Activation of a Metabolic Gene Regulatory Network Downstream of mTOR Complex 1

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1α) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease

Potent, selective, and orally bioavailable inhibitors of VPS34 provide chemical tools to modulate autophagy in vivo

Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools but selective inhib-itors are now available to modulate the PI 3-kinase VPS34 which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models

Relieving autophagy and 4EBP1 from rapamycin resistance

The mammalian target of rapamycin complex 1 (mTORC1) is a multiprotein signaling complex regulated by oncogenes and tumor suppressors. Outputs downstream of mTORC1 include ribosomal protein S6 kinase 1 (S6K1), eukaryotic translation initiation factor 4E (eIF4E) and autophagy, and their modulation leads to changes in cell growth, proliferation and metabolism. Rapamycin, an allosteric mTORC1 inhibitor, does not antagonize equally these outputs, but the reason for this is unknown. Here, we show that the ability of rapamycin to activate autophagy in different cell lines correlates with mTORC1 stability. Rapamycin exposure destabilizes mTORC1, but in cell lines where autophagy is drug-insensitive, higher levels of mTOR-bound raptor are detected, as compared to cells where rapamycin stimulates autophagy. Using siRNA we find that knockdown of raptor relieves autophagy and the eIF4E effector pathway from rapamycin resistance. Importantly, non-efficacious concentrations of an ATP-competitive mTOR inhibitor can be combined with rapamycin to synergistically inhibit mTORC1, activate autophagy, but leave mTORC2 signaling intact. These data suggest that partial inhibition of mTORC1 by rapamycin can be overcome using combination strategies and offer a therapeutic avenue to achieve complete and selective inhibition of mTORC1

Down-regulation of class II phosphoinositide 3-kinase alpha expression below a critical threshold induces apoptotic cell death.

Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy

Activation of a metabolic gene regulatory network downstream of mTOR complex 1

Aberrant activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a common molecular event in a large variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease

Potent, Selective, and Orally Bioavailable Inhibitors of VPS34 Provide Chemical Tools to Modulate Autophagy <i>in Vivo</i>

Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools, but selective inhibitors are now available to modulate the PI 3-kinase VPS34, which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models

Bidirectional transport of amino acids regulates mTOR and autophagy

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.Peer reviewe