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.

Targeting of neuroblastoma cells through Kynurenine-AHR pathway inhibition.

Neuroblastoma poses significant challenges in clinical management. Despite its relatively low incidence, this malignancy contributes disproportionately to cancer-related childhood mortality. Tailoring treatments based on risk stratification, including MYCN oncogene amplification, remains crucial, yet high-risk cases often confront therapeutic resistance and relapse. Here, we explore the aryl hydrocarbon receptor (AHR), a versatile transcription factor implicated in diverse physiological functions such as xenobiotic response, immune modulation, and cell growth. Despite its varying roles in malignancies, AHR's involvement in neuroblastoma remains elusive. Our study investigates the interplay between AHR and its ligand kynurenine (Kyn) in neuroblastoma cells. Kyn is generated from tryptophan (Trp) by the activity of the enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2). We found that neuroblastoma cells displayed sensitivity to the TDO2 inhibitor 680C91, exposing potential vulnerabilities. Furthermore, combining TDO2 inhibition with retinoic acid or irinotecan (two chemotherapeutic agents used to treat neuroblastoma patients) revealed synergistic effects in select cell lines. Importantly, clinical correlation analysis using patient data established a link between elevated expression of Kyn-AHR pathway genes and adverse prognosis, particularly in older children. These findings underscore the significance of the Kyn-AHR pathway in neuroblastoma progression, emphasizing its potential role as a therapeutic target.

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.

Nucleolar stress: From development to cancer.

The nucleolus is a large nuclear membraneless organelle responsible for ribosome biogenesis. Ribosomes are cytoplasmic macromolecular complexes comprising RNA and proteins that link amino acids together to form new proteins. The biogenesis of ribosomes is an intricate multistep process that involves the transcription of ribosomal DNA (rDNA), the processing of ribosomal RNA (rRNA), and the assembly of rRNA with ribosomal proteins to form active ribosomes. Nearly all steps necessary for ribosome production and maturation occur in the nucleolus. Nucleolar shape, size, and number are directly linked to ribosome biogenesis. Errors in the steps of ribosomal biogenesis are sensed by the nucleolus causing global alterations in nucleolar function and morphology. This phenomenon, known as nucleolar stress, can lead to molecular changes such as stabilization of p53, which in turn activates cell cycle arrest or apoptosis. In this review, we discuss recent work on the association of nucleolar stress with degenerative diseases and developmental defects. In addition, we highlight the importance of de novo nucleotide biosynthesis for the enhanced nucleolar activity of cancer cells and discuss targeting nucleotide biosynthesis as a strategy to activate nucleolar stress to specifically target cancer cells.

Regulation of Nucleolar Activity by MYC.

The nucleolus harbors the machinery necessary to produce new ribosomes which are critical for protein synthesis. Nucleolar size, shape, and density are highly dynamic and can be adjusted to accommodate ribosome biogenesis according to the needs for protein synthesis. In cancer, cells undergo continuous proliferation; therefore, nucleolar activity is elevated due to their high demand for protein synthesis. The transcription factor and universal oncogene MYC promotes nucleolar activity by enhancing the transcription of ribosomal DNA (rDNA) and ribosomal proteins. This review summarizes the importance of nucleolar activity in mammalian cells, MYC's role in nucleolar regulation in cancer, and discusses how a better understanding (and the potential inhibition) of aberrant nucleolar activity in cancer cells could lead to novel therapeutics.

A novel role of MNT as a negative regulator of REL and the NF-κB pathway.

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix-loop-helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT-REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT-REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells.

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.

The transcription factors aryl hydrocarbon receptor and MYC cooperate in the regulation of cellular metabolism.

The transcription factor AHR (aryl hydrocarbon receptor) drives the expression of genes involved in detoxification pathways in cells exposed to pollutants and other small molecules. Moreover, AHR supports transcriptional programs that promote ribosome biogenesis and protein synthesis in cells stimulated to proliferate by the oncoprotein MYC. Thus, AHR is necessary for the proliferation of MYC-overexpressing cells. To define metabolic pathways in which AHR cooperates with MYC in supporting cell growth, here we used LC-MS-based metabolomics to examine the metabolome of MYC-expressing cells upon AHR knockdown. We found that AHR knockdown reduced lactate, S-lactoylglutathione, N-acetyl-l-alanine, 2-hydroxyglutarate, and UMP levels. Using our previously obtained RNA sequencing data, we found that AHR mediates the expression of the UMP-generating enzymes dihydroorotate dehydrogenase (quinone) (DHODH) and uridine monophosphate synthetase (UMPS), as well as lactate dehydrogenase A (LDHA), establishing a mechanism by which AHR regulates lactate and UMP production in MYC-overexpressing cells. AHR knockdown in glioblastoma cells also reduced the expression of LDHA (and lactate), DHODH, and UMPS but did not affect UMP levels, likely because of compensatory mechanisms in these cells. Our results indicate that AHR contributes to the regulation of metabolic pathways necessary for the proliferation of transformed cells.

The MNT transcription factor autoregulates its expression and supports proliferation in MYC-associated factor X (MAX)-deficient cells.

The MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the basic helix-loop-helix (bHLH) family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E-boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC-MAX dimer activates gene expression. MNT also binds to the MAX dimerization protein MLX (MLX), and MNT-MLX and MNT-MAX dimers co-exist. However, all MNT functions have been attributed to MNT-MAX dimers, and no functions of the MNT-MLX dimer have been described. MNT's biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT-MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E-boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-sequencing experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT-MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.

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.

NUMB inactivation confers resistance to imatinib in chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase, where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and its derivatives. NUMB is an evolutionary well-conserved protein initially described as a functional antagonist of NOTCH function. NUMB is an endocytic protein associated with receptor internalization, involved in multiple cellular functions. It has been reported that MSI2 protein, a NUMB inhibitor, is upregulated in CML blast crisis, whereas NUMB itself is downregulated. This suggest that NUMB plays a role in the malignant progression of CML. Here we have generated K562 cells (derived from CML in blast crisis) constitutively expressing a dominant negative form of NUMB (dnNUMB). We show that dnNUMB expression confers a high proliferative phenotype to the cells. Importantly, dnNUMB triggers a partial resistance to imatinib in these cells, antagonizing the apoptosis mediated by the drug. Interestingly, imatinib resistance is not linked to p53 status or NOTCH signaling, as K562 lack p53 and imatinib resistance is reproduced in the presence of NOTCH inhibitors. Taken together, our data support the hypothesis that NUMB activation could be a new therapeutic target in CML.