distribution, oligomerization state and detergent resistance of transiently overexpressed Cav1 labeled with three different C-terminal tags (EGFP, mCherry and myc). We show that addition of fluorescent protein tags enhances the aggregation and/or degradation of both wild-type Cav1 and an oligomerization defective P132L mutant. Strikingly, complexes formed by overexpressed Cav1 fusion proteins excluded endogenous Cav1 and Cav2, and the properties of native caveolins were largely preserved even when abnormal aggregates were present in cells. These findings suggest that differences in tagging strategies may be a source of variation in previously published studies of Cav1 and that overexpressed Cav1 may exert functional effects outside of caveolae. They also highlight the need for a critical re-evaluation of current knowledge based on transient overexpression of tagged Cav1.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The relevance of changes to the coding sequence of the c-MYC oncogene to malignancy is controversial. Overexpression of a pristine form of MYC is observed in many cancers and is sufficient to drive tumorigenesis in most contexts. Yet missense changes to MYC are found in ~50% of Burkitt's lymphomas, aggregate within an amino-terminal degron important for proteasomal destruction of MYC, and where examined profoundly enhance the tumorigenic properties of MYC in vitro and in vivo. Much of the controversy surrounding these mutants stems from the limited number of mutations that have been evaluated and their clustering within a single region of the MYC protein; the highly-conserved Myc box I (MbI) element. Here, by analysis of extant genomic data sets, we identify a previously unrecognized hotspot for tumor-associated MYC mutations, located in a conserved central portion of the protein. We show that, despite their distal location in MYC, mutations in this region precisely phenocopy those in MbI in terms of stability, in vitro transformation, growth-promoting properties, in vivo tumorigenesis and ability to escape p53-dependent tumor surveillance mechanisms. The striking parallels between the behavior of tumor-derived mutations in disparate regions of the MYC protein reveals that a common molecular process is disrupted by these mutations, implying an active role for these mutations in tumorigenesis and suggesting that different therapeutic strategies may be needed for treatment of lymphomas expressing wild type versus mutant forms of MYC protein.

Despite its involvement in most human cancers, MYC continues to pose a challenge as a readily tractable therapeutic target. Here we identify the MYC transcriptional cofactors TIP48 and TIP49 and MYC as novel binding partners of Mdm2-binding protein (MTBP), a functionally undefined protein that we show is oncogenic and overexpressed in many human cancers. MTBP associated with MYC at promoters and increased MYC-mediated transcription, proliferation, neoplastic transformation, and tumor development. In breast cancer specimens, we determined overexpression of both MYC and MTBP was associated with a reduction in 10-year patient survival compared with MYC overexpression alone. MTBP was also frequently co-amplified with MYC in many human cancers. Mechanistic investigations implicated associations with TIP48/TIP49 as well as MYC in MTBP function in cellular transformation and the growth of human breast cancer cells. Taken together, our findings show MTBP functions with MYC to promote malignancy, identifying this protein as a novel general therapeutic target in human cancer.
©2014 American Association for Cancer Research.

PURPOSE - Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication.
PATIENTS AND METHODS - Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models.
RESULTS - Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas.
CONCLUSION - Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.

BACKGROUND - Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.
OBJECTIVE - To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.
DESIGN, SETTING, AND PARTICIPANTS - Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.
OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS - The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.
RESULTS AND LIMITATIONS - Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.
CONCLUSIONS - IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.
Copyright © 2013 European Association of Urology. Published by Elsevier B.V. All rights reserved.

PURPOSE - Glioblastoma is refractory to conventional therapies. The bromodomain and extraterminal domain (BET) proteins are epigenetic readers that selectively bind to acetylated lysine residues on histone tails. These proteins recently emerged as important therapeutic targets in NUT midline carcinoma and several types of hematopoietic cancers. In this study, the therapeutic potential of a novel BET bromodomain inhibitor, JQ1, was assessed in a panel of genetically heterogeneous glioblastoma samples.
EXPERIMENTAL DESIGN - The antineoplastic effects of JQ1 were shown using ex vivo cultures derived from primary glioblastoma xenograft lines and surgical specimens of different genetic background. The in vivo efficacy was assessed in orthotopic glioblastoma tumors.
RESULTS - We showed that JQ1 induced marked G1 cell-cycle arrest and apoptosis, which was phenocopied by knockdown of individual BET family members. JQ1 treatment resulted in significant changes in expression of genes that play important roles in glioblastoma such as c-Myc, p21(CIP1/WAF1), hTERT, Bcl-2, and Bcl-xL. Unlike the observations in some hematopoietic cancer cell lines, exogenous c-Myc did not significantly protect glioblastoma cells against JQ1. In contrast, ectopically expressed Bcl-xL partially rescued cells from JQ1-induced apoptosis, and knockdown of p21(CIP1/WAF1) attenuated JQ1-induced cell-cycle arrest. Cells genetically engineered for Akt hyperactivation or p53/Rb inactivation did not compromise JQ1 efficacy, suggesting that these frequently mutated signaling pathways may not confer resistance to JQ1. Furthermore, JQ1 significantly repressed growth of orthotopic glioblastoma tumors.
CONCLUSION - Our results suggest potentially broad therapeutic use of BET bromodomain inhibitors for treating genetically diverse glioblastoma tumors.
©2013 AACR.

The oncogenic transcription factor c-Myc causes transformation and tumorigenesis, but it can also induce apoptotic cell death. Although tumor suppressors are necessary for c-Myc to induce apoptosis, the pathways and mechanisms are unclear. To further understand how c-Myc switches from an oncogenic protein to an apoptotic protein, we examined the mechanism of p53-independent c-Myc-induced apoptosis. We show that the tumor suppressor protein ARF mediates this switch by inhibiting ubiquitylation of the c-Myc transcriptional domain (TD). Whereas TD ubiquitylation is critical for c-Myc canonical transcriptional activity and transformation, inhibition of ubiquitylation leads to the induction of the noncanonical c-Myc target gene, Egr1, which is essential for efficient c-Myc-induced p53-independent apoptosis. ARF inhibits the interaction of c-Myc with the E3 ubiquitin ligase Skp2. Overexpression of Skp2, which occurs in many human tumors, inhibits the recruitment of ARF to the Egr1 promoter, leading to inhibition of c-Myc-induced apoptosis. Therapeutic strategies could be developed to activate this intrinsic apoptotic activity of c-Myc to inhibit tumorigenesis.

We assessed several methods of (13)C metabolic flux analysis (MFA) and found that isotopically nonstationary MFA achieved maximum flux resolution in cultured P493-6 B-cells, which have been engineered to provide tunable expression of the Myc oncoprotein. Comparison of metabolic flux maps obtained under oncogenic (High) and endogenous (Low) Myc expression levels revealed network-wide reprogramming in response to ectopic Myc expression. High Myc cells relied more heavily on mitochondrial oxidative metabolism than Low Myc cells and globally upregulated their consumption of amino acids relative to glucose. TCA cycle and amphibolic mitochondrial pathways exhibited 2- to 4-fold flux increases in High Myc cells, in contrast to modest increases in glucose uptake and lactate excretion. Because our MFA approach relied exclusively upon isotopic measurements of protein-bound amino acids and RNA-bound ribose, it is readily applicable to more complex tumor models that are not amenable to direct extraction and isotopic analysis of free intracellular metabolites.
Copyright © 2012 Elsevier Inc. All rights reserved.