Joyce V. Lee

ATM Couples Replication Stress and Metabolic Reprogramming during Cellular Senescence

Replication stress induced by nucleotide deficiency plays an important role in cancer initiation. Replication stress in primary cells typically activates the cellular senescence tumor-suppression mechanism. Senescence bypass correlates with development of cancer, a disease characterized by metabolic reprogramming. However, the role of metabolic reprogramming in the cellular response to replication stress has been little explored. Here, we report that ataxia telangiectasia mutated (ATM) plays a central role in regulating the cellular response to replication stress by shifting cellular metabolism. ATM inactivation bypasses senescence induced by replication stress triggered by nucleotide deficiency. This was due to restoration of deoxyribonucleotide triphosphate (dNTP) levels through both upregulation of the pentose phosphate pathway via increased glucose-6-phosphate dehydrogenase (G6PD) activity and enhanced glucose and glutamine consumption. These phenotypes were mediated by a coordinated suppression of p53 and upregulation of c-MYC downstream of ATM inactivation. Our data indicate that ATM status couples replication stress and metabolic reprogramming during senescence.

Nutrient sensor O -GlcNAc transferase controls cancer lipid metabolism via SREBP-1 regulation

Elevated O-GlcNAcylation is associated with disease states such as diabetes and cancer. O-GlcNAc transferase (OGT) is elevated in multiple cancers and inhibition of this enzyme genetically or pharmacologically inhibits oncogenesis. Here we show that O-GlcNAcylation modulates lipid metabolism in cancer cells. OGT regulates expression of the master lipid regulator the transcription factor sterol regulatory element binding protein 1 (SREBP-1) and its transcriptional targets both in cancer and lipogenic tissue. OGT regulates SREBP-1 protein expression via AMP-activated protein kinase (AMPK). SREBP-1 is critical for OGT-mediated regulation of cell survival and of lipid synthesis, as overexpression of SREBP-1 rescues lipogenic defects associated with OGT suppression, and tumor growth in vitro and in vivo. These results unravel a previously unidentified link between O-GlcNAcylation, lipid metabolism and the regulation of SREBP-1 in cancer and suggests a crucial role for O-GlcNAc signaling in transducing nutritional state to regulate lipid metabolism.

Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+–NFAT signaling

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca2+ signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca2+-NFAT signaling.

A cancerous web: signaling, metabolism, and the epigenome

Histone acetylation is sensitive to the availability of acetyl-CoA. However, the extent to which metabolic alterations in cancer cells impact tumor histone acetylation has been unclear. Here, we discuss our recent findings that oncogenic AKT1 activation regulates histone acetylation levels in tumors through regulation of acetyl-CoA metabolism.

Abstract B40: Oncogenic Kras induces histone acetylation in pancreatic ductal adenocarcinoma

Deregulation of cellular epigenetics is essential for malignant transformation. However, the mechanisms that cause epigenetic alterations in cancer cells are incompletely understood. Recent evidence has shown that cellular metabolism has a direct impact on the epigenome, since many chromatin-modifying enzymes rely on intracellular metabolites as cofactors or donor substrates. We have previously shown that the metabolic enzyme ATP-citrate lyase (ACLY), which generates nuclear-cytoplasmic acetyl-CoA from glucose, is required for maintaining histone acetylation levels in multiple mammalian cell types, suggesting that alterations in acetyl-CoA metabolism in cancer cells might also impact histone acetylation levels. Cellular metabolism is massively rewired in multiple cancer types, including pancreatic cancer, although the impact of metabolic alterations on the tumor epigenome is poorly understood. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. In this study, we demonstrate that acetyl-CoA abundance in cancer cells is dynamically regulated by glucose availability and that histone acetylation levels are responsive to the ratio of acetyl-CoA:coenzyme A within the nucleus. To test whether oncogene activation could mediate changes in histone acetylation in vivo, we performed immunohistochemical analysis comparing pancreata from mice expressing Kras G12D with those from mice with WT Kras. Whereas the acinar cells of WT mice exhibited very low levels of histone H4 (K5/8/12/16) acetylation, in KPC (LSL-KrasG12D; p53L/+; Pdx1-Cre) mice, acinar H4 acetylation was markedly increased, prior to the appearance of histological abnormalities or aberrant cell proliferation. High H4 acetylation persisted in pancreatic intraepithelial neoplasia (PanIN) and PDA. Histone acetylation in PanIN-derived primary cells was selectively impaired by PI3K and Akt inhibitors, correlating with suppression of glucose consumption and cellular acetyl-CoA levels. Moreover, addition of supraphysiological doses of acetate, a source of acetyl-CoA alternative to glucose, restored histone acetylation levels. These data suggest that oncogenic Kras promotes elevated histone acetylation preceding tumor development through Akt-dependent regulation of cellular acetyl-CoA levels. Further mechanistic analysis suggests that Akt promotes elevated histone acetylation through combined effects on promoting glucose uptake and phosphorylation and activation of ATP-citrate lyase, a metabolic enzyme that produces nuclear-cytoplasmic acetyl-CoA. The aberrant activation of the PI3K/Akt pathway occurs in broad variety of human malignancies. pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors, suggesting that PI3K-Akt-dependent promotion of acetyl-CoA metabolism may contribute to histone acetylation levels in multiple cancer types. Our data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in tumors and offer novel insights on Kras-induced pancreatic carcinogenesis. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nathaniel W. Snyder, Ellen Jackson, Nicole M. Aiello, Benjamin A. Garcia, Lewis A. Chodosh, Ben Z. Stanger, Ian A. Blair, Kathryn E. Wellen. Oncogenic Kras induces histone acetylation in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B40.

Abstract B40: AKT-ACLY signaling preferentially maintains histone acetylation over lipid synthesis during nutrient limitation

Metabolic reprogramming by oncogenes is a mechanism to support biosynthesis and growth in cancer cells. Activation of the PI3K/AKT pathway increases glucose consumption and glycolysis, leading to higher acetyl-CoA production. Extra-mitochondrial acetyl-CoA is utilized in lipid synthesis and protein acetylation. We previously showed that tumor cell histone acetylation is regulated in a glucose-dependent manner, through ATP-citrate-lyase (ACLY) conversion of glucose-derived citrate into acetyl-CoA. In low glucose conditions, acetyl-CoA and histone acetylation levels decrease. However, AKT-dependent phosphorylation of ACLY at serine 455 promotes acetyl-CoA production to allow maintenance of global levels of histone acetylation under glucose limitation. It is unclear whether partitioning of acetyl-CoA between lipid synthesis and histone acetylation in the setting of nutrient limitation impacts cell growth or survival. In this study, we examined whether acetyl-CoA utilization for lipid synthesis is impacted by AKT-ACLY signaling during nutrient limitation. Using 13C-glucose labeling and mass spectrometry, we determined that de novo lipid synthesis is suppressed to a much greater extent than histone acetylation in low glucose conditions. Moreover, phosphorylation of ACLY does not sustain fatty acid synthesis under glucose limitation, in contrast to its effects on histone acetylation. These data indicate that the PI3K-AKT-ACLY axis preferentially maintains histone acetylation over lipid synthesis during nutrient limitation. This regulation is likely due to AMPK-dependent phosphorylation of acetyl-CoA carboxylase (p-ACC), blocking acetyl-CoA commitment to fatty acid synthesis. This study reveals that cancer cells employ robust mechanisms to maintain histone acetylation in nutrient limited environments. Citation Format: Joyce V. Lee, Zuo-Fei Yuan, Shichong Liu, Benjamin A. Garcia, Kathryn E. Wellen. AKT-ACLY signaling preferentially maintains histone acetylation over lipid synthesis during nutrient limitation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B40.

Abstract PR03: Exploring the link between Kras and histone acetylation

KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) have been shown to extensively rewire cellular metabolism to promote macromolecular biosynthesis and maintain redox homeostasis. Recently, compelling evidence has emerged demonstrating that many epigenetic modifications are sensitive to the availability of cellular metabolites. For example, acetyl-CoA is the donor substrate for lysine acetylation, and histone acetylation is responsive to levels of acetyl-CoA, which is produced largely from glucose metabolism. We thus hypothesized that oncogenic metabolic reprogramming alters metabolite levels in a way that impacts the epigenome and could thus contribute to tumorigenesis. Indeed, we find that oncogenic Akt activation promotes elevated histone acetylation, and that this effect is mediated by ATP-citrate-lyase (ACLY), a nucleocytoplasmic enzyme that converts glucose-derived citrate into acetyl-CoA. In order to understand what cellular processes are affected by such AKT-ACLY-mediated increase in histone acetylation, we employed a mouse model of pancreatic ductal adenocarcinoma, driven by oncogenic Kras (Pdx1-Cre; LSL-KrasG12D; TP53L/+). In this model, the PI3K/Akt pathway is activated downstream of Kras. By immunohistochemistry we found that, in mice with WT Kras, pancreatic acinar cells exhibit very low levels of histone H4 (K5/8/12/16) acetylation (AcH4), although AcH4 is clearly detectable in ductal epithelial cells and islets. By contrast, in mice expressing oncogenic Kras in the pancreas (LSL-KrasG12D; p53L/+; Pdx1-Cre), acinar H4 acetylation is dramatically increased. Remarkably, this overt increase in histone acetylation precedes the appearance of histological abnormalities and persists during several steps in tumor progression. Moreover, Kras and Akt-dependent reuglation of histone acetylation levels can be recapitulated in ex vivo acinar cell culture, suggesting that this is a primary effect of oncogene activation in these cells. Akt inhibition also significantly reduced glucose consumption and phosphorylation of ACLY and ultimately decreased acetyl-CoA levels. Akt inhibition in vitro reduces Kras-induced acinar cell hyperacetylation and block acinar-to-ductal metaplasia, an initiating event of pancreatic carcinoma. Our data indicate that Akt orchestrates a metabolic rewiring in pancreatic tumorigenesis, which promotes histone hyperacetylation, a phenotype evident before the manifestation of the disease and that might conceivably contribute to cell plasticity and tumor progression. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nicole M. Aiello, Nathaniel W. Snyder, Andrew J. Worth, Ian A. Blair, Ben Z. Stanger, Kathryn E. Wellen. Exploring the link between Kras and histone acetylation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR03.

Abstract A31: Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation

Histone acetylation plays important roles in gene regulation, DNA replication, and the response to DNA damage, and it is frequently deregulated in tumors. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. Here, we demonstrate that acetyl-CoA is dynamically regulated by glucose availability in cancer cells and that the ratio of acetyl-CoA: coenzyme A within the nucleus modulates global histone acetylation levels. In vivo, expression of oncogenic Kras or Akt stimulates histone acetylation changes that precede tumor development. Furthermore, we show that Akt9s effects on histone acetylation are mediated through the metabolic enzyme ATP-citrate lyase (ACLY), and that pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors. The data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in cancer cells. Citation Format: Supriya Shah, Joyce V. Lee, Alessandro Carrer, Nathaniel W. Snyder, Kathryn E. Wellen. Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A31.