Markus Hartl

Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC, and silenced BASP1 , we generated stable cell lines overexpressing BASP1 , either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR-based system. BASP1 encodes a neuronal signaling protein and transcriptional corepressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.

The proto-oncogene myc has been intensively studied primarily in vertebrate cell culture systems. Myc transcription factors control fundamental cellular processes such as cell proliferation, cell cycle control and stem cell maintenance. Myc interacts with the Max protein and Myc/Max heterodimers regulate thousands of target genes. The genome of the freshwater polyp Hydra encodes four myc genes (myc1-4). Previous structural and biochemical characterization showed that the Hydra Myc1 and Myc2 proteins share high similarities with vertebrate c-Myc, and their expression patterns suggested a function in adult stem cell maintenance. In contrast, an additional Hydra Myc protein termed Myc3 is highly divergent, lacking the common N-terminal domain and all conserved Myc-boxes. Single cell transcriptome analysis revealed that the myc3 gene is expressed in a distinct population of interstitial precursor cells committed to nerve- and gland-cell differentiation, where the Myc3 protein may counteract the stemness actions of Myc1 and Myc2 and thereby allow the implementation of a differentiation program. In vitro DNA binding studies showed that Myc3 dimerizes with Hydra Max, and this dimer efficiently binds to DNA containing the canonical Myc consensus motif (E-box). In vivo cell transformation assays in avian fibroblast cultures further revealed an unexpected high potential for oncogenic transformation in the conserved Myc3 C-terminus, as compared to Hydra Myc2 or Myc1. Structure modeling of the Myc3 protein predicted conserved amino acid residues in its bHLH-LZ domain engaged in Myc3/Max dimerization. Mutating these amino acid residues in the human c-Myc (MYC) sequence resulted in a significant decrease in its cell transformation potential. We discuss our findings in the context of oncogenic transformation and cell differentiation, both relevant for human cancer, where Myc represents a major driver.

The MYC oncoprotein functions as a master regulator of cellular transcription and executes non-transcriptional tasks relevant to DNA replication and cell cycle regulation, thereby interacting with multiple proteins. MYC is required for fundamental cellular processes triggering proliferation, growth, differentiation, or apoptosis and also represents a major cancer driver being aberrantly activated in most human tumors. Due to its non-enzymatic biochemical functions and largely unstructured surface, MYC has remained difficult for specific inhibitor compounds to directly address, and consequently, alternative approaches leading to indirect MYC inhibition have evolved. Nowadays, multiple organic compounds, nucleic acids, or peptides specifically interfering with MYC activities are in preclinical or early-stage clinical studies, but none of them have been approved so far for the pharmacological treatment of cancer patients. In addition, specific and efficient delivery technologies to deliver MYC-inhibiting agents into MYC-dependent tumor cells are just beginning to emerge. In this review, an overview of direct and indirect MYC-inhibiting agents and their modes of MYC inhibition is given. Furthermore, we summarize current possibilities to deliver appropriate drugs into cancer cells containing derailed MYC using viral vectors or appropriate nanoparticles. Finding the right formulation to target MYC-dependent cancers and to achieve a high intracellular concentration of compounds blocking or attenuating oncogenic MYC activities could be as important as the development of novel MYC-inhibiting principles.

Azides are versatile bioorthogonal reporter moieties that are commonly used for site-specific labeling and functionalization of RNA to probe its biology. The preparation of azido modified nucleic acids by solid-phase synthesis is problematic due to the inherent reactivity of P(III) species with azides according to the Staudinger reaction. Various strategies have been developed to bypass this limitation and are often time-consuming, low-yielding and labor-intensive. In particular, the synthesis of RNA with internal 2′-azido modifications is restricted to a single approach that employs P(V) chemistry instead of the widely used P(III) phosphoramidite chemistry. To fill this methodological gap, we present a novel convenient path toward 2′-azido RNA from readily accessible 2′-amino RNA through treatment with the diazotizing reagent fluorosulfuryl azide (FSO₂N₃). A diazotransfer reaction was established for oligoribonucleotides of different lengths and secondary structures. The robustness of the approach was further demonstrated for RNAs containing multiple 2′-azido moieties and for RNAs containing other sensitive modifications such as thiouridine or methylated nucleobases with a positive charge. The synthetic ease of generating 2′-azido RNA will pave the way for biotechnological applications, in particular for siRNA technologies and for referencing the growing number of RNA metabolic labeling approaches that rely on 2′-azido nucleosides.

Background: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. Results: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. Conclusions: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

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 c-Myc protein (MYC) is a transcription factor with strong oncogenic potential controlling fundamental cellular processes. In most human tumors, MYC is overexpressed by enhanced transcriptional activation, gene amplification, chromosomal rearrangements, or increased protein stabilization. To pharmacologically suppress oncogenic MYC functions, multiple approaches have been applied either to inhibit transcriptional activation of the endogenous MYC gene, or to interfere with biochemical functions of aberrantly activated MYC. Other critical points of attack are targeted protein modification, or destabilization leading to a non-functional MYC oncoprotein. It has been claimed that the natural compound curcumin representing the principal curcumoid of turmeric (Curcuma longa) has anticancer properties although its specificity, efficacy, and the underlying molecular mechanisms have been controversially discussed. Here, we have tested curcumin’s effect on MYC-dependent cell transformation and transcriptional activation, and found that this natural compound interferes with both of these MYC activities. Furthermore, in curcumin-treated cells, the endogenous 60-kDa MYC protein is covalently and specifically cross-linked to one of its transcriptional interaction partners, namely the 434-kDa transformation/transcription domain associated protein (TRRAP). Thereby, endogenous MYC levels are strongly reduced and cells stop to proliferate. TRRAP is a multidomain adaptor protein of the phosphoinositide 3-kinase-related kinases (PIKK) family and represents an important component of many histone acetyltransferase (HAT) complexes. TRRAP is important to mediate transcriptional activation executed by the MYC oncoprotein, but on the other hand TRRAP also negatively regulates protein stability of the tumor suppressor p53 (TP53). Curcumin-mediated covalent binding of MYC to TRRAP reduces the protein amounts of both interaction partners but does not downregulate TP53, so that the growth-arresting effect of wild type TP53 could prevail. Our results elucidate a molecular mechanism of curcumin action that specifically and irreversibly targets two crucial multifunctional cellular players. With regard to their broad impact in cancer, our findings contribute to explain the pleiotropic functions of curcumin, and suggest that this natural spice, or more bioavailable derivatives thereof, may constitute useful adjuvants in the therapy of MYC-dependent and TRRAP-associated human tumors.

Significance: The pharmaceutical targeting of mutated BRAF has shown promising clinical outcomes in patients with melanoma. However, more than 300 reported BRAF patient mutations and the occurrence of a kinase-drug resistance mechanism hamper patient-oriented therapies. We have developed an extendable and cell-based kinase conformation reporter platform (KinCon) to predict and compare the effect of kinase drugs. We systematically evaluated drug efficacies by assessing KinCon reporter dynamics in response to different drugs and patient mutations. Our findings suggest that FDA-approved melanoma inhibitors may have the potential to block non-V600E–mutated BRAF activities also in non–small-cell lung cancers (NSCLC). We assume that widespread analyses of KinCon:drug interactions may assist in the future in identifying patient mutation–specific and thus more effective kinase inhibitors.