Date of Degree

2-2017

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor(s)

David Foster

Committee Members

Diego Loayza

Derrick Brazill

Hans-Guido Wendel

Nancy Du

Subject Categories

Biochemistry

Keywords

Metabolism, Cell cycle, Glutamine, Lipids, Checkpoints

Abstract

During the G1 phase of the cell cycle, normal cells respond to growth factors and nutrients prior to entering S-phase to replicate its genome. We previously reported that the growth factor-mediated restriction point is distinguishable from a series of late G1 metabolic checkpoints mediated by essential amino acids (EAAs), the conditionally essential amino acid glutamine (Gln), and mTOR – the mammalian target of rapamycin. Mutations in genes encoding proteins that regulate G1 cell cycle progression are observed in virtually all cancers. We observed that cancer cells with K-Ras mutations bypass the late G1 Gln checkpoint when deprived of Gln and instead arrest in S-phase. Significantly, this created a synthetic lethality for rapamycin. Whereas rapamycin arrests cells in late G1, in S-phase rapamycin induces apoptosis. While depriving cells of Gln can be achieved in culture, this is not a viable option in an animal. However, K-Ras-driven cancer cells utilize Gln via a novel transaminase reaction whereby Gln is first deaminated to glutamate and then glutamate is deamidated to a-ketoglutarate, with the concomitant conversion of oxaloacetate to aspartate. This transamination reaction can be inhibited by aminooxyacetate, which mimics Gln deprivation and causes S-phase arrest – creating synthetic lethality for rapamycin.

Since S-phase arrest created a synthetic lethality for rapamycin, we investigated the molecular basis for the S-phase arrest. We found that S-phase arrest was due to an inability to generate deoxynucleotides needed for DNA synthesis in the absence of glutamine. The lack of Gln suppressed deoxynucleotides biosynthesis, which in turn induced replicative stress. The replicative stress activated the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which caused S-phase arrest. Of significance, aspartate, which is a critical metabolite for deoxynucleotides biosynthesis and is generated by the transaminase reaction between glutamate and oxaloacetate, rescued the S-phase arrest caused by inhibition of glutamine utilization.

The presence of distinct metabolic checkpoints in late G1 for EAAs and Gln led us to look for additional checkpoints that monitor the presence of critical nutrients. Since lipids are critical for membrane biosynthesis, we investigated whether serum lipids were important for G1 cell cycle progression. We found that when put in delipidated serum, cells arrest in late G1 at a distinct site downstream from the Gln checkpoint and upstream from the mTOR site. Intriguingly, this checkpoint is dysregulated in clear cell renal carcinoma cells. These cells continue to replicate in the absence of lipids until they ultimately starve themselves to death.

In summary, we have identified a series of late G1 metabolic checkpoints that are dysregulated in specific cancer cell lines. It is speculated that when these checkpoints are dysregulated in cancer cells, there are novel opportunities for therapeutic intervention.

Included in

Biochemistry Commons

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