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The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.

The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.

Wnt/β-Catenin signaling plays crucial roles in regenerative processes in eumetazoans. It also acts in regeneration and axial patterning in the simple freshwater polyp Hydra, whose morphallactic regenerative capacity is unparalleled in the animal kingdom. Previous studies have identified β-catenin as an early response gene activated within the first 30min in Hydra head regeneration. Here, we have studied the role of β-Catenin in more detail. First, we show that nuclear β-Catenin signaling is required for head and foot regeneration. Loss of nuclear β-Catenin function blocks head and foot regeneration. Transgenic Hydra tissue, in which β-Catenin is over-expressed, regenerates more heads and feet. In addition, we have identified a set of putative β-Catenin target genes by transcriptional profiling, and these genes exhibit distinct expression patterns in the hypostome, in the tentacles, or in an apical gradient in the body column. All of them are transcriptionally up-regulated in the tips of early head and foot regenerates. In foot regenerates, this is a transient response, and expression starts to disappear after 12-36h. ChIP experiments using an anti-HydraTcf antibody show Tcf binding at promoters of these targets. We propose that gene regulatory β-Catenin activity in the pre-patterning phase is generally required as an early regeneration response. When regenerates are blocked with iCRT14, initial local transcriptional activation of β-catenin and the target genes occurs, and all these genes remain upregulated at the site of both head and foot regeneration for the following 2-3 days. This indicates that the initial regulatory network is followed by position-specific programs that inactivate fractions of this network in order to proceed to differentiation of head or foot structures. brachyury1 (hybra1) has previously been described as early response gene in head and foot regeneration. The HyBra1 protein, however, appears in head regenerating tips not earlier than about twelve hours after decapitation, and HyBra1 translation does not occur in iCRT14-treated regenerates. Foot regenerates never show detectable levels of HyBra1 protein at all. These results suggest that translational control mechanisms may play a decisive role in the head- and foot-specific differentiation phase, and HyBra1 is an excellent candidate for such a key regulator of head specification.

// Philipp Raffeiner 1, 3 , Andrea Schraffl 1 , Thomas Schwarz 2 , Ruth Rock 1 , Karin Ledolter 2 , Markus Hartl 1 , Robert Konrat 2 , Eduard Stefan 1 , Klaus Bister 1 1 Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria 2 Max F. Perutz Laboratories, Department of Structural and Computational Biology, University of Vienna, A-1030 Vienna, Austria 3 Present address: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA Correspondence to: Eduard Stefan, email: eduard.stefan@uibk.ac.at Klaus Bister, email: klaus.bister@uibk.ac.at Keywords: oncogene, transcription factor, signal transduction, protein-protein interaction, second messenger signaling Received: August 08, 2016 Accepted: November 21, 2016 Published: December 01, 2016 ABSTRACT The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca 2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca 2+ -dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca 2+ -loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15 N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca 2+ sensing CaM in the fine-tuning of Myc function.

The architectures of central signaling hubs are precisely organized by static and dynamic protein-protein interactions (PPIs). Upon deregulation, these PPI platforms are capable to propagate or initiate pathophysiological signaling events. This causes the acquisition of molecular features contributing to the etiology or progression of many diseases, including cancer, where deregulated molecular interactions of signaling proteins have been best studied. The reasons for PPI-dependent reprogramming of cancer-initiating cells are manifold; in many cases, mutations perturb PPIs, enzyme activities, protein abundance, or protein localization. Consequently, the pharmaceutical targeting of PPIs promises to be of remarkable therapeutic value. For this review we have selected three key players of oncogenic signaling which are differently affected by PPI deregulation: two (the small G proteins of the RAS family and the transcription factor MYC) are considered "undruggable" using classical drug discovery approaches and in the case of the third protein discussed here, PKA, standard kinase inhibitors, may be unsuitable in the clinic. These circumstances require alternative strategies, which may lie in pharmaceutical drug interference of critical PPIs accountable for oncogenic signaling.

The architectures of central signaling hubs are precisely organized by static and dynamic protein-protein interactions (PPIs). Upon deregulation, these PPI platforms are capable to propagate or initiate pathophysiological signaling events. This causes the acquisition of molecular features contributing to the etiology or progression of many diseases, including cancer, where deregulated molecular interactions of signaling proteins have been best studied. The reasons for PPI-dependent reprogramming of cancer-initiating cells are manifold; in many cases, mutations perturb PPIs, enzyme activities, protein abundance, or protein localization. Consequently, the pharmaceutical targeting of PPIs promises to be of remarkable therapeutic value. For this review we have selected three key players of oncogenic signaling which are differently affected by PPI deregulation: two (the small G proteins of the RAS family and the transcription factor MYC) are considered "undruggable" using classical drug discovery approaches and in the case of the third protein discussed here, PKA, standard kinase inhibitors, may be unsuitable in the clinic. These circumstances require alternative strategies, which may lie in pharmaceutical drug interference of critical PPIs accountable for oncogenic signaling.

Exchange of Genetic Sequences Between Viruses and Host.- Virus-Host Gene Interactions that Define HIV Disease Progression.- Sequencing the Biology of Entry: The Retroviral env Gene.- Infectious Agents in Bovine Red Meat and Milk and Their Potential Role in Cancer and Other Chronic Diseases.- MYC and RAF: Key Effectors in Cellular Signaling and Major Drivers in Human Cancer.- Oncogenic Roles of the PI3K/AKT/mTOR Axis.- MicroRNAs and Cancer.- The Evolution of Tumor Formation in Humans and Mice with Inherited Mutations in the P53 Gene.

The prototypes of the human MYC and RAF gene families are orthologs of animal proto-oncogenes that were originally identified as transduced alleles in the genomes of highly oncogenic retroviruses. MYC and RAF genes are now established as key regulatory elements in normal cellular physiology, but also as major cancer driver genes. Although the predominantly nuclear MYC proteins and the cytoplasmic RAF proteins have different biochemical functions, they are functionally linked in pivotal signaling cascades and circuits. The MYC protein is a transcription factor and together with its dimerization partner MAX holds a central position in a regulatory network of bHLH-LZ proteins. MYC regulates transcription conducted by all RNA polymerases and controls virtually the entire transcriptome. Fundamental cellular processes including distinct catabolic and anabolic branches of metabolism, cell cycle regulation, cell growth and proliferation, differentiation, stem cell regulation, and apoptosis are under MYC control. Deregulation of MYC expression by rearrangement or amplification of the MYC locus or by defects in kinase-mediated upstream signaling, accompanied by loss of apoptotic checkpoints, leads to tumorigenesis and is a hallmark of most human cancers. The critically controlled serine/threonine RAF kinases are central nodes of the cytoplasmic MAPK signaling cascade transducing converted extracellular signals to the nucleus for reshaping transcription factor controlled gene expression profiles. Specific mutations of RAF kinases, such as the prevalent BRAF(V600E) mutation in melanoma, or defects in upstream signaling or feedback loops cause decoupled kinase activities which lead to tumorigenesis. Different strategies for pharmacological interference with MYC- or RAF-induced tumorigenesis are being developed and several RAF kinase inhibitors are already in clinical use.

In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2'-azido cytidine and 2'-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2'-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2'-azido uridine and 2'-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2'-azido group supports the C3'-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2'-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2'-azido modifications are compatible with 2'-fluoro and/or 2'-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2'-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2'-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72 % for Melittin, which lacks carboxylic acid functionalities, to 19 % for an acidic 147-residue protein, to 12 % for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO2. Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

Avian fibroblasts transformed simultaneously by the v-myc and v-mil(raf) oncogenes of acute leukemia and carcinoma virus MH2 contain elevated levels of c-Fos and c-Jun, major components of the transcription factor complex AP-1. To define specific transcriptional targets in these cells, subtractive hybridization techniques were employed leading to the identification of strongly upregulated genes including OPN (osteopontin), 126MRP, and rac2. OPN is a cytokine and cell attachment protein which has been implicated in human tumor progression and metastasis, the calcium binding 126MRP protein is related to the human S100 protein family involved in invasive cell growth, and the Rac2 protein belongs to the Rho family of small GTPases regulating actin reorganization and cell migration. Promoter analysis indicated that OPN activation is mediated by a non-consensus AP-1 binding site located close to the transcription start site. Electrophoretic mobility shift assays, chromatin immunoprecipitation and transcriptional reporter gene analyses showed that c-Fos and c-Jun bind specifically to this site and that c-Fos efficiently transactivates the OPN promoter. High-level expression of OPN, 126MRP, or Rac2 proteins from a retroviral vector led to partial cell transformation, documented by morphological changes and anchorage-independent growth. The specific activation in v-myc/v-mil(raf)-transformed cells of target genes with intrinsic oncogenic potential may provide an explanation for the longstanding observation that concomitant expression of these oncogenes leads to strongly enhanced oncogenicity in vivo and in vitro compared to cell transformation by v-myc or v-mil(raf) alone.

The Jun oncoprotein is a major component of the transcription factor complex AP-1, which regulates the expression of multiple genes essential for cell proliferation, differentiation and apoptosis. Constitutive activation of endogenous AP-1 is required for tumor formation in avian and mammalian cell transformation systems, and also occurs in distinct human tumor cells suggesting that AP-1 plays an important role in human oncogenesis. The highly oncogenic v-jun allele capable of inducing neoplastic transformation in avian fibroblasts and fibrosarcomas in chicken as a single oncogenic event, was generated by mutation of the cellular c-jun gene during retroviral transduction. Hence, avian cells represent an excellent model system to investigate molecular mechanisms underlying jun-induced cell transformation. Approaches aimed at the identification of genes specifically deregulated in jun-transformed fibroblasts have led to the identification of several genes targeted by oncogenic Jun. Some of the activated genes represent direct transcriptional targets of Jun encoding proteins, which are presumably involved in cell growth and differentiation. Genes suppressed in v-jun-transformed cells include several extracellular proteins like components of the extracellular matrix or proteins involved in extracellular signalling. Due to aberrant regulation of multiple genes by the Jun oncoprotein, it is assumed that only the combined differential expression of Jun target genes or of a subset thereof contributes to the conversion of a normal fibroblast into a tumor cell displaying a phenotype typical of jun-induced cell transformation. It has already been shown that distinct activated targets exhibit partial transforming activity upon over-expression in avian fibroblasts. Also, distinct target genes silenced by v-Jun inhibit tumor formation when re-expressed in v-jun-transformed cells. The protein products of these transformation-relevant genes may thus represent potential drug targets for interference with jun-induced tumorigenesis.

In their classic paper on the identification of the transforming principle of Rous sarcoma virus (RSV) published 1970 in PNAS (1), Peter Duesberg at the University of California, Berkeley, and Peter Vogt, then at the University of Washington, Seattle, drew a seemingly simple yet groundbreaking conclusion. When they analyzed the genomic RNAs of transforming, acutely oncogenic RSV and of transformation-defective (td) mutant derivatives, they found that all transforming virus stocks contained two classes of RNA subunits, a larger one (a) and a smaller one (b), whereas the nontransforming yet replication-competent mutants contained the smaller b subunits only. Duesberg and Vogt concluded that the larger a subunit contained the transforming principle of RSV. Based on this and on subsequent structural comparisons of the a and b subunits of biologically cloned viruses, the transforming principle was defined by the remarkably simple equation a − b = x and was later termed src (for sarcoma). The first biochemical identification of a cancer gene was achieved, initially in a chicken virus. However, the principal proof of a physical underpinning of the cancer gene hypothesis had tremendous impact on a fundamental challenge of medicine, decoding the molecular basis of human carcinogenesis.

The solution structure of quail CRP2(LIM2) was significantly improved by using an increased number of NOE constraints obtained from a 13 C, 15 N-labeled protein sample and by applying a recently developed triple-resonance cross-correlated relaxation experiment for the determination of the backbone dihedral angle a. Additionally, the relative orientation of the 15 N(i)- 1 H N (i) dipole and the 13 CO(i) CSA tensor, which is related to both backbone angles E and a, was probed by nitrogen-carbonyl multiple- quantum relaxation and used as an additional constraint for the refinement of the local geometry of the metal-coordination sites in CRP2(LIM2). The backbone dynamics of residues located in the folded part of CRP2(LIM2) have been characterized by proton-detected 13 C'(i-1)- 15 N(i) and 15 N(i)- 1 H N (i) multiple- quantum relaxation, respectively. We show that regions having cross-correlated time modulation of backbone isotropic chemical shifts on the millisecond to microsecond time scale correlate with residues that are structurally altered in the mutant protein CRP2(LIM2)R122A (disruption of the CCHC zinc- finger stabilizing side-chain hydrogen bond) and that these residues are part of an extended hydrogen- bonding network connecting the two zinc-binding sites. This indicates the presence of long-range collective motions in the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recognition motif.

The protein product (c-Myc) of the protooncogene c-myc is a transcriptional regulator playing a key role in cellular growth, differentiation, and apoptosis. Deregulated myc genes, like the transduced retroviral v-myc allele, are oncogenic and cause cell transformation. The C-terminal bHLHZip domain of v-Myc, encompassing protein dimerization (helix-loop-helix, leucine zipper) and DNA contact (basic region) surfaces, was expressed in bacteria as a highly soluble p15(v-myc )recombinant protein. Dissociation constants (K(d)) for the heterodimer formed with the recombinant bHLHZip domain of the Myc binding partner Max (p14(max)) and for the Myc-Max-DNA complex were estimated using circular dichroism (CD) spectroscopy and quantitative electrophoretic mobility shift assay (EMSA). Multi-dimensional NMR spectroscopy was used to characterize the solution structural and dynamic properties of the v-Myc bHLHZip domain. Significant secondary chemical shifts indicate the presence of two separated alpha-helical regions. The C-terminal leucine zipper region forms a compact alpha-helix, while the N-terminal basic region exhibits conformational averaging with substantial alpha-helical content. Both helices lack stabilizing tertiary side-chain interactions and represent exceptional examples for loosely coupled secondary structural segments in a native protein. These results and CD thermal denaturation data indicate a monomeric state of the v-Myc bHLHZip domain. The (15)N relaxation data revealed backbone mobilities which corroborate the existence of a partially folded state, and suggest a "beads-on-a-string" motional behaviour of the v-Myc bHLHZip domain in solution. The preformation of alpha-helical regions was confirmed by CD thermal denaturation studies, and quantification of the entropy changes caused by the hydrophobic effect and the reduction of conformational entropy upon protein dimerization. The restricted conformational space of the v-Myc bHLHZip domain reduces the entropy penalty associated with heterodimerization and allows rapid and accurate recognition by the authentic Myc binding partner Max.

The c-myc protooncogene encodes the Myc transcription factor, a global regulator of fundamental cellular processes. Deregulation of c-myc leads to tumorigenesis, and c-myc is an important driver in human cancer. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins involved in transcriptional regulation of target genes. Non-transcriptional functions have also been attributed to the Myc protein, notably direct interaction with the pre-replicative complex (pre-RC) controlling the initiation of DNA replication. A key component of the pre-RC is the Cdt1 protein, an essential factor in origin licensing. Here we present data suggesting that the CDT1 gene is a transcriptional target of the Myc-Max complex. Expression of the CDT1 gene in v-myc-transformed cells directly correlates with myc expression. Also, human tumor cells with elevated c-myc expression display increased CDT1 expression. Occupation of the CDT1 promoter by Myc-Max is demonstrated by chromatin immunoprecipitation, and transactivation by Myc-Max is shown in reporter assays. Ectopic expression of CDT1 leads to cell transformation. Our results provide a possible direct mechanistic link of Myc's canonical function as a transcription factor to DNA replication. Furthermore, we suggest that aberrant transcriptional activation of CDT1 by deregulated myc alleles contributes to the genomic instabilities observed in tumor cells.

The LIM domain is a conserved cysteine and histidine-containing structural module of two tandemly arranged zinc fingers. It has been identified in single or multiple copies in a variety of regulatory proteins, either in combination with defined functional domains, like homeodomains, or alone, like in the CRP family of LIM proteins. Structural studies of CRP proteins have allowed a detailed evaluation of interactions in LIM-domains at the molecular level. The packing interactions in the hydrophobic core have been identified as a significant contribution to the LIM domain fold, whereas hydrogen bonding within each single zinc binding site stabilizes zinc finger geometry in a so-called "outer" or "indirect" coordination sphere. Here we report the solution structure of a point-mutant of the carboxyl-terminal LIM domain of quail cysteine and glycine-rich protein CRP2, CRP2(LIM2)R122A, and discuss the structural consequences of the disruption of the hydrogen bond formed between the guanidinium side-chain of Arg122 and the zinc-coordinating cysteine thiolate group in the CCHC rubredoxin-knuckle. The structural analysis revealed that the three-dimensional structure of the CCHC zinc binding site in CRP2(LIM2)R122A is adapted as a consequence of the modified hydrogen bonding pattern. Additionally, as a result of the conformational rearrangement of the zinc binding site, the packing interactions in the hydrophobic core region are altered, leading to a change in the relative orientation of the two zinc fingers with a concomitant change in the solvent accessibilities of hydrophobic residues located at the interface of the two modules. The backbone dynamics of residues located in the folded part of CRP2(LIM2)R122A have been characterized by proton-detected(15)N NMR spectroscopy. Analysis of the R2/R1ratios revealed a rotational correlation time of approximately 6.2 ns and tumbling with an axially symmetric diffusion tensor (D parallel/D perpendicular=1.43). The relaxation data were also analyzed using a reduced spectral density mapping approach. As in wild-type CRP2(LIM2), significant mobility on a picosecond/nanosecond time-scale was detected, and conformational exchange on a microsecond time-scale was identified for residues located in loop regions between secondary structure elements. In summary, the relative orientation of the two zinc binding sites and the accessibility of hydrophobic residues is not only determined by hydrophobic interactions, but can also be modified by the formation and/or breakage of hydrogen bonds. This may be important for the molecular interactions of an adaptor-type LIM domain protein in macromolecular complexes, particularly for the modulation of protein-protein interactions.

OPN is an RGD-containing protein overexpressed in cells transformed by v-myc and v-mil(raf) oncogenes. Here we report the resonance assignment of recombinant quail OPN and provide NMR evidence that quail OPN is an intrinsically unstructured protein in solution.

The BKJ gene was originally identified based on its specific transcriptional activation in jun-transformed avian fibroblasts. We now show that BKJ is a direct transcriptional target of the AP-1 transcription factor components Jun and Fos. The complete structural organization of the quail BKJ gene was determined by nucleotide sequence analysis and transcriptional mapping. The gene contains three exons with the coding region confined to the third exon. A major mRNA species of 0.8 kb and a minor one of 1.3 kb are produced by variable usage of two transcriptional initiation sites. The BKJ promoter region contains two authentic AP-1 binding sites. By transactivation of reporter gene constructs and direct binding of Jun recombinant protein, the proximal AP-1 element was shown to be essential for BKJ promoter activation. Using polyclonal antiserum directed against recombinant BKJ protein, the activation of BKJ in jun-transformed avian fibroblasts was also demonstrated at the protein level. BKJ is a novel gene related to the avian beta-keratin gene family whose members display highly specific expression patterns during embryogenesis and epidermal development. Activation of BKJ in fibroblasts by retroviral or deregulated cellular jun or fos alleles may contribute to cell transformation.

MYC is a transcriptional regulator that in order to function has to dimerize with a specific partner protein, MAX. The MYC-MAX dimer regulates the expression of thousands of genes involved in fundamental cellular processes including growth, proliferation, differentiation, biosynthesis, energy metabolism, and apoptosis [1-3]. The discovery that MYC becomes overexpressed as a result of chromosomal re-arrangements in Burkitt's lymphoma was the first implication of MYC in human cancer, and today deregulated MYC expression is considered to be the crucial driving force in most if not all cancers [1-3]. Because of this prevalent role in carcinogenesis, MYC is an important target for therapeutic intervention. However, there are both principal and practical obstacles in targeting MYC. Inhibition of a gene that is essential for fundamental cellular processes could cause unacceptable side effects. Yet inhibition of MYC by expression of a dominant-negative MYC construct in an animal model caused regression of tumor growth but no lasting damage to rapidly proliferating normal tissues [4]. Practical problems in directly targeting MYC or the MYC-MAX heterodimer with small molecules (Figure ​(Figure1)1) stem from the disordered state of the MYC monomer in solution and from the general nature of protein-protein interactions. These commonly involve large interacting surfaces that present no well-defined pockets or grooves for high-energy binding of small ligands. However, proof of principle for overcoming these difficulties was provided by the identification of small-molecule antagonists for MYC-MAX dimerization that reduced MYC-driven cell transformation in tissue culture [5]. Figure 1 Elevated levels of MYC-MAX complexes drive cell proliferation and carcinogenesis Two recent publications in PNAS and Oncotarget now report the identification and characterization of novel small-molecule inhibitors of MYC-MAX dimerization (Figure ​(Figure1)1) that are active in a pharmacologically relevant nanomolar range [6,7]. The MYC-MAX antagonists were isolated from a Krohnke combinatorial library of 2,4,6-trisubstituted pyridines designed for drug discovery. These lead compounds inhibit MYC-MAX dimerization, specifically interfere with MYC-induced oncogenic transformation in cell culture, reduce the MYC-specific transcriptional signature, and block MYC-driven tumor growth in a xenotransplant of human cancer cells [6]. These data were complemented with a specific protein-fragment complementation assay (PCA). In this assay, Renilla luciferase (Rluc) is rationally dissected into two fragments, one of these is fused to MYC, the other to MAX. When the MYC and MAX components of these hybrid proteins dimerize, luciferase activity is restored. This PCA allows direct recording of the interplay of MYC and MAX in living cells [7]. The studies documented inhibition of MYC-MAX dimerization by the small-molecule inhibitors, showed the expected nuclear localization of MYC-MAX complexes, and demonstrated the effect of inactivating MYC mutations on the nuclear MYC-MAX complex levels as well as sensitivity of MYC-MAX dimerization to limiting levels of available MAX. The degree to which MYC-MAX levels are reduced by the small-molecule antagonists correlates with the cytocidal and cytostatic activity of the inhibitors for MYC-driven human or avian tumor cells. The Rluc PCA is a specific and sensitive reporter assay broadly applicable to the analysis of protein-protein interactions, including screening and optimization of small-molecule inhibitors. The promising features of the MYC inhibitors described in the two recent reports [6,7] will initiate further efforts to improve their pharmacokinetic properties, and to unveil their precise binding mode and molecular mechanism of interference with MYC-MAX function.

Members of the cysteine- and glycine-rich protein family (CRP1, CRP2, and CRP3) contain two zinc-binding LIM domains, LIM1 (amino-terminal) and LIM2 (carboxyl-terminal), and are implicated in diverse cellular processes linked to differentiation, growth control, and pathogenesis. Here we report the solution structure of full-length recombinant quail CRP2 as determined by multi-dimensional triple-resonance NMR spectroscopy. The structural analysis revealed that the global fold of the two LIM domains in the context of the full-length protein is identical to the recently determined solution structures of the isolated individual LIM domains of quail CRP2. There is no preference in relative spatial orientation of the two domains. This supports the view that the two LIM domains are independent structural and presumably functional modules of CRP proteins. This is also reflected by the dynamic properties of CRP2 probed by15N relaxation values (T 1,T 2, and nuclear Overhauser effect). A model-free analysis revealed local variations in mobility along the backbone of the two LIM domains in the native protein, similar to those observed for the isolated domains. Interestingly, fast and slow motions observed in the 58-amino acid linker region between the two LIM domains endow extensive motional freedom to CRP2. The dynamic analysis indicates independent backbone mobility of the two LIM domains and rules out correlated LIM domain motion in full-length CRP2. The finding that the LIM domains in a protein encompassing multiple LIM motifs are structurally and dynamically independent from each other supports the notion that these proteins may function as adaptor molecules arranging two or more protein constituents into a macromolecular complex.

Members of the cysteine and glycine-rich protein (CRP) family (CRP1, CRP2, and CRP3) contain two zinc-binding LIM domains, LIM1 and LIM2, and are implicated in diverse cellular processes linked to differentiation, growth control and pathogenesis. The solution structure of an 81-amino acid recombinant peptide encompassing the amino-terminal LIM1 domain of quail CRP2 has been determined by 2D and 3D homo- and heteronuclear NMR spectroscopy. The LIM1 domain consists of two zinc binding sites of the CCHC and the CCCC type, respectively, which both contain two orthogonally arranged antiparallel beta-sheets and which are packed together by a hydrophobic core composed of residues from the zinc finger loop regions. The CCCC zinc finger is followed by a short alpha-helical stretch. The structural analysis revealed that the global fold of LIM1 closely resembles the recently determined solution structures of the carboxyl-terminal LIM2 domains of quail CRP2 and chicken CRP1, and that LIM1 and LIM2 are independently folded structural and presumably functional domains of CRP proteins. To explore the dynamical properties of CRP proteins, we have used 15N relaxation values (T1, T2, and nuclear Overhauser effect (NOE) to describe the dynamical behavior of a LIM domain. A model-free analysis revealed local variations in mobility along the backbone of the quail CRP2 LIM1 motif. Slow motions are evident in turn regions located between the various antiparallel beta-sheets or between their strands. By use of an extended motional model, fast backbone motions were detected for backbone amide NH groups of hydrophobic residues located in the core region of the LIM1 domain. These findings point to a flexible hydrophobic core in the LIM1 domain allowing residual relative mobility of the two zinc fingers, which might be important to optimize the LIM1 interface for interaction with its physiological target molecule(s) and to compensate enthalpically for the entropy loss upon binding.

The CSRP2 gene encoding the LIM domain protein CRP2 was originally identified in quail based on its strong transcriptional suppression in transformed avian fibroblasts. Here we have isolated a human CSRP2 cDNA clone encoding a 193-amino-acid human CRP2 (hCRP2) protein with 96.4% amino acid sequence identity to the avian homolog. The CSRP2 cDNA clone was used to isolate CSRP2-related clones from gamma EMBL3 and P1 libraries of human genomic DNA. The complete organization of the CSRP2 gene was determined by nucleic acid hybridization, transcriptional mapping, and nucleotide sequence analysis. The gene spans a total of approximately 22 kb and contains six exons. The coding region is confined to exons 2-6 and predicts a hCRP2 protein identical in its amino acid sequence to the protein deduced from the CSRP2 cDNA clone. By fluorescence in situ hybridization using both lambda EMBL3 and P1 library clones as hybridization probes and a new method for computerized signal localization, CSRP2 was mapped to chromosome subband 12q21.1, a region frequently affected by deletion or breakage events in various tumor types. The library screens also led to the isolation of a CSRP2-related pseudogene, CSRP2P, which carried several extensive deletions and nucleotide substitutions but no intervening sequences in comparison to the CSRP2 cDNA sequence. By physical linkage and fluorescence in situ hybridization, CSRP2P was mapped to chromosome subband 3q21.1.

Proteins of the cysteine-rich protein (CRP) family (CRP1, CRP2, and CRP3) are implicated in diverse processes linked to cellular differentiation and growth control. CRP proteins contain two LIM domains, each formed by two zinc-binding modules of the CCHC and CCCC type, respectively. The solution structure of the carboxyl-terminal LIM domain (LIM2) from recombinant quail CRP2 was determined by multidimensional homo- and heteronuclear magnetic resonance spectroscopy. The folding topology retains both independent zinc binding modules (CCHC and CCCC). Each module consists of two orthogonally arranged antiparallel beta-sheets, and the carboxyl-terminal CCCC module is terminated by an alpha-helix. 15N magnetic relaxation data indicate that the modules differ in terms of conformational flexibility. They pack together via a hydrophobic core region. In addition, Arg122 in the CCHC module and Glu155 in the CCCC module are linked by an intermodular hydrogen bond and/or salt bridge. These residues are absolutely conserved in the CRP family of LIM proteins, and their interaction might contribute to the relative orientation of the two zinc-binding modules in CRP LIM2 domains. The global fold of quail CRP2 LIM2 is very similar to that of the carboxyl-terminal LIM domain of the related but functionally distinct CRP family member CRP1, analyzed recently. The carboxyl-terminal CCCC module is structurally related to the DNA-binding domain of the erythroid transcription factor GATA-1. In the two zinc-binding modules of quail CRP2 LIM2, flexible loop regions made up of conserved amino acid residues are located on the same side of the LIM2 domain and may cooperate in macromolecular recognition.

The myc oncogene was originally identified as a transduced allele (v-myc) in the genome of a highly oncogenic avian retrovirus. The protein product (Myc) of the cellular c-myc protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. Myc regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of c-myc leading to increased levels of the Myc protein is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to monitor and quantify the oncogenic potential of myc alleles, and to isolate and characterize transcriptional targets of Myc that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of myc and to analyze structure and function of the ancestral myc protooncogenes.

MC29 avian myelocytomatosis virus is an acute leukemia virus and the prototype of the MC29-subgroup of the avian leukosis-sarcoma genus of type C retroviruses. It causes myelocytomatosis, endotheliomas, liver and kidney carcinomas, and sarcomas in chickens. Biochemical and genetic analyses of the RNA genome of MC29 led to the discovery of the myc oncogene. The MC29 genome contains partial complements of the essential virion genes gag and env , and v- myc as the transforming principle. The single protein product of MC29, p110 gag-myc , is a hybrid translational product of the partial gag gene and the v- myc sequences. The retroviral v- myc oncogene is a transduced mutant allele of the cellular protooncogene c- myc . The protein product of c- myc (Myc) is a bHLH-Zip protein, forms heterodimers with the bHLH-Zip protein Max, binds to specific DNA sequence elements (E-boxes), and is the central part of a transcription factor network controlling fundamental cellular processes like growth, metabolism, proliferation, differentiation, and apoptosis. Mutation and deregulation of c- myc is a frequent event in human tumorigenesis, occurring in about 30% of all human cancers. Myc and Max proteins were identified in early diploblastic organisms, suggesting that the principal functions of the Myc master regulator arose very early in metazoan evolution.

We have analyzed differential gene expression in normal versus jun-transformed avian fibroblasts by using subtracted nucleic acid probes and differential nucleic acid hybridization techniques for the isolation of cDNA clones. One clone corresponded to a gene that was strongly expressed in a previously established quail (Coturnix japonica) embryo fibroblast line (VCD) transformed by a chimeric jun oncogene but whose expression was undetectable in normal quail embryo fibroblasts. Furthermore, the gene was expressed in quail or chicken fibroblast cultures that were freshly transformed by retroviral constructs carrying various viral or cellular jun alleles and in chicken fibroblasts transformed by the avian retrovirus ASV17 carrying the original viral v-jun allele. However, its expression was undetectable in a variety of established avian cell lines or freshly prepared avian fibroblast cultures transformed by other oncogenes or a chemical carcinogen. The nucleotide and deduced amino acid sequences of the cDNA clone were not identical to any sequence entries in the data bases but revealed significant similarities to avian beta-keratin genes; the highest degree of amino acid sequence identity was 63%. The gene, which we termed bkj, may represent a direct or indirect target for jun function.

A chimeric construct (VCD) consisting of parts from viral jun, chicken c-jun, and chicken junD was cloned into the replication-competent retroviral RCAS vector. This construct, RCAS-VCD, was found to have a higher focus forming potential in quail fibroblasts than the equivalent construct RCAS-VJ-1, expressing viral jun. DNAs from RCAS-VCD and RCAS-VJ-1 were transfected into primary quail embryo fibroblasts. Cells derived from one RCAS-VCD-induced focus survived cell crisis, which became manifest after 15 passages, and could be expanded into a long-term culture. This cell line, termed VCD, has been passaged for over 100 times so far. The cells grow to very high densities and then pile up into clumps of rounded cells. The culture releases a transforming virus with a titer of 105 FFU/ml, as tested on primary quail embryo fibroblasts. The transformed phenotype of VCD cells was verified by agar colony formation. VCD cells are capable of anchorage-independent growth with a cloning efficiency of 10%. Southern blot analysis of genomic DNA from VCD cells showed proviral integration of the RCAS construct without detectable rearrangements. Northern and Western blot analyses confirmed correct expression from integrated RCAS-VCD of predicted RNAs and of the chimeric Jun(VCD) protein. jun (VCD)-transformed cells provide a constant source of homogeneous cellular material for the investigation of the molecular mechanisms of jun -induced cell transformation and for the identification of direct and indirect targets of Jun protein function.

Myc binds to a 6 bp 2-fold symmetric DNA site: 5'-C-3A-2C-1G+1T+2G+3-3'. Using site-specific 5-bromouracil mediated photocrosslinking, we show that His336 of Myc contacts, or is close to, the thymine 5-methyl group at 2-fold symmetry-related positions -2 and +2 of the DNA site in the Myc-DNA complex. Our results strongly suggest that homologous amino acids of Myc and Max make equivalent contacts in the respective protein-DNA complexes.

LIM domain proteins are implicated in fundamental developmental processes, and the functional hallmark of all LIM domains is presumably the mediation of protein–protein interactions. Although originally identified in LIM-homeodomain proteins, LIM domains have now been identified in a variety of proteins that lack homeodomains, either being entirely composed of LIM domains or containing additional functional domains of various types (e.g. kinase domains). The LIM domain contains a distinct cysteine-rich motif that was first identified in the protein products of three genes – lin-1, isl1, and mec-3. The LIM motif is composed of two zinc finger structures separated by a two-amino acid spacer and comprises the conserved sequence CX2CX16 − 23HX2CX2CX2CX16 − 21CX2(C/H/D). 3D Structure Keywords: protein–protein interaction module; multidomain protein; cell differentiation; transcription factor activation; zinc finger; NMR spectroscopy

Siderocalins are particular lipocalins that participate in the innate immune response by interfering with bacterial siderophore-mediated iron uptake. Additionally, siderocalins are involved in several physiological and pathological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. Here we show that siderocalin Q83 displays an unexpected dual ligand binding mode as it can bind enterobactin and unsaturated fatty acids simultaneously. The solution structure of the siderocalin Q83 in complex with arachidonic acid and enterobactin reveals molecular details of this novel dual binding mode and the determinants of fatty acid binding specificity. Our results suggest that Q83 is a metabolic hub linking iron and fatty acid pathways. This unexpected coupling might contribute to the pleiotropic functions of siderocalins.

Osteopontin (OPN) is an acidic hydrophilic glycophosphoprotein that was first identified as a major sialoprotein in bones. It functions as a cell attachment protein displaying a RGD cell adhesion sequence and as a cytokine that signals through integrin and CD44 cell adhesion molecules. OPN is also implicated in human tumor progression and cell invasion. OPN has intrinsic transforming activity, and elevated OPN levels promote metastasis. OPN gene expression is also strongly activated in avian fibroblasts simultaneously transformed by the v-myc and v-mil(raf) oncogenes. Here we have investigated the solution structure of a 220-amino acid recombinant OPN protein by an integrated structural biology approach employing bioinformatic sequence analysis, multidimensional nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism spectroscopy, and small-angle X-ray scattering. These studies suggest that OPN is an intrinsically unstructured protein in solution. Although OPN does not fold into a single defined structure, its conformational flexibility significantly deviates from random coil-like behavior. OPN comprises distinct local secondary structure elements with reduced conformational flexibility and substantially populates a compact subspace displaying distinct tertiary contacts. These compacted regions of OPN encompass the binding sites for α(V)β(III) integrin and heparin. The conformational flexibility combined with the modular architecture of OPN may represent an important structural prerequisite for its functional diversity.

One of the most important scientific discoveries in the recent past concerns RNA interference (RNAi), which is a post-transcriptional gene-silencing mechanism induced by small interfering RNA (siRNA) and micro-RNA (miRNA).[1] RNAi has opened up new avenues in the development of siRNA and miRNA as therapeutic agents for various diseases.[2] The reason for the large number of reports about chemically modified siRNA is their potential to enhance nuclease resistance, to prevent immune activation, to decrease off-target effects, and to improve pharmacokinetic and pharmacodynamic properties, all of which are important for the application of siRNA as therapeutic agents.[3] Another substantial challenge is siRNA delivery, because these reagents cannot easily traverse cell membranes because of their size and negative charge.[4] To date, the most promising therapeutic approach based on RNAi involves chemically modified siRNA that can resolve some of the issues mentioned above.

Siderocalins are atypical lipocalins able to capture siderophores with high affinity. They contribute to the innate immune response by interfering with bacterial siderophore-mediated iron uptake but are also involved in numerous physiological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. The Q83 lipocalin was originally identified based on its overexpression in quail embryo fibroblasts transformed by the v-myc oncogene. We show here that Q83 is a siderocalin, binding the siderophore enterobactin with an affinity and mode of binding nearly identical to that of neutrophil gelatinase-associated lipocalin (NGAL), the prototypical siderocalin. This strengthens the role of siderocalins in cancer progression and inflammation. In addition, we also present the solution structure of Q83 in complex with intact enterobactin and a detailed analysis of the Q83 binding mode, including mutagenesis of the critical residues involved in enterobactin binding. These data provide a first insight into the molecular details of siderophore binding and delineate the common molecular properties defining the siderocalin protein family.

The c- myc protooncogene encodes a transcription factor (Myc) with oncogenic potential. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins controlling fundamental cellular processes. Deregulation of c- myc leads to tumorigenesis and is a hallmark of many human cancers. We have identified and extensively characterized ancestral forms of myc and max genes from the early diploblastic cnidarian Hydra , the most primitive metazoan organism employed so far for the structural, functional, and evolutionary analysis of these genes. Hydra myc is specifically activated in all stem cells and nematoblast nests which represent the rapidly proliferating cell types of the interstitial stem cell system and in proliferating gland cells. In terminally differentiated nerve cells, nematocytes, or epithelial cells, myc expression is not detectable by in situ hybridization. Hydra max exhibits a similar expression pattern in interstitial cell clusters. The ancestral Hydra Myc and Max proteins display the principal design of their vertebrate derivatives, with the highest degree of sequence identities confined to the bHLH-Zip domains. Furthermore, the 314-amino acid Hydra Myc protein contains basic forms of the essential Myc boxes I through III. A recombinant Hydra Myc/Max complex binds to the consensus DNA sequence CACGTG with high affinity. Hybrid proteins composed of segments from the retroviral v-Myc oncoprotein and the Hydra Myc protein display oncogenic potential in cell transformation assays. Our results suggest that the principal functions of the Myc master regulator arose very early in metazoan evolution, allowing their dissection in a simple model organism showing regenerative ability but no senescence.

Myc proteins have a tripartite carboxyl-terminal domain containing specific amino acid sequence motifs: a basic motif, a helix-loop-helix motif, and a leucine heptad repeat. Similar sequence motifs have been identified in several eukaryotic transcription factors and were shown to facilitate protein-DNA and protein-protein interactions. By using recombinant v-Myc proteins obtained by bacterial expression of full-length or partially deleted avian v-myc alleles, the functional relevance of these sequence motifs for Myc protein oligomerization and for DNA binding was investigated. All recombinant v-Myc proteins that have retained the carboxyl-terminal domain dimerize and specifically bind to double-stranded DNA containing the palindromic core sequence CACGTG. This and a closely related DNA sequence element have been defined previously as part of the binding sites for human transcription factors USF and TFE3, which specifically bind to the adenovirus major late promoter or the muE3 motif within the immunoglobulin heavy-chain enhancer, respectively. It is shown that a 61-amino-acid peptide sequence containing only the bipartite basic motif/helix-loop-helix domain of Myc is necessary and sufficient for dimerization and sequence-specific DNA binding of v-Myc recombinant proteins.

We have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. KJ-Pyr-9 functions as an inhibitor of MYC in biochemical assays, in cells and in xenograft experiments. In vitro, KJ-Pyr-9 binds to MYC as well as MYC-MAX dimers and can disrupt binding of MYC-MAX dimers with E-box DNA. In cells, MYC-MAX dimerization can be disrupted in cells as demonstrated by protein fragment complementation assays. KJ-Pyr-9 disrupts the transcriptional targets of MYC. The P493-6 cell line has tetracycline regulatable MYC expression. For this cell line, the transcriptional targets of MYC can be readily observed upon the addition of 0.1 ug/mL doxycycline. Both doxycycline and KJ-Pyr-9 negatively regulate MYC transcriptional signatures as determined by GSEA. The growth of a variety of different MYC-dependent cell lines is inhibited, including MYC-translocated Burkitt's lymphoma cell lines expressing high levels of MYC as well as solid tumor cell lines expressing moderate amounts of MYC. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells. Acknowledgements: This work was supported by the National Cancer Institute under award number R01 CA078230 (PKV), The Skaggs Institute for Chemical Biology (KDJ) and by the Austrian Science Fund (FWF) grant P23652 (KB). This is manuscript 25089 of The Scripps Research Institute. Citation Format: Jonathan R. Hart, Klaus Bister, Kim D. Janda, Peter K. Vogt. Identification of KJ-Pyr-9 as a potent MYC inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A23.

We have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. KJ-Pyr-9 functions as an inhibitor of MYC in biochemical assays, in cells and in xenograft experiments. In vitro, KJ-Pyr-9 binds to MYC as well as MYC-MAX dimers and can disrupt binding of MYC-MAX dimers with E-box DNA. In cells, MYC-MAX dimerization can be disrupted in cells as demonstrated by protein fragment complementation assays. KJ-PYR-9 disrupts the transcriptional targets of MYC. The P493-6 cell line has tetracycline regulatable MYC expression. For this cell line, the transcriptional targets of MYC can be readily observed upon the addition of 0.1 ug/mL doxycycline. Both doxycycline and KJ-PYR-9 negatively regulate MYC transcriptional signatures as determined by GSEA. The growth of a variety of different MYC-dependent cell lines are inhibited, including MYC translocated Burkitt's lymphoma cell lines expressing high levels of MYC as well as solid tumor cell lines expressing moderate amounts of MYC. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells. Citation Format: Jonathan R. Hart, Klaus Bister, Kim D. Janda, Peter K. Vogt. Identification of KJ-Pyr-9 as a potent MYC inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2015-965

Using differential hybridization techniques, a cDNA clone ( Q83 ) was isolated that corresponds to a highly abundant mRNA in quail embryo fibroblasts transformed by the v- myc oncogene. The deduced 178 amino acid protein product of Q83 contains an N-terminal signal sequence and a lipocalin sequence motif, the hallmark of a family of secretory proteins binding and transporting small hydrophobic molecules of diverse biological function, including retinoids and steroids. The quail Q83 protein displays 87% sequence identity with a developmentally regulated chicken protein, termed p20K or Ch21. Cell transformation specifically by v- myc , but not by other oncogenic agents, induces high-level expression of Q83 mRNA and of the Q83 protein. Nucleotide sequence analysis and transcriptional mapping revealed that the Q83 gene encompasses seven exons with the coding region confined to exons 1 through 6. The promoter region contains consensus binding sites for the transcriptional regulators Myc and C/EBPβ. Transcriptional activation of Q83 is principally dependent on C/EBPβ, but is blocked in normal cells by the endogenous c-Myc/Max/Mad transcription factor network. In v- myc -transformed cells, high-level expression of the v-Myc protein and formation of highly stable v-Myc/Max heterodimers leads to abrogation of Q83 gene suppression and activation by C/EBPβ. A 157 amino acid residue recombinant protein representing the secreted form of Q83 was used for structure determination by nuclear magnetic resonance spectroscopy. Q83 folds into a single globular domain of the lipocalin-type. The central part consists of an eight-stranded up-and-down β-barrel core flanked by an N-terminal 3 10 -like helix and a C-terminal α-helix. The orientation of the C-terminal α-helix is partially determined by a disulfide bridge between Cys59 and Cys152. The three-dimensional structure determination of the Q83 protein will facilitate the identification of its authentic ligand and the assessment of its biological function, including the putative role in myc -induced cell transformation.

Using subtractive hybridization techniques, we have isolated a gene termed JAC that is strongly and specifically activated in avian fibroblasts transformed by the v- jun oncogene of avian sarcoma virus 17 (ASV17), but not in cells transformed by other oncogenic agents. Furthermore, JAC is highly expressed in cell lines derived from jun -induced avian fibrosarcomas. Kinetic analysis using a doxycycline-controlled conditional cell transformation system showed that expression of the 0.8-kb JAC mRNA is induced rapidly upon activation of the oncogenic v- jun allele. Nucleotide sequence analysis and transcriptional mapping revealed that the JAC gene contains two exons, with the longest ORF confined to exon 2. The deduced 68-amino acid chicken JAC protein is rich in cysteine residues and displays 37% sequence identity to mammalian high-sulfur keratin-associated proteins. The promoter region of JAC contains a consensus (5′-TGACTCA-3′) and a nonconsensus (5′-TGAGTAA-3′) AP-1 binding site in tandem, which are both specifically bound by the Gag-Jun hybrid protein encoded by ASV17. Mutational analysis revealed that the two AP-1 sites confer strong transcriptional activation by Gag-Jun in a synergistic manner. Ectopic expression of JAC in avian fibroblasts leads to anchorage-independent growth, strongly suggesting that deregulation of JAC is an essential event in jun -induced cell transformation and tumorigenesis.

Using the established quail cell line Q/d3 conditionally transformed by the v-jun oncogene, cDNA clones (TOJ2, TOJ3, TOJ5, TOJ6) were isolated by representational difference analysis (RDA) that correspond to genes which were induced immediately upon conditional activation of v-jun. One of these genes, TOJ3, is immediately and specifically activated after doxycycline-mediated v-jun induction, with kinetics similar to the induction of well characterized direct AP-1 target genes. TOJ3 is neither activated upon conditional activation of v-myc, nor in cells or cell lines non-conditionally transformed by oncogenes other than v-jun. Sequence analysis revealed that the TOJ3-specific cDNA encodes a 530-amino acid protein with significant sequence similarities to the murine or human microspherule protein 1 (MCRS1, MSP58), a nucleolar protein that directly interacts with the ICP22 regulatory protein from herpes simplex virus 1 or with p120, a proliferation-related protein expressed at high levels in most human malignant tumor cells. Similar to its mammalian counterparts, the TOJ3 protein contains a bipartite nuclear localization motif and a forkhead associated domain (FHA). Using polyclonal antibodies directed against a recombinant amino-terminal TOJ3 protein segment, the activation of TOJ3 in jun-transformed fibroblasts was also demonstrated at the protein level by specific detection of a polypeptide with an apparent M(r) of 65 000. Retroviral expression of the TOJ3 gene in quail or chicken embryo fibroblasts induces anchorage-independent growth, indicating that the immediate activation of TOJ3 in fibroblasts transformed by the v-jun oncogene contributes to cell transformation.

To investigate the molecular basis of oncogenesis induced by the v-jun oncogene of avian sarcoma virus 17 (ASV17), we developed a conditional cell transformation system in which transcription of the ASV17 v-jun allele is controlled by a doxycycline-sensitive transactivator (tTA) or a reverse (doxycycline-dependent) transactivator (rtTA), respectively. Permanent cell lines of quail embryo fibroblasts conditionally transformed by a doxycycline-controlled v-jun allele revert to the normal phenotype within 3 days and lose their ability to grow in soft agar, strictly dependent on the addition or removal of the drug, respectively. The reverted cells are rapidly retransformed on conditional activation of v-jun. While full-level synthesis of v-jun mRNA and v-Jun protein in these cells is established within 2 and 14 h, respectively, after switching to the permissive conditions, the first morphological alterations are observed after 24 h, and as early as 2 days later the morphology has changed entirely from flat cells resembling normal fibroblasts to spindle-shaped fusiform cells showing a typical jun-transformed phenotype. Kinetic expression analysis revealed that transcriptional activation of the direct jun target gene BKJ precisely coincides with the establishment of full-level v-Jun protein synthesis. Furthermore, we have analyzed the expression of a novel candidate v-jun target gene, termed JAC, which shows no sequence homology to known genes. Similar to BKJ, JAC is specifically activated in jun-transformed fibroblasts, and induction of JAC is tightly linked to the conditional expression of oncogenic v-Jun. These results demonstrate the high stringency of the doxycycline-controlled v-jun expression system, and they also indicate that expression of v-jun in these cells is indispensable for enhanced proliferation, cell transformation, and the induction of specific expression patterns of downstream target genes.

To investigate the molecular basis of oncogenesis induced by the v- myc oncogene of avian myelocytomatosis virus MC29, we developed a conditional cell transformation system in which expression of the MC29 v- myc allele is dependent on a doxycycline-sensitive transactivator (tTA). Clonal lines of quail embryo fibroblasts transformed by doxycycline-controlled v- myc revert to the normal phenotype and lose their ability to grow in soft agar after the addition of doxycycline. Repression of v- myc causes the cells to withdraw from the cell cycle, and long-term survival in culture requires reexpression of v- myc . Although complete repression of v- myc mRNA and v-Myc protein in these cells occurs within 14 h after the addition of doxycycline, the first morphological alterations are observed after 24 h, and after 3 days, the morphology changed entirely from small rounded cells showing a typical myc -transformed phenotype to large flat cells resembling normal fibroblasts. Cells exposed to doxycycline for 3 days reexpressed v- myc within 24 h after withdrawal of the drug from the culture medium, partial retransformation occurred after 2 days, and complete morphological transformation was reestablished after 6 days. Analogous results were obtained with a cell line in which expression of the v- myc allele is dependent on a reverse transactivator (rtTA) that is activated by doxycycline. The striking differential expression of known transformation-sensitive genes and of new candidate v- myc target genes revealed the tightness of the doxycycline-controlled v- myc expression system. The data also indicate that expression of v- myc in these cells is indispensable for enhanced proliferation, transformation, and immortalization.

Gene expression patterns in normal and v-myc-transformed quail embryo fibroblasts were compared by mRNA differential display. Displaying approximately 2500 mRNA species by reverse transcription/PCR, reamplification of 73 differential cDNA fragments and rescreening by Northern analysis led to the isolation of a clone, termed CO6, that hybridized to an mRNA species present only in the normal but not in the transformed fibroblasts. Further analyses revealed that the 0.95-kb CO6 mRNA was present in all normal quail and chicken embryo fibroblasts tested, but that it was undetectable in a variety of established quail cell lines transformed by the v-myc, v-myc/v-mil, v-jun/junD or v-src oncogenes or by a chemical carcinogen. Furthermore, CO6 mRNA was not detectable in fibroblasts newly transformed by retroviral constructs carrying v-myc or v-jun alleles or by the avian sarcoma virus ASV17. In fibroblasts transformed by a temperature-sensitive v-src mutant, expression of CO6 was strongly induced at the non-permissive temperature and reduced at the permissive temperature. Nucleotide sequence analysis of quail CO6 cDNA indicated that the corresponding gene encodes a 200-amino acid protein with 46 to 48% amino acid sequence identity to the regulatory beta subunits (K(VCa)beta) of the bovine, human and canine high conductance Ca2+-activated K+ channels. No sequence homology to other ion channel subunits or to any other proteins in the databases was found. Like the K(VCa)beta subunits, the CO6 protein contains two putative transmembrane segments. Based on the relationship to mammalian K(VCa)beta both in primary structure and domain topology, the CO6 protein may represent the regulatory subunit of a yet unidentified avian Ca2+-activated potassium channel or a related membrane protein possibly involved in the regulation of cell proliferation.