MYC-Mediated Inhibition of ARNT2 Uncovers a Key Tumor Suppressor in Glioblastoma.

Tumor initiation and progression rely on intricate cellular pathways that promote proliferation while suppressing differentiation, yet the importance of pathways inhibiting differentiation in cancer remains incompletely understood. Here, we reveal a novel mechanism centered on the repression of the neuronal-specific transcription factor ARNT2 by the MYC oncogene that governs the balance between proliferation and differentiation. We found that MYC coordinates the transcriptional repression of ARNT2 through the activity of polycomb repressive complex 2 (PRC2). Notably, ARNT2, highly and specifically expressed in the central nervous system, is diminished in glioblastoma, inversely correlating with patient survival. Utilizing in vitro and in vivo models, we demonstrate that ARNT2 knockout (KO) exerts no discernible effect on the in vitro proliferation of glioblastoma cells, but significantly enhances the growth of glioblastoma cells in vivo. Conversely, ARNT2 overexpression severely dampens the growth of fully transformed glioblastoma cells subcutaneously or orthotopically xenografted in mice. Mechanistically, ARNT2 depletion diminishes differentiation and enhances stemness of glioblastoma cells. Our findings provide new insights into the complex mechanisms used by oncogenes to limit differentiation in cancer cells and define ARNT2 as a tumor suppressor in glioblastoma.

Tryptophan fuels MYC-dependent liver tumorigenesis through indole 3-pyruvate synthesis.

Cancer cells exhibit distinct metabolic activities and nutritional dependencies compared to normal cells. Thus, characterization of nutrient demands by individual tumor types may identify specific vulnerabilities that can be manipulated to target the destruction of cancer cells. We find that MYC-driven liver tumors rely on augmented tryptophan (Trp) uptake, yet Trp utilization to generate metabolites in the kynurenine (Kyn) pathway is reduced. Depriving MYC-driven tumors of Trp through a No-Trp diet not only prevents tumor growth but also restores the transcriptional profile of normal liver cells. Despite Trp starvation, protein synthesis remains unhindered in liver cancer cells. We define a crucial role for the Trp-derived metabolite indole 3-pyruvate (I3P) in liver tumor growth. I3P supplementation effectively restores the growth of liver cancer cells starved of Trp. These findings suggest that I3P is a potential therapeutic target in MYC-driven cancers. Developing methods to target this metabolite represents a potential avenue for liver cancer treatment.

ZNF692 regulates nucleolar morphology by interacting with NPM1 and modifying its self-assembly properties.

The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells.

ZNF692 organizes a hub specialized in 40S ribosomal subunit maturation enhancing translation in rapidly proliferating cells.

Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.

The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of ß-catenin.

The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated ß-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of ß-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of ß-catenin. This article has an associated First Person interview with the first authors of the paper.

Induction of LEF1 by MYC activates the WNT pathway and maintains cell proliferation.

BACKGROUND: While regulated WNT activity is required for normal development and stem cell maintenance, mutations that lead to constitutive activation of the WNT pathway cause cellular transformation and drive colorectal cancer. Activation of the WNT pathway ultimately leads to the nuclear translocation of ß-catenin which, in complex with TCF/LEF factors, promotes the transcription of genes necessary for growth. The proto-oncogene MYC is one of the most critical genes activated downstream the WNT pathway in colon cancer. Here, we investigate the converse regulation of the WNT pathway by MYC. METHODS: We performed RNA-seq analyses to identify genes regulated in cells expressing MYC. We validated the regulation of genes in the WNT pathway including LEF1 by MYC using RT-qPCR, Western blotting, and ChIP-seq. We investigated the importance of LEF1 for the viability of MYC-expressing cells in in fibroblasts, epithelial cells, and colon cells. Bioinformatic analyses were utilized to define the expression of MYC-regulated genes in human colon cancer and metabolomics analyses were used to identify pathways regulated by LEF1 in MYC expressing cells. RESULTS: MYC regulates the levels of numerous WNT-related genes, including the ß-catenin co-transcription factor LEF1. MYC activates the transcription of LEF1 and is required for LEF1 expression in colon cancer cells and in primary colonic cells transformed by APC loss of function, a common mutation in colon cancer patients. LEF1 caused the retention of ß-catenin in the nucleus, leading to the activation of the WNT pathway in MYC-expressing cells. Consequently, MYC-expressing cells were sensitive to LEF1 inhibition. Moreover, we describe two examples of genes induced in MYC-expressing cells that require LEF1 activity: the peroxisome proliferator activated receptor delta (PPARδ) and the Acyl CoA dehydrogenase 9 (ACAD9). CONCLUSIONS: We demonstrated that MYC is a transcriptional regulator of LEF1 in colonic cells. Our work proposes a novel pathway by which MYC regulates proliferation through activating LEF1 expression which in turn activates the WNT pathway.

MYC promotes tryptophan uptake and metabolism by the kynurenine pathway in colon cancer.

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.

The aryl hydrocarbon receptor regulates nucleolar activity and protein synthesis in MYC-expressing cells.

MYC enhances protein synthesis by regulating genes involved in ribosome biogenesis and protein translation. Here, we show that MYC-induced protein translation is mediated by the transcription factor aryl hydrocarbon receptor (AHR), which is induced by MYC in colonic cells. AHR promotes protein synthesis by activating the transcription of genes required for ribosome biogenesis and protein translation, including OGFOD1 and NOLC1. Using surface sensing of translation (SUnSET) to measure global protein translation, we found that silencing AHR or its targets diminishes protein synthesis. Therefore, targeting AHR or its downstream pathways could provide a novel approach to limit biomass production in MYC-driven tumors.

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays.

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.

Characterization of a rabbit polyclonal antibody against threonine-AMPylation.

An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.