Morris E. Feldman

Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.

The translational landscape of mTOR signalling steers cancer initiation and metastasis

The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the ‘cancerous’ translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.

Genetic dissection of the oncogenic mTOR pathway reveals druggable addiction to translational control via 4EBP-eIF4E.

We genetically dissect the contribution of the most prominent downstream translational components of mTOR signaling toward Akt-driven lymphomagenesis. While phosphorylation of rpS6 is dispensable for cancer formation, 4EBP-eIF4E exerts significant control over cap-dependent translation, cell growth, cancer initiation, and progression. This effect is mediated at least in part through 4EBP-dependent control of Mcl-1 expression, a key antiapoptotic protein. By using an active site inhibitor of mTOR, PP242, we show a marked therapeutic response in rapamycin-resistant tumors. The therapeutic benefit of PP242 is mediated through inhibition of mTORC1-dependent 4EBP-eIF4E hyperactivation. Thus, the 4EBP-eIF4E axis downstream of mTOR is a druggable mediator of translational control and Akt-mediated tumorigenesis that has important implications for the treatment of human cancers.

Akt and Autophagy Cooperate to Promote Survival of Drug-Resistant Glioma

Combined inhibition of PI3K, mTOR, and autophagy promotes glioma cell death. Blocking All Escape Routes Many cancers, including glioma, are associated with increased signaling through the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway, which promotes cell growth, proliferation, and survival. This suggests that pharmacological inhibition of key kinases in this pathway could provide an approach to antineoplastic therapy. Disappointingly, however, inhibitors of PI3K, Akt, or mTOR typically block cancer cell growth rather than eliciting the death of malignant cells, limiting their utility as antineoplastic agents. Noting that autophagy, a process of autodigestion that can enable cells to endure periods of stress and nutrient deprivation, could provide a survival mechanism under conditions of decreased PI3K-Akt-mTOR signaling, Fan et al. explored the effects of various combinations of kinase and autophagy inhibitors on glioma cell survival. Inhibition of mTOR complex 1 (mTORC1) with rapamycin induced autophagy; however, cells survived the combination of rapamycin with inhibitors of autophagy by activating Akt signaling. In contrast, the combined inhibition of mTORC1, PI3K, and autophagy, or that of mTORC1, mTORC2, and autophagy, triggered apoptosis—the process of programmed cell death. The authors elicited cell death with combinations of drugs that are either now in use in patients or in clinical trials, raising the hope that this approach could be readily translatable to human therapy. Although the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway promotes survival signaling, inhibitors of PI3K and mTOR induce minimal cell death in PTEN (phosphatase and tensin homolog deleted from chromosome 10) mutant glioma. Here, we show that the dual PI3K-mTOR inhibitor PI-103 induces autophagy in a form of glioma that is resistant to therapy. Inhibitors of autophagosome maturation cooperated with PI-103 to induce apoptosis through the mitochondrial pathway, indicating that the cellular self-digestion process of autophagy acted as a survival signal in this setting. Not all inhibitors of mTOR synergized with inhibitors of autophagy. Rapamycin delivered alone induced autophagy, yet cells survived inhibition of autophagosome maturation because of rapamycin-mediated activation of Akt. In contrast, adenosine 5′-triphosphate–competitive inhibitors of mTOR stimulated autophagy more potently than did rapamycin, with inhibition of mTOR complexes 1 and 2 contributing independently to induction of autophagy. We show that combined inhibition of PI3K and mTOR, which activates autophagy without activating Akt, cooperated with inhibition of autophagy to cause glioma cells to undergo apoptosis. Moreover, the PI3K-mTOR inhibitor NVP-BEZ235, which is in clinical use, synergized with the lysosomotropic inhibitor of autophagy, chloroquine, another agent in clinical use, to induce apoptosis in glioma xenografts in vivo, providing a therapeutic approach potentially translatable to humans.

Constitutive mTORC1 activation by a herpesvirus Akt surrogate stimulates mRNA translation and viral replication

All viruses require cellular ribosomes to translate their mRNAs. Viruses producing methyl-7 (m⁷) GTP-capped mRNAs, like Herpes Simplex Virus-1 (HSV-1), stimulate cap-dependent translation by activating mTORC1 to inhibit the translational repressor 4E-binding protein 1 (4E-BP1). Here, we establish that the HSV-1 kinase Us3 masquerades as Akt to activate mTORC1. Remarkably, Us3 displays no sequence homology with the cellular kinase Akt, yet directly phosphorylates tuberous sclerosis complex 2 (TSC2) on the same sites as Akt. TSC2 depletion rescued Us3-deficient virus replication, establishing that Us3 enhances replication by phosphorylating TSC2 to constitutively activate mTORC1, effectively bypassing S6K-mediated feedback inhibition. Moreover, Us3 stimulated Akt substrate phosphorylation in infected cells, including FOXO1 and GSK3. Thus, HSV-1 encodes an Akt surrogate with overlapping substrate specificity to activate mTORC1, stimulating translation and virus replication. This establishes Us3 as a unique viral kinase with promising drug development potential.

mTOR Complex-2 Activates ENaC by Phosphorylating SGK1

The serum- and glucocorticoid-induced kinase 1 (SGK1) plays a central role in hormone regulation of epithelial sodium (Na+) channel (ENaC)-dependent Na+ transport in the distal nephron. Phosphorylation within a carboxy-terminal domain, designated the hydrophobic motif (HM), determines the activity of SGK1, but the identity of the HM kinase is unknown. Here, we show that the highly conserved serine-threonine kinase mammalian target of rapamycin (mTOR) is essential for the phosphorylation of the HM of SGK1 and the activation of ENaC. We observed that mTOR, in conjunction with rictor (mTORC2), phosphorylated SGK1 and stimulated ENaC. In contrast, when mTOR assembled with raptor in the rapamycin-inhibited complex (mTORC1), it did not phosphorylate SGK1 or stimulate ENaC. Inhibition of mTOR blocked both SGK1 phosphorylation and ENaC-mediated Na+ transport, whereas specific inhibition of mTORC1 had no effect. Similarly, small hairpin RNA-mediated knockdown of rictor inhibited SGK1 phosphorylation and Na+ current, whereas knockdown of raptor had no effect. Finally, in co-immunoprecipitation experiments, SGK1 interacted selectively with rictor but not with raptor, suggesting selective recruitment of SGK1 to mTORC2. We conclude that mTOR, specifically mTORC2, is the HM kinase for SGK1 and is required for ENaC-mediated Na+ transport, thereby extending our understanding of the molecular mechanisms underlying Na+ balance.

New Inhibitors of the PI3K-Akt-mTOR Pathway: Insights into mTOR Signaling from a New Generation of Tor Kinase Domain Inhibitors (TORKinibs)

mTOR (mammalian Target of Rapamycin) is the hub of the phosphoinositide 3-Kinase (PI3-K)→Akt→mTOR pathway, which is one of the most commonly mutated pathways in cancer. PI3-Ks and mTOR are related kinases which share an evolutionarily related kinase domain, although the former is a lipid kinase and the latter is a protein kinase. As a result of their similar ATP sites, the prototypical PI3-K inhibitors LY294002 and wortmannin inhibit both kinases, although the compounds have been primarily thought of as inhibitors of PI3-Ks. The widespread use of these reagents to understand PI3-K signaling and the likelihood that many of their effects are confounded by dual inhibition of PI3-K and mTOR make it essential to develop selective mTOR inhibitors in part to understand the unique cellular effects of inhibition of this key downstream component in the growth factor pathway. Rapamycin has historically provided a means for selective mTOR inhibition, yet it is not a typical ATP competitive inhibitor, making its effects difficult to reconcile with LY294002 and wortmannin. Several groups have recently reported pharmacological agents which inhibit mTOR but not PI3-K, providing a new pharmacological approach to selective mTOR inhibition. The TOR kinase domain inhibitors of mTOR have been termed TORKinibs to distinguish their mode of action from rapamycin and its analogs (rapalogs). These inhibitors bind to the ATP binding site of the kinase domain of mTOR and as a result inhibit both mTOR complexes, TORC1 (rapamycin sensitive) and TORC2 (rapamycin resistant). These molecules have allowed a reinvestigation of mTOR and in particular a reinvestigation of the mechanistic basis for incomplete proliferative arrest of cells by Rapamycin. A consensus has quickly emerged from the study of various TORKinibs that Rapamycin is ineffective at blocking cell proliferation because it only partially inhibits the activity of mTORC1. The profound anti-proliferative effect of TORKinibs suggests that as the molecules enter the clinic they may be successful in the treatment of cancers where rapamycin has failed.

Combination of ATP-competitive mammalian target of rapamycin inhibitors with standard chemotherapy for colorectal cancer

ATP-competitive mammalian target of rapamycin (mTOR) inhibitors are in early phase clinical trials. These novel targeted agents, including PP242, are mechanistically distinct from the allosteric, partial mTOR inhibitor, rapamycin. The goal of this study was to evaluate how PP242 best combines with standard chemotherapies for colorectal cancer (CRC), and which subsets of patients are most likely to benefit. The combination index for PP242 plus 5-fluorouracil, oxaliplatin, or irinotecan was determined in CRC cell lines with different mutational backgrounds. In KRAS mutant CRC cell lines, sensitivity to PP242 increases with co-mutation of PIK3CA. Mutation of p53 predicts resistance to chemotherapy, but not PP242. Efficacy of PP242 was comparable to that of standard chemotherapies over the dose range tested. Sensitivity or resistance to PP242 dictates relative synergy or antagonism, respectively, when PP242 is combined with 5-fluorouracil. The same trend exists for PP242 + oxaliplatin, but with a narrower dynamic range. Conversely potency of PP242 and the combination index for PP242 + irinotecan were unrelated, but synergy exists across all dose levels in PP242 and irinotecan sensitive, p53 wild-type cell lines. Overall, our in vitro analysis predicts that mutational status can be used to rank sensitivity to PP242 and standard chemotherapies. Single agent potency can in turn be used to predict the combination index in a drug-specific manner. Our data suggest a clinical trial to determine whether ATP-competitive mTOR inhibitors provide benefit in combination with standard chemotherapies for patients with PIK3CA mutant metastatic CRC, stratified by the presence or absence of KRAS co-mutation.