Lipid metabolism is frequently perturbed in cancers, but the underlying mechanism is unclear. We present comprehensive evidence that oncogene MYC, in collaboration with transcription factor sterol-regulated element-binding protein (SREBP1), regulates lipogenesis to promote tumorigenesis. We used human and mouse tumor-derived cell lines, tumor xenografts, and four conditional transgenic mouse models of MYC-induced tumors to show that MYC regulates lipogenesis genes, enzymes, and metabolites. We found that MYC induces SREBP1, and they collaborate to activate fatty acid (FA) synthesis and drive FA chain elongation from glucose and glutamine. Further, by employing desorption electrospray ionization mass spectrometry imaging (DESI-MSI), we observed in vivo lipidomic changes upon MYC induction across different cancers, for example, a global increase in glycerophosphoglycerols. After inhibition of FA synthesis, tumorigenesis was blocked, and tumors regressed in both xenograft and primary transgenic mouse models, revealing the vulnerability of MYC-induced tumors to the inhibition of lipogenesis.

The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stress-adaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. In this study, we report that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, and we probe the mechanism by which this activation occurs. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived from Gcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. Compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.

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The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, ∼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism.

The molecular circadian clock, which controls rhythmic 24-hour oscillation of genes, proteins, and metabolites, is disrupted across many human cancers. Deregulated expression of MYC oncoprotein has been shown to alter expression of molecular clock genes, leading to a disruption of molecular clock oscillation across cancer types. It remains unclear what benefit cancer cells gain from suppressing clock oscillation, and how this loss of molecular clock oscillation impacts global gene expression and metabolism in cancer. We hypothesized that MYC suppresses oscillation of gene expression and metabolism to instead upregulate pathways involved in biosynthesis in a static, non-oscillatory fashion. To test this, cells from distinct cancer types with inducible MYC or the closely related N-MYC were examined, using detailed time-series RNA-sequencing and metabolomics, to determine the extent to which MYC activation disrupts global oscillation of genes, gene expression, programs, and metabolites. We focused our analyses on genes, pathways, and metabolites that changed in common across multiple cancer cell line models. We report here that MYC disrupted over 85% of oscillating genes, while instead promoting enhanced ribosomal and mitochondrial biogenesis and suppressed cell attachment pathways. Notably, when MYC is activated, biosynthetic programs that were formerly circadian flipped to being upregulated in an oscillation-free manner. Further, activation of MYC ablates the oscillation of nutrient transporter glycosylation while greatly upregulating transporter expression, cell surface localization, and intracellular amino acid pools. Finally, we report that MYC disrupts metabolite oscillations and the temporal segregation of amino acid metabolism from nucleotide metabolism. Our results demonstrate that MYC disruption of the molecular circadian clock releases metabolic and biosynthetic processes from circadian control, which may provide a distinct advantage to cancer cells.

Disrupted circadian rhythmicity is a prominent feature of modern society and has been designated as a probable carcinogen by the World Health Organization. However, the biological mechanisms that connect circadian disruption and cancer risk remain largely undefined. We demonstrate that exposure to chronic circadian disruption [chronic jetlag (CJL)] increases tumor burden in a mouse model of KRAS-driven lung cancer. Molecular characterization of tumors and tumor-bearing lung tissues revealed that CJL enhances the expression of heat shock factor 1 (HSF1) target genes. Consistently, exposure to CJL disrupted the highly rhythmic nuclear trafficking of HSF1 in the lung, resulting in an enhanced accumulation of HSF1 in the nucleus. HSF1 has been shown to promote tumorigenesis in other systems, and we find that pharmacological or genetic inhibition of HSF1 reduces the growth of KRAS-mutant human lung cancer cells. These findings implicate HSF1 as a molecular link between circadian disruption and enhanced tumorigenesis.

Deletion of the paralogs ZDS1 and ZDS2 in the budding yeast Saccharomyces cerevisiae causes a mis-regulation of polarized cell growth. Here we show a function for these genes as regulators of the Swe1p (Wee1p) kinase-dependent G2/M checkpoint. We identified a conserved domain in the C-terminus of Zds2p consisting of amino acids 813-912 (hereafter referred to as ZH4 for Zds homology 4) that is required for regulation of Swe1p-dependent polarized bud growth. ZH4 is shown by protein affinity assays to be necessary and sufficient for interaction with Cdc55p, a regulatory subunit of protein phosphatase 2A (PP2A). We hypothesized that the Zds proteins are in a pathway that negatively regulates the Swe1p-dependent G2/M checkpoint via Cdc55p. Supporting this model, deletion of CDC55 rescues the aberrant bud morphology of a zds1Δzds2Δ strain. We also show that expression of ZDS1 or ZDS2 from a strong galactose-inducible promoter can induce mitosis even when the Swe1p-dependent G2/M checkpoint is activated by mis-organization of the actin cytoskeleton. This negative regulation requires the CDC55 gene. Together these data indicate that the Cdc55p/Zds2p module has a function in the regulation of the Swe1p-dependent G2/M checkpoint.

The unfolded protein response (UPR) is a stress-activated signalling pathway that regulates cell proliferation, metabolism and survival. The circadian clock coordinates metabolism and signal transduction with light/dark cycles. We explore how UPR signalling interfaces with the circadian clock. UPR activation induces a 10 h phase shift in circadian oscillations through induction of miR-211, a PERK-inducible microRNA that transiently suppresses both Bmal1 and Clock, core circadian regulators. Molecular investigation reveals that miR-211 directly regulates Bmal1 and Clock via distinct mechanisms. Suppression of Bmal1 and Clock has the anticipated impact on expression of select circadian genes, but we also find that repression of Bmal1 is essential for UPR-dependent inhibition of protein synthesis and cell adaptation to stresses that disrupt endoplasmic reticulum homeostasis. Our data demonstrate that c-Myc-dependent activation of the UPR inhibits Bmal1 in Burkitt's lymphoma, thereby suppressing both circadian oscillation and ongoing protein synthesis to facilitate tumour progression.

Terminal differentiation opposes proliferation in the vast majority of tissue types. As a result, loss of lineage differentiation is a hallmark of aggressive cancers, including soft tissue sarcomas (STS). Consistent with these observations, undifferentiated pleomorphic sarcoma (UPS), an STS subtype devoid of lineage markers, is among the most lethal sarcomas in adults. Though tissue-specific features are lost in these mesenchymal tumors they are most commonly diagnosed in skeletal muscle, and are thought to develop from transformed muscle progenitor cells. We have found that a combination of HDAC (Vorinostat) and BET bromodomain (JQ1) inhibition partially restores differentiation to skeletal muscle UPS cells and tissues, enforcing a myoblast-like identity. Importantly, differentiation is partially contingent upon downregulation of the Hippo pathway transcriptional effector Yes-associated protein 1 (YAP1) and nuclear factor (NF)-κB. Previously, we observed that Vorinostat/JQ1 inactivates YAP1 and restores oscillation of NF-κB in differentiating myoblasts. These effects correlate with reduced tumorigenesis, and enhanced differentiation. However, the mechanisms by which the Hippo/NF-κB axis impact differentiation remained unknown. Here, we report that YAP1 and NF-κB activity suppress circadian clock function, inhibiting differentiation and promoting proliferation. In most tissues, clock activation is antagonized by the unfolded protein response (UPR). However, skeletal muscle differentiation requires both Clock and UPR activity, suggesting the molecular link between them is unique in muscle. In skeletal muscle-derived UPS, we observed that YAP1 suppresses PERK and ATF6-mediated UPR target expression as well as clock genes. These pathways govern metabolic processes, including autophagy, and their disruption shifts metabolism toward cancer cell-associated glycolysis and hyper-proliferation. Treatment with Vorinostat/JQ1 inhibited glycolysis/MTOR signaling, activated the clock, and upregulated the UPR and autophagy via inhibition of YAP1/NF-κB. These findings support the use of epigenetic modulators to treat human UPS. In addition, we identify specific autophagy, UPR, and muscle differentiation-associated genes as potential biomarkers of treatment efficacy and differentiation.

Cancer-related fatigue is a prevalent, debilitating condition that can persist for months or years after treatment. In a single-arm clinical trial, the feasibility and safety of a time-restricted eating (TRE) intervention were evaluated among cancer survivors, and initial estimates of within-person change in cancer-related fatigue were obtained.

The c-Myc oncogene drives malignant progression and induces robust anabolic and proliferative programmes leading to intrinsic stress. The mechanisms enabling adaptation to MYC-induced stress are not fully understood. Here we reveal an essential role for activating transcription factor 4 (ATF4) in survival following MYC activation. MYC upregulates ATF4 by activating general control nonderepressible 2 (GCN2) kinase through uncharged transfer RNAs. Subsequently, ATF4 co-occupies promoter regions of over 30 MYC-target genes, primarily those regulating amino acid and protein synthesis, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation. 4E-BP1 relieves MYC-induced proteotoxic stress and is essential to balance protein synthesis. 4E-BP1 activity is negatively regulated by mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation and inhibition of mTORC1 signalling rescues ATF4-deficient cells from MYC-induced endoplasmic reticulum stress. Acute deletion of ATF4 significantly delays MYC-driven tumour progression and increases survival in mouse models. Our results establish ATF4 as a cellular rheostat of MYC activity, which ensures that enhanced translation rates are compatible with survival and tumour progression.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

The molecular circadian clock, which controls rhythmic 24-hour oscillation of genes, proteins, and metabolites in healthy tissues, is disrupted across many human cancers. Deregulated expression of the MYC oncoprotein has been shown to alter expression of molecular clock genes, leading to a disruption of molecular clock oscillation across cancer types. It remains unclear what benefit cancer cells gain from suppressing clock oscillation, and how this loss of molecular clock oscillation impacts global gene expression and metabolism in cancer. We hypothesized that MYC or its paralog N-MYC (collectively termed MYC herein) suppress oscillation of gene expression and metabolism to upregulate pathways involved in biosynthesis in a static, non-oscillatory fashion. To test this, cells from distinct cancer types with inducible MYC were examined, using time-series RNA-sequencing and metabolomics, to determine the extent to which MYC activation disrupts global oscillation of genes, gene expression pathways, and metabolites. We focused our analyses on genes, pathways, and metabolites that changed in common across multiple cancer cell line models. We report here that MYC disrupted over 85% of oscillating genes, while instead promoting enhanced ribosomal and mitochondrial biogenesis and suppressed cell attachment pathways. Notably, when MYC is activated, biosynthetic programs that were formerly circadian flipped to being upregulated in an oscillation-free manner. Further, activation of MYC ablates the oscillation of nutrient transporter proteins while greatly upregulating transporter expression, cell surface localization, and intracellular amino acid pools. Finally, we report that MYC disrupts metabolite oscillations and the temporal segregation of amino acid metabolism from nucleotide metabolism. Our results demonstrate that MYC disruption of the molecular circadian clock releases metabolic and biosynthetic processes from circadian control, which may provide a distinct advantage to cancer cells.

Macrophages are prime therapeutic targets due to their pro-tumorigenic and immunosuppressive functions in tumors, but varying efficacy of therapeutic approaches targeting macrophages highlights our incomplete understanding of how the tumor microenvironment (TME) can influence regulation of macrophages. The circadian clock is a key internal regulator of macrophage function, but how circadian rhythms of macrophages may be influenced by the tumor microenvironment remains unknown. We found that conditions associated with the TME such as polarizing stimuli, acidic pH, and elevated lactate concentrations can each alter circadian rhythms in macrophages. Circadian rhythms were enhanced in pro-resolution macrophages but suppressed in pro-inflammatory macrophages, while acidic pH had divergent effects on circadian rhythms depending on macrophage phenotype. While cyclic AMP (cAMP) has been reported to play a role in macrophage response to acidic pH, our results indicate that pH-driven changes in circadian rhythms are not mediated solely by the cAMP signaling pathway. Remarkably, clock correlation distance analysis of tumor-associated macrophages (TAMs) revealed evidence of circadian disorder in TAMs. This is the first report providing evidence that circadian rhythms of macrophages are altered within the TME. Our data suggest that heterogeneity in circadian rhythms at the population level may underlie this circadian disorder. Finally, we sought to determine how circadian regulation of macrophages impacts tumorigenesis, and found that tumor growth was suppressed when macrophages had a functional circadian clock. Our work demonstrates a novel mechanism by which the tumor microenvironment can influence macrophage biology through altering circadian rhythms, and the contribution of circadian rhythms in macrophages to suppressing tumor growth.