Abstract SY12-03: MYC-mediated metabolic vulnerabilities and the circadian clock
Authors: Dang Chi Van 
Published: July 2016

The MYC oncogene belongs to the family of MYC genes, including MYCN (N-MYC) and MYCL (L-MYC), which are linked to human cancers such as Burkitt's lymphoma, neuroblastoma and lung cancer, respectively. MYC proteins belong to the MAX-MLX network of heterodimeric transcription factors that bind ‘E-boxes’ (5’-CACGTG-3’) to regulate genes involved in cell proliferation, differentiation and metabolism. MYC is downstream of many signal transduction pathways in normal cells; when stimulated through pathways such as tyrosine receptor kinases, MYC is induced to produce the MYC protein that dimerizes with MAX to bind DNA and regulate a transcriptional program of metabolism, cell growth and proliferation (1). MYC is under the control of growth factor signaling as well as nutrient availability, such that nutrient deprivation could result in diminished normal MYC expression and cell growth arrest. By contrast, deregulation of MYC in normal cells results in MYC binding not only to high affinity physiological binding sites but also invading into low-affinity binding sites and enhancers, resulting in an imbalance amplification of gene expression that triggers stress and cell death through activation of checkpoints such as p53. Elimination of p53 during tumorigenesis, however, can unleash MYC's transcriptional power to drive deregulated cell growth that renders cells addicted to nutrients, such that deprivation of glucose or glutamine results in cell death. The conceptual framework has been exploited to target metabolism for therapy against MYC-driven cancers, particularly aiming at glycolysis and glutaminolysis - metabolic processes that are increased by MYC. While lactate dehydrogenase A (LDHA) inhibition could curb MYC-mediated tumorigenesis (2), glutaminolysis remains an alternative survival pathway that could be targeted through inhibition of glutaminase (3-9). Glutaminase is normally expressed from two different genes: GLS that is normally expressed in kidney and brain, and GLS2 that is normally expressed in liver. Indeed, we have shown that knockdown of glutaminase (GLS) or inhibition with a small molecule (BPTES) diminishes the progression of a MYC-inducible human lymphoma xenograft model (3-7). We further documented that glutaminase (Gls) is induced by MYC and is required for early tumor development in a MYC-inducible model of mouse liver cancer (8), which displayed a decrease in the expression of normal liver Gls2. In this regard, the isoform switch from Gls2 to Gls1 in mouse (and also human) liver cancer renders tumors vulnerable to loss of one copy of Gls, which delayed tumorigenesis. We further showed that treatment with BPTES as a single agent was sufficient to prolong survival of mice bearing these MYC-induced liver cancers, providing proof-of-concept that targeting a single enzyme, in this case GLS, could change the course of the disease (8).

Targeting GLS, however, has limitations, since the oncogenotypes of cancers result in different re-wiring of metabolism that is best illustrated by metabolomics studies of different mouse models of cancer (10). The MET oncogene-driven liver cancer model largely relies on glucose, whereas MYC oncogene-driven liver and lung cancers rely on both glutamine and glucose. As such, glutaminase inhibition should be considered in the context of the oncogenotype and metabolic profile of specific cancer types. Further, targeting metabolism could be constrained by the circadian regulation of cellular metabolism, rendering proliferating normal cells more vulnerable to inhibition of metabolism at specific times of the day. In this regard, we tested the hypothesis that high oncogenic MYC would ectopically invade the circadian Clock regulated genes that are also driven by E-boxes (11). The cell intrinsic clock machinery comprises of the central Clock-Bmal1 transcription factor, which induces Rev-erb’s, Cry’s, and Per's that in turn negatively regulated Bmal1 expression or Clock-Bmal1 levels, resulting in a circadian oscillation of Clock-Bmal1 function. Oscillation of the central clock transcription, which drives metabolic genes with E-boxes, results in oscillatory metabolism. Indeed, using an inducible MYC-ER system, we demonstrate that MYC could activate the negative clock regulators PER, CRY, and REV-ERBs and documented that the suppression of Bmal1 expression by MYC is mediated transcriptionally through REV-ERBs. Metabolic profiling in time-series experiments reveals oscillation of glucose and glutamine metabolic in the MYC-OFF state in U2OS cells that have very low basal endogenous MYC expression. In the MYC-ON state, intracellular glucose levels cease to oscillate and are barely detectable by NMR. Additional evidence suggests that it is converted toward lactate and biomass for cell growth. MYC, hence, can invade and disrupt the molecular circadian clock as well as metabolism, in favor of an anabolic growth program.

These observations have key implications for targeting metabolic enzymes that have been documented as being induced by Clock-Bmal1 and MYC. For example, ornithine decarboxylase (ODC) and nicotinamide phosphoribosyltransferase (NAMPT) are common targets of MYC and Clock-Bmal1. Hence, we hypothesize that the dose-limiting toxicity of NAMPT inhibition, being thrombocytopenia (12), could be curbed in a lymphoma xenograft model by applying chronotherapy. In this regard, our preliminary studies illustrate that while NAMPT inhibition at two different times of the day resulted in similar efficacy in reducing lymphoma xenograft growth, one of administration time resulted in thrombocytopenia while the other time of drug administration was indistinguishable from the control treated animals (unpublished). These observations indicate that the combination of identifying metabolic vulnerabilities of MYC-driven cancer along with understanding circadian could strategically guide the use of metabolism-targeted drugs in the clinic through oncogenotyping and potentially chronotherapy.

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Citation Format: Chi Van Dang. MYC-mediated metabolic vulnerabilities and the circadian clock. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY12-03.