MYC Binding Near Transcriptional End Sites Regulates Basal Gene Expression, Read-Through Transcription and Intragenic Contacts.

The MYC oncoprotein regulates numerous genes involved in cellular processes such as cell cycle and mitochondrial and ribosomal structure and function. This requires heterodimerization with its partner, MAX, and binding to specific promoter and enhancer elements. Here, we show that MYC and MAX also bind near transcriptional end sites (TESs) of over one-sixth of all annotated genes. These interactions are dose-dependent, evolutionarily conserved, stabilize the normally short-lived MYC protein and regulate expression both in concert with and independent of MYC's binding elsewhere. MYC's TES binding occurs in association with other transcription factors, alters the chromatin landscape, increases nuclease susceptibility and can alter transcriptional read-through, particularly in response to certain stresses. MYC-bound TESs can directly contact promoters and may fine-tune gene expression in response to both physiologic and pathologic stimuli. Collectively, these findings support a previously unrecognized role for MYC in regulating transcription and its read-through via direct intragenic contacts between TESs and promoters.

Inhibition of DUSP18 impairs cholesterol biosynthesis and promotes anti-tumor immunity in colorectal cancer.

Tumor cells reprogram their metabolism to produce specialized metabolites that both fuel their own growth and license tumor immune evasion. However, the relationships between these functions remain poorly understood. Here, we report CRISPR screens in a mouse model of colo-rectal cancer (CRC) that implicates the dual specificity phosphatase 18 (DUSP18) in the establishment of tumor-directed immune evasion. Dusp18 inhibition reduces CRC growth rates, which correlate with high levels of CD8+ T cell activation. Mechanistically, DUSP18 dephosphorylates and stabilizes the USF1 bHLH-ZIP transcription factor. In turn, USF1 induces the SREBF2 gene, which allows cells to accumulate the cholesterol biosynthesis intermediate lanosterol and release it into the tumor microenvironment (TME). There, lanosterol uptake by CD8+ T cells suppresses the mevalonate pathway and reduces KRAS protein prenylation and function, which in turn inhibits their activation and establishes a molecular basis for tumor cell immune escape. Finally, the combination of an anti-PD-1 antibody and Lumacaftor, an FDA-approved small molecule inhibitor of DUSP18, inhibits CRC growth in mice and synergistically enhances anti-tumor immunity. Collectively, our findings support the idea that a combination of immune checkpoint and metabolic blockade represents a rationally-designed, mechanistically-based and potential therapy for CRC.

Body-Wide Inactivation of the Myc-Like Mlx Transcription Factor Network Accelerates Aging and Increases the Lifetime Cancer Incidence.

The "Mlx" and "Myc" transcription factor networks cross-communicate and share many common gene targets. Myc's activity depends upon its heterodimerization with Max, whereas the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. The current work demonstrates that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability, and aging. Whereas "MycKO" mice have an extended lifespan because of a lower cancer incidence, "MlxKO" mice have normal lifespans and a higher cancer incidence. Like Myc, the expression of Mlx, MondoA, and ChREBP and their control over their target genes deteriorate with age in both mice and humans. Collectively, these findings underscore the importance of lifelong and balanced cross-talk between the two networks to maintain proper function and regulation of the many factors that can affect normal aging.

The Myc-Like Mlx Network Impacts Aging and Metabolism.

The "Mlx" and "Myc" Networks share many common gene targets. Just as Myc's activity depends upon its heterodimerization with Max, the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. We show here that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability and aging. Whereas "MycKO" mice have an extended lifespan because of a lower cancer incidence, "MlxKO" mice have normal lifespans and a somewhat higher cancer incidence. Like Myc, Mlx, MondoA and ChREBP expression and that of their target genes, deteriorate with age in both mice and humans, underscoring the importance of life-long and balanced cross-talk between the two Networks to maintain normal aging.

Cholesterol esterification and p53-mediated tumor suppression.

Many human cancers carry missense mutations in or deletions of the tumor protein 53 (TP53) tumor suppressor gene. TP53's product, p53 regulates many biological processes, including cell metabolism. Cholesterol is a key lipid needed for the maintenance of membrane function and tissue homeostasis while also serving as a precursor for steroid hormone and bile acid synthesis. An over-abundance of cholesterol can lead to its esterification and storage as cholesterol esters. The recent study has shown that the loss of p53 leads to excessive cholesterol ester biosynthesis, which promotes hepatocellular carcinoma in mice. Blocking cholesterol esterification improves treatment outcomes, particularly for liver cancers with p53 deletions/mutations that originate in a background of non-alcoholic fatty liver disease.

A Novel Transgenic Mouse Model Implicates Sirt2 as a Promoter of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths globally. Incidence rates are steadily increasing, creating an unmet need for new therapeutic options. Recently, the inhibition of sirtuin-2 (Sirt2) was proposed as a potential treatment for HCC, despite contradictory findings of its role as both a tumor promoter and suppressor in vitro. Sirt2 functions as a lysine deacetylase enzyme. However, little is known about its biological influence, despite its implication in several age-related diseases. This study evaluated Sirt2's role in HCC in vivo using an inducible c-MYC transgene in Sirt2+/+ and Sirt2-/- mice. Sirt2-/- HCC mice had smaller, less proliferative, and more differentiated liver tumors, suggesting that Sirt2 functions as a tumor promoter in this context. Furthermore, Sirt2-/- HCCs had significantly less c-MYC oncoprotein and reduction in c-MYC nuclear localization. The RNA-seq showed that only three genes were significantly dysregulated due to loss of Sirt2, suggesting the underlying mechanism is due to Sirt2-mediated changes in the acetylome, and that the therapeutic inhibition of Sirt2 would not perturb the oncogenic transcriptome. The findings of this study suggest that Sirt2 inhibition could be a promising molecular target for slowing HCC growth.

Lessons in aging from Myc knockout mouse models.

Despite MYC being among the most intensively studied oncogenes, its role in normal development has not been determined as Myc-/- mice do not survival beyond mid-gestation. Myc ± mice live longer than their wild-type counterparts and are slower to accumulate many age-related phenotypes. However, Myc haplo-insufficiency likely conceals other important phenotypes as many high-affinity Myc targets genes continue to be regulated normally. By delaying Myc inactivation until after birth it has recently been possible to study the consequences of its near-complete total body loss and thus to infer its normal function. Against expectation, these "MycKO" mice lived significantly longer than control wild-type mice but manifested a marked premature aging phenotype. This seemingly paradoxical behavior was potentially explained by a >3-fold lower lifetime incidence of cancer, normally the most common cause of death in mice and often Myc-driven. Myc loss accelerated the accumulation of numerous "Aging Hallmarks", including the loss of mitochondrial and ribosomal structural and functional integrity, the generation of reactive oxygen species, the acquisition of genotoxic damage, the detrimental rewiring of metabolism and the onset of senescence. In both mice and humans, normal aging in many tissues was accompaniued by the downregulation of Myc and the loss of Myc target gene regulation. Unlike most mouse models of premature aging, which are based on monogenic disorders of DNA damage recognition and repair, the MycKO mouse model directly impacts most Aging Hallmarks and may therefore more faithfully replicate the normal aging process of both mice and humans. It further establishes that the strong association between aging and cancer can be genetically separated and is maintained by a single gene.

Premature aging and reduced cancer incidence associated with near-complete body-wide Myc inactivation.

MYC proto-oncogene dysregulation alters metabolism, translation, and other functions in ways that support tumor induction and maintenance. Although Myc+/- mice are healthier and longer-lived than control mice, the long-term ramifications of more complete Myc loss remain unknown. We now describe the chronic consequences of body-wide Myc inactivation initiated postnatally. "MycKO" mice acquire numerous features of premature aging, including altered body composition and habitus, metabolic dysfunction, hepatic steatosis, and dysregulation of gene sets involved in functions that normally deteriorate with aging. Yet, MycKO mice have extended lifespans that correlate with a 3- to 4-fold lower lifetime cancer incidence. Aging tissues from normal mice and humans also downregulate Myc and gradually alter many of the same Myc target gene sets seen in MycKO mice. Normal aging and its associated cancer predisposition are thus highly linked via Myc.

Phosphomevalonate Kinase Controls ß-Catenin Signaling via the Metabolite 5-Diphosphomevalonate.

ß-catenin signaling is abnormally activated in cancer. Here, this work screens the mevalonate metabolic pathway enzyme PMVK to stabilize ß-catenin signaling using a human genome-wide library. On the one hand, PMVK-produced MVA-5PP competitively binds to CKIα to prevent ß-catenin Ser45 phosphorylation and degradation. On the other hand, PMVK functions as a protein kinase to directly phosphorylate ß-catenin Ser184 to increase its protein nuclear localization. This synergistic effect of PMVK and MVA-5PP together promotes ß-catenin signaling. In addition, PMVK deletion impairs mouse embryonic development and causes embryonic lethal. PMVK deficiency in liver tissue alleviates DEN/CCl4 -induced hepatocarcinogenesis. Finally, the small molecule inhibitor of PMVK, PMVKi5, is developed and PMVKi5 inhibits carcinogenesis of liver and colorectal tissues. These findings reveal a non-canonical function of a key metabolic enzyme PMVK and a novel link between the mevalonate pathway and ß-catenin signaling in carcinogenesis providing a new target for clinical cancer therapy.

p53 promotes peroxisomal fatty acid ß-oxidation to repress purine biosynthesis and mediate tumor suppression.

The metabolic pathways through which p53 functions as a potent tumor suppressor are incompletely understood. Here we report that, by associating with the Vitamin D receptor (VDR), p53 induces numerous genes encoding enzymes for peroxisomal fatty acid ß-oxidation (FAO). This leads to increased cytosolic acetyl-CoA levels and acetylation of the enzyme 5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase (ATIC), which catalyzes the last two steps in the purine biosynthetic pathway. This acetylation step, mediated by lysine acetyltransferase 2B (KAT2B), occurs at ATIC Lys 266, dramatically inhibits ATIC activity, and inversely correlates with colorectal cancer (CRC) tumor growth in vitro and in vivo, and acetylation of ATIC is downregulated in human CRC samples. p53-deficient CRCs with high levels of ATIC is more susceptible to ATIC inhibition. Collectively, these findings link p53 to peroxisomal FAO, purine biosynthesis, and CRC pathogenesis in a manner that is regulated by the levels of ATIC acetylation.

P53 deficiency affects cholesterol esterification to exacerbate hepatocarcinogenesis.

BACKGROUND AND AIMS: Cholesterol ester (CE) biosynthesis and homeostasis play critical roles in many cancers, including HCC, but their exact mechanistic contributions to HCC disease development require further study. APPROACH AND RESULTS: Here, we report on a proposed role of tumor suppressor P53 in its repressing ubiquitin-specific peptidase 19 (USP19) and sterol O-acyltransferase (SOAT) 1, which maintains CE homeostasis. USP19 enhances cholesterol esterification and contributes to hepatocarcinogenesis (HCG) by deubiquitinating and stabilizing SOAT1. Loss of either SOAT1 or USP19 dramatically attenuates cholesterol esterification and HCG in P53-deficient mice fed with either a normal chow diet or a high-cholesterol, high-fat diet (HCHFD). SOAT1 inhibitor avasimibe has more inhibitory effect on HCC progression in HCHFD-maintained P53-deficient mice when compared to the inhibitors of de novo cholesterol synthesis. Consistent with our findings in the mouse model, the P53-USP19-SOAT1 signaling axis is also dysregulated in human HCCs. CONCLUSIONS: Collectively, our findings demonstrate that SOAT1 participates in HCG by increasing cholesterol esterification, thus indicating that SOAT1 is a potential biomarker and therapeutic target in P53-deficient HCC.

Regulation of Normal and Neoplastic Proliferation and Metabolism by the Extended Myc Network.

The Myc Network, comprising a small assemblage of bHLH-ZIP transcription factors, regulates many hundreds to thousands of genes involved in proliferation, energy metabolism, translation and other activities. A structurally and functionally related set of factors known as the Mlx Network also supervises some of these same functions via the regulation of a more limited but overlapping transcriptional repertoire. Target gene co-regulation by these two Networks is the result of their sharing of three members that suppress target gene expression as well as by the ability of both Network's members to cross-bind one another's consensus DNA sites. The two Networks also differ in that the Mlx Network's control over transcription is positively regulated by several glycolytic pathway intermediates and other metabolites. These distinctive properties, functions and tissue expression patterns potentially allow for sensitive control of gene regulation in ways that are differentially responsive to environmental and metabolic cues while allowing for them to be both rapid and of limited duration. This review explores how such control might occur. It further discusses how the actual functional dependencies of the Myc and Mlx Networks rely upon cellular context and how they may differ between normal and neoplastic cells. Finally, consideration is given to how future studies may permit a more refined understanding of the functional interrelationships between the two Networks.

Disruption of Multiple Overlapping Functions Following Stepwise Inactivation of the Extended Myc Network.

Myc, a member of the "Myc Network" of bHLH-ZIP transcription factors, supervises proliferation, metabolism, and translation. It also engages in crosstalk with the related "Mlx Network" to co-regulate overlapping genes and functions. We investigated the consequences of stepwise conditional inactivation of Myc and Mlx in primary and SV40 T-antigen-immortalized murine embryonic fibroblasts (MEFs). Myc-knockout (MycKO) and Myc × Mlx "double KO" (DKO)-but not MlxKO-primary MEFs showed rapid growth arrest and displayed features of accelerated aging and senescence. However, DKO MEFs soon resumed proliferating, indicating that durable growth arrest requires an intact Mlx network. All three KO MEF groups deregulated multiple genes and functions pertaining to aging, senescence, and DNA damage recognition/repair. Immortalized KO MEFs proliferated in Myc's absence while demonstrating variable degrees of widespread genomic instability and sensitivity to genotoxic agents. Finally, compared to primary MycKO MEFs, DKO MEFs selectively downregulated numerous gene sets associated with the p53 and retinoblastoma (Rb) pathways and G2/M arrest. Thus, the reversal of primary MycKO MEF growth arrest by either Mlx loss or SV40 T-antigen immortalization appears to involve inactivation of the p53 and/or Rb pathways.

Posttranslational control of lipogenesis in the tumor microenvironment.

Metabolic reprogramming of cancer cells within the tumor microenvironment typically occurs in response to increased nutritional, translation and proliferative demands. Altered lipid metabolism is a marker of tumor progression that is frequently observed in aggressive tumors with poor prognosis. Underlying these abnormal metabolic behaviors are posttranslational modifications (PTMs) of lipid metabolism-related enzymes and other factors that can impact their activity and/or subcellular localization. This review focuses on the roles of these PTMs and specifically on how they permit the re-wiring of cancer lipid metabolism, particularly within the context of the tumor microenvironment.

Coordinated Cross-Talk Between the Myc and Mlx Networks in Liver Regeneration and Neoplasia.

BACKGROUND & AIMS: The c-Myc (Myc) Basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factor is deregulated in most cancers. In association with Max, Myc controls target genes that supervise metabolism, ribosome biogenesis, translation, and proliferation. This Myc network crosstalks with the Mlx network, which consists of the Myc-like proteins MondoA and ChREBP, and Max-like Mlx. Together, this extended Myc network regulates both common and distinct gene targets. Here, we studied the consequence of Myc and/or Mlx ablation in the liver, particularly those pertaining to hepatocyte proliferation, metabolism, and spontaneous tumorigenesis. METHODS: We examined the ability of hepatocytes lacking Mlx (MlxKO) or Myc+Mlx (double KO [DKO]) to repopulate the liver over an extended period of time in a murine model of type I tyrosinemia. We also compared this and other relevant behaviors, phenotypes, and transcriptomes of the livers with those from previously characterized MycKO, ChrebpKO, and MycKO × ChrebpKO mice. RESULTS: Hepatocyte regenerative potential deteriorated as the Extended Myc Network was progressively dismantled. Genes and pathways dysregulated in MlxKO and DKO hepatocytes included those pertaining to translation, mitochondrial function, and hepatic steatosis resembling nonalcoholic fatty liver disease. The Myc and Mlx Networks were shown to crosstalk, with the latter playing a disproportionate role in target gene regulation. All cohorts also developed steatosis and molecular evidence of early steatohepatitis. Finally, MlxKO and DKO mice showed extensive hepatic adenomatosis. CONCLUSIONS: In addition to showing cooperation between the Myc and Mlx Networks, this study showed the latter to be more important in maintaining proliferative, metabolic, and translational homeostasis, while concurrently serving as a suppressor of benign tumorigenesis. GEO accession numbers: GSE181371, GSE130178, and GSE114634.

Normal and Neoplastic Growth Suppression by the Extended Myc Network.

Among the first discovered and most prominent cellular oncogenes is MYC, which encodes a bHLH-ZIP transcription factor (Myc) that both activates and suppresses numerous genes involved in proliferation, energy production, metabolism and translation. Myc belongs to a small group of bHLH-ZIP transcriptional regulators (the Myc Network) that includes its obligate heterodimerization partner Max and six "Mxd proteins" (Mxd1-4, Mnt and Mga), each of which heterodimerizes with Max and largely opposes Myc's functions. More recently, a second group of bHLH-ZIP proteins (the Mlx Network) has emerged that bears many parallels with the Myc Network. It is comprised of the Myc-like factors ChREBP and MondoA, which, in association with the Max-like member Mlx, regulate smaller and more functionally restricted repertoires of target genes, some of which are shared with Myc. Opposing ChREBP and MondoA are heterodimers comprised of Mlx and Mxd1, Mxd4 and Mnt, which also structurally and operationally link the two Networks. We discuss here the functions of these "Extended Myc Network" members, with particular emphasis on their roles in suppressing normal and neoplastic growth. These roles are complex due to the temporal- and tissue-restricted expression of Extended Myc Network proteins in normal cells, their regulation of both common and unique target genes and, in some cases, their functional redundancy.

Ceramide-mediated gut dysbiosis enhances cholesterol esterification and promotes colorectal tumorigenesis in mice.

Colorectal cancer (CRC) severely threatens human health and life span. An effective therapeutic strategy has not been established because we do not clearly know its pathogenesis. Here, we report that ceramide and sterol O-acyltransferase 1 (SOAT1) have roles in both spontaneous and chemical-induced intestinal cancers. We first found that miRNA-148a deficiency dramatically increased mouse gut dysbiosis through upregulating ceramide synthase 5 (Cers5) expression, which promoted ceramide synthesis afterward. The newly generated ceramide further promoted both azoxymethane/dextran sodium sulfate-induced (AOM/DSS-induced) and ApcMin/+ spontaneous intestinal tumorigenesis via increasing mouse gut dysbiosis. Meanwhile, increased level of ceramide correlated with the significant enhancements of both ß-catenin activity and colorectal tumorigenesis in a TLR4-dependent fashion. Next, we found a direct binding of ß-catenin to SOAT1 promoter to activate transcriptional expression of SOAT1, which further induced cholesterol esterification and colorectal tumorigenesis. In human patients with CRC, the same CERS5/TLR4/ß-catenin/SOAT1 axis was also found to be dysregulated. Finally, the SOAT1 inhibitor (avasimibe) showed significant levels of therapeutic effects on both AOM/DSS-induced and ApcMin/+ spontaneous intestinal cancer. Our study clarified that ceramide promoted CRC development through increasing gut dysbiosis, further resulting in the increase of cholesterol esterification in a SOAT1-dependent way. Treatment with avasimibe to specifically decrease cholesterol esterification could be considered as a clinical strategy for effective CRC therapy in a future study.

USP19 exacerbates lipogenesis and colorectal carcinogenesis by stabilizing ME1.

Lipogenesis plays a critical role in colorectal carcinogenesis, but precisely how remains unclear. Here, we show that ERK2 phosphorylates ME1 at T103, thereby inhibiting its polyubiquitination and proteasomal degradation and enhancing its interaction with USP19. USP19 antagonizes RNF1-mediated ME1 degradation by deubiquitination, which in turn promotes lipid metabolism and NADPH production and suppresses ROS. Meanwhile, ROS dramatically increases PD-L1 mRNA levels through accelerating expression of the transcription factor NRF2. Increased lipid metabolism is correlated with ERK2 activity and colorectal carcinogenesis in human patients. Therefore, the combination of ERK2 inhibitor and anti-PD-L1 antibody significantly inhibits spontaneous and chemically induced colorectal carcinogenesis. Collectively, the USP19-ME1 axis plays a vital role in colorectal carcinogenesis and may also provide a potential therapeutic target.