Defining Anabolic Functions of ATF4 as a Downstream Effector of mTORC1 Signaling

Abstract

In response to a variety of upstream growth and oncogenic signals, the mechanistic target of rapamycin complex 1 (mTORC1) promotes anabolic metabolism, in part, through activation of downstream transcription factors. Previous studies have found that mTORC1 induces an increase in activating transcription factor 4 (ATF4), which is required for mTORC1-stimulated purine synthesis. Though ATF4 is classically known as a component of the integrated stress response (ISR) downstream of the phosphorylation of eukaryotic initiation factor 2α (eIF2α), the mTORC1-mediated induction of ATF4 is independent of this canonical pathway. The goals of this dissertation were to define the target genes and biological significance of the mTORC1-ATF4 axis and better understand how the mTORC1-ATF4 transcriptional program compared to the ISR-ATF4 response. Therefore, we used RNA-Seq in wild-type and ATF4-knockout MEFs to identify and compare the insulin-stimulated, rapamycin-sensitive transcripts to those induced by the ER stress-inducing agent tunicamycin. These analyses revealed a subset of the ATF4-dependent transcriptional targets induced downstream of the ISR was regulated by mTORC1. The mTORC1-ATF4 target genes were involved in amino acid uptake, biosynthesis, and tRNA charging. Interestingly, we find that ATF4 is required for mTORC1-stimulated protein synthesis, highlighting the importance of these ATF4-dependent transcriptional changes to the canonical function of mTORC1. Additionally, we observe increases in the uptake of oxidized cysteine and subsequent synthesis of glutathione downstream of mTORC1 that are driven by its regulation of ATF4. I show that mTORC1 regulates glutathione synthesis in PTEN-deficient cell lines and xenografts. This dissertation also explores the potential role of ATF4 in tumors, where both aberrant mTORC1 signaling and nutrient deprivation often occur. Collectively, these data indicate that the transcriptional targets of ATF4 related to amino acid acquisition and utilization, which are induced as part of an adaptive response to stress, are also harnessed as part of a broader anabolic program downstream of mTORC1 in response to growth signals, including those from oncogenic signaling pathways.