Search

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. Implications: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

The altered metabolism of cancer cells, first recognized by Warburg as aerobic glycolysis, has often been ascribed to answering increased needs for macromolecular biosynthesis to support rapid tumor growth. Recent discoveries that mutations leading to accumulation of fumarate, succinate or 2-hydroxyglutarate (2-HG) may drive cancer via targeting chromatin and DNA modifying enzymes have forced a reevaluation. Indeed, multiple metabolic intermediates can modulate chromatin modifiers, suggesting that altered metabolism may drive epigenetic changes enabling carcinogenesis. Our work has implicated a distinct mechanism, whereby metabolic reprogramming and altered chromatin promote genomic instability and cellular immortality, two hallmarks of cancer. This work began with efforts to target the Warburg effect with small-molecule glucose uptake inhibitors. While targeting glycolysis failed to affect cell proliferation on its own, treated cells were sensitized to radiation, displaying a loss of DNA repair and increased cellular senescence. Working downstream, we found much of this effect was mediated by the hexosamine biosynthetic pathway (HBP) which brings together glucose and glutamine to produce N-acetyl glucosamine (GlcNAc) to support protein glycosylation. Thousands of nuclear, cytoplasmic and mitochondrial protein serine and threonine residues can be modified by O-GlcNAc transferase (OGT) and the resulting O-GlcNAcylation is linked to stress and DNA damage tolerance. Our data implicated the polycomb repressive complexes PRC1 and 2 and their catalytic subunits BMI1 and EZH2 as key OGT targets. Small molecule inhibitors and/or RNAi targeting the HBP, OGT, BMI1 or EZH2 similarly delayed DSB repair and induced senescence after irradiation. In light of others' work linking the histone H3 K27 methyltransferase activity of EZH2 to 53BP1 binding at DSBs to drive repair by non-homologous end-joining (NHEJ), these studies suggest a pathway from the Warburg effect to O-GlcNAcylation and rapid, error-prone DSB repair. Working downstream to validate this mechanism, we discovered that glycolysis and the HBP balanced an antagonistic effect of the TCA cycle on DSB repair. Excess α-ketoglutarate (2-OG) serves as a cofactor for diverse dioxygenases, including the lysine demethylases (KDMs) that antagonize histone methyltransferases such as EZH2. Activating the TCA cycle or providing exogenous 2-OG blocked DSB repair after irradiation and increased senescence while inhibiting all 2-OG-dependent dioxygenases or only the KDM enzymes that remove histone modifications by EZH2 at H3 K27 or G9A at H3 K9 restored DSB repair. This led us to examine the effects of treating cells with the oncometabolite 2-HG, known to mediate its effects by serving as a 2-OG-dependent dioxygenase inhibitor. While prior attention has largely focused on TET DNA demethylases as 2-HG targets and epigenetic mechanisms for carcinogenesis, we observed 2-HG blocking KDM enzymes to accelerate error-prone NHEJ repair, suggesting a role in genomic instability in IDH mutant cells. Toward translation, we have examined the effects of blocking O-GlcNAcylation in vivo as a means to sensitize tumors to radiation. Animals bearing tumors expressing shRNA targeting OGT or treated with OGT inhibitors and then irradiated displayed decreased protein O-GlcNAcylation, a delay in DSB repair, greater cell death, delayed cell recovery and increased senescence. In turn, when animals were treated with GlcNAc or with shRNA or inhibitors targeting O-GlcNAcase, their tumors displayed increased O-GlcNAcylation and dramatic radiation resistance, indicating accelerated DSB repair in vivo. These results implicate OGT and O-GlcNAcyation as a new mechanism by which the Warburg effect may mediate resistance to radiation therapy. Further, our studies point to dietary interventions and/or drugs targeting OGT as a route to improving outcomes from radiotherapy in the clinic. Taken together, an implication of this work is that cancer metabolism may help drive carcinogenesis by shifting repair to NHEJ and away from HR, leading to genomic instability, as well as by overcoming barriers to transformation such as accelerated senescence to promote cell immortality. Our studies argue for renewed efforts to explore cancer metabolism as a target for prevention and therapy, with a focus on leveraging crosstalk with pathways regulating cellular responses to DNA damage. Citation Format: Elena Efimova, Yue Liu, Julian Lutze, Tamica Collins, Amy Flor, Jacqueline Brinkman, Don Wolfgeher, Sara Warrington, Stephen J. Kron. Shutting off cancer's fountain of youth: Targeting metabolism to block repair and reverse immortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY21-03.

The elevation of glucose and glutamine metabolism characteristic of cancer has been ascribed to increased demand for metabolic intermediates that provide building blocks for rapid cell growth. However, recent discoveries have highlighted complementary effects mediated by metabolites serving as cofactors for chromatin modifying enzymes and providing metabolic intermediates for proteins posttranslational modification. Along with epigenetic regulation of gene expression, multiple chromatin modifiers modulate DNA damage repair, raising the question of whether metabolic reprogramming may thereby promote genomic instability and cell immortality. In our prior work, we uncovered a role for the glycolysis hexosamine biosynthetic pathway (HBP) in DNA repair, ionizing radiation induced foci (IRIF) resolution, and prevention of radiation induced cancer cell senescence. Here we report that the targeting of key enzymes in the HBP pathway, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) or O-GlcNAcase (OGA/ MGEA5), have contrary effects on the DNA damage response in tumors. shRNA knockdown of OGA resulted in increased GlcNAc protein modification, protection of cancer cells from irradiation through improved double strand break (DSB) repair and IRIF resolution in tumor cells, increased proliferation, and prevented radiation-induced senescence. In turn, silencing of OGT diminished O-GlcNAc modification, prevented DSB repair, increased IRIF persistence, reduced proliferation, and promoted radiation-induced senescence in tumor xenografts. These findings reveal an important relationship between cancer cell metabolism, DSB repair, and senescence and provide a rationale for targeting metabolic reprogramming, specifically O-GlcNAc modification, to block cancer cell immortality and overcome resistance to genotoxic stress. Citation Format: Elena V. Efimova, Oliver K. Appelbe, Steve Seung-Young Lee, Natalia Ricco, Aishwarya Ramamarthy, Nicolas Rymut, Tamica Collins, Donald J. Wolfgeher, Sara Warrington, Stephen J. Kron. Tumor radiosensitization by suppression of hexosamine biosynthetic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3505.