The TOR complex 1 is a direct target of Rho1 GTPase.

The TOR complex 1 (TORC1) in yeast is regulated by various stress conditions. However, the underlying mechanism is poorly understood. In this study, we show that stresses affect TORC1 function through Rho1, a member of Rho family GTPases. Upon activation by stresses, Rho1 binds directly to Kog1, a unique component of TORC1, resulting in downregulation of TORC1 activity and disruption of its membrane association. The binding also triggers the release and activation of the Tap42-2A phosphatase, a major effector of TORC1 that resides on the complex. Rapamycin and caffeine also induce Rho1 activation. While the two agents inhibit TOR directly, their effects on TORC1 signaling are largely dependent on Rho1 activation. Our findings demonstrate that TORC1 acts both upstream and downstream of Rho1 GTPase, unveiling a mechanism that integrates stress and nutrient signals to coordinate Rho1-mediated spatial expansion and TORC1-dependent mass increase.

General Control Nonderepressible 2 (GCN2) Kinase Inhibits Target of Rapamycin Complex 1 in Response to Amino Acid Starvation in Saccharomyces cerevisiae.

In eukaryotic cells, two conserved protein kinases, Gcn2 and TOR complex 1 (TORC1), couple amino acid conditions to protein translation. Gcn2 functions as an amino acid sensor and is activated by uncharged tRNAs that accumulate when intracellular amino acids are limited. Activated Gcn2 phosphorylates and inhibits eukaryotic initiation factor-2α (eIF2α), resulting in repression of general protein synthesis. Like Gcn2, TORC1 is also involved in sensing amino acid conditions. However, the underlying mechanism remains unclear. In the present study, we show that TORC1 is a direct target of Gcn2 kinase in the yeast Saccharomyces cerevisiae In response to amino acid starvation, Gcn2 binds to TORC1 and phosphorylates Kog1, the unique regulatory subunit of TORC1, resulting in down-regulation of TORC1 kinase activity. In the absence of Gcn2, TORC1 signaling activity increases and becomes unresponsive to amino acid starvation. Our findings demonstrate that TORC1 is an effector of Gcn2 in amino acid signaling, hence defining a novel mechanism by which TORC1 senses amino acid starvation.

Target of rapamycin complex 1 and Tap42-associated phosphatases are required for sensing changes in nitrogen conditions in the yeast Saccharomyces cerevisiae.

In yeast target of rapamycin complex 1 (TORC1) and Tap42-associated phosphatases regulate expression of genes involved in nitrogen limitation response and the nitrogen discrimination pathway. However, it remains unclear whether TORC1 and the phosphatases are required for sensing nitrogen conditions. Utilizing temperature sensitive mutants of tor2 and tap42, we examined the role of TORC1 and Tap42 in nuclear entry of Gln3, a key transcription factor in yeast nitrogen metabolism, in response to changes in nitrogen conditions. Our data show that TORC1 is essential for Gln3 nuclear entry upon nitrogen limitation and downshift in nitrogen quality. However, Tap42-associated phosphatases are required only under nitrogen limitation condition. In cells grown in poor nitrogen medium, the nitrogen permease reactivator kinase (Npr1) inhibits TORC1 activity and alters its association with Tap42, rendering Tap42-associated phosphatases unresponsive to nitrogen limitation. These findings demonstrate a direct role for TORC1 and Tap42-associated phosphatases in sensing nitrogen conditions and unveil an Npr1-dependent mechanism that controls TORC1 and the phosphatases in response to changes in nitrogen quality.

Tumor Suppressor Folliculin Regulates mTORC1 through Primary Cilia.

Folliculin (FLCN) is the tumor suppressor associated with Birt-Hogg-Dubé (BHD) syndrome that predisposes patients to incident of hamartomas and cysts in multiple organs. Its inactivation causes deregulation in the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. However, the underlying mechanism is poorly defined. In this study, we show that FLCN is a ciliary protein that functions through primary cilia to regulate mTORC1. In response to flow stress, FLCN associates with LKB1 and recruits the kinase to primary cilia for activation of AMPK resided at basal bodies, which causes mTORC1 down-regulation. In cells depleted of FLCN, LKB1 fails to accumulate in primary cilia and AMPK at the basal bodies remains inactive, thus nullifying the inhibitory effect of flow stress on mTORC1 activity. Our results demonstrate that FLCN is part of a flow sensory mechanism that regulates mTORC1 through primary cilia.

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth-related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12-rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2(W2041R) ) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non-covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.

Regulation of mammalian target of rapamycin complex 1 by Bcl-2 and Bcl-XL proteins.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and metabolism. Its activity is controlled by various types of signals, including growth factors, nutrients, and stresses. In this study, we show that changes in expression levels of two antiapoptotic proteins, Bcl-2 and Bcl-XL, also affect mTORC1 signaling activity. In cells overexpressing Bcl-XL, mTORC1 activity is increased and becomes less sensitive to growth factor or nutrient conditions. In contrast, reduction in expression levels of the two antiapoptotic proteins inhibits mTORC1 signaling activity. Our results suggest that the effect of Bcl-2 and Bcl-XL on mTORC1 is mediated by FKBP38, an inhibitor of mTORC1. The two proteins compete with mTORC1 for FKBP38 binding and hence alter mTORC1 activity. This study reveals a novel cross-talk between Bcl-2/XL and mTORC1 signaling, which is likely to contribute to cancer development.

Precision Combination Therapies Based on Recurrent Oncogenic Coalterations.

Cancer cells depend on multiple driver alterations whose oncogenic effects can be suppressed by drug combinations. Here, we provide a comprehensive resource of precision combination therapies tailored to oncogenic coalterations that are recurrent across patient cohorts. To generate the resource, we developed Recurrent Features Leveraged for Combination Therapy (REFLECT), which integrates machine learning and cancer informatics algorithms. Using multiomic data, the method maps recurrent coalteration signatures in patient cohorts to combination therapies. We validated the REFLECT pipeline using data from patient-derived xenografts, in vitro drug screens, and a combination therapy clinical trial. These validations demonstrate that REFLECT-selected combination therapies have significantly improved efficacy, synergy, and survival outcomes. In patient cohorts with immunotherapy response markers, DNA repair aberrations, and HER2 activation, we have identified therapeutically actionable and recurrent coalteration signatures. REFLECT provides a resource and framework to design combination therapies tailored to tumor cohorts in data-driven clinical trials and preclinical studies. SIGNIFICANCE: We developed the predictive bioinformatics platform REFLECT and a multiomics- based precision combination therapy resource. The REFLECT-selected therapies lead to significant improvements in efficacy and patient survival in preclinical and clinical settings. Use of REFLECT can optimize therapeutic benefit through selection of drug combinations tailored to molecular signatures of tumors. See related commentary by Pugh and Haibe-Kains, p. 1416. This article is highlighted in the In This Issue feature, p. 1397.

BET inhibition induces vulnerability to MCL1 targeting through upregulation of fatty acid synthesis pathway in breast cancer.

Therapeutic options for treatment of basal-like breast cancers remain limited. Here, we demonstrate that bromodomain and extra-terminal (BET) inhibition induces an adaptive response leading to MCL1 protein-driven evasion of apoptosis in breast cancer cells. Consequently, co-targeting MCL1 and BET is highly synergistic in breast cancer models. The mechanism of adaptive response to BET inhibition involves the upregulation of lipid synthesis enzymes including the rate-limiting stearoyl-coenzyme A (CoA) desaturase. Changes in lipid synthesis pathway are associated with increases in cell motility and membrane fluidity as well as re-localization and activation of HER2/EGFR. In turn, the HER2/EGFR signaling results in the accumulation of and vulnerability to the inhibition of MCL1. Drug response and genomics analyses reveal that MCL1 copy-number alterations are associated with effective BET and MCL1 co-targeting. The high frequency of MCL1 chromosomal amplifications (>30%) in basal-like breast cancers suggests that BET and MCL1 co-targeting may have therapeutic utility in this aggressive subtype of breast cancer.

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress.

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.

Signal-Oriented Pathway Analyses Reveal a Signaling Complex as a Synthetic Lethal Target for p53 Mutations.

Defining processes that are synthetic lethal with p53 mutations in cancer cells may reveal possible therapeutic strategies. In this study, we report the development of a signal-oriented computational framework for cancer pathway discovery in this context. We applied our bipartite graph-based functional module discovery algorithm to identify transcriptomic modules abnormally expressed in multiple tumors, such that the genes in a module were likely regulated by a common, perturbed signal. For each transcriptomic module, we applied our weighted k-path merge algorithm to search for a set of somatic genome alterations (SGA) that likely perturbed the signal, that is, the candidate members of the pathway that regulate the transcriptomic module. Computational evaluations indicated that our methods-identified pathways were perturbed by SGA. In particular, our analyses revealed that SGA affecting TP53, PTK2, YWHAZ, and MED1 perturbed a set of signals that promote cell proliferation, anchor-free colony formation, and epithelial-mesenchymal transition (EMT). These proteins formed a signaling complex that mediates these oncogenic processes in a coordinated fashion. Disruption of this signaling complex by knocking down PTK2, YWHAZ, or MED1 attenuated and reversed oncogenic phenotypes caused by mutant p53 in a synthetic lethal manner. This signal-oriented framework for searching pathways and therapeutic targets is applicable to all cancer types, thus potentially impacting precision medicine in cancer. Cancer Res; 76(23); 6785-94. ©2016 AACR.

TOR under stress: targeting TORC1 by Rho1 GTPase.

In the yeast Saccharomyces cerevisiae, small GTPase Rho1 controls polarized actin distribution and cell wall expansion in response to many different environmental and intracellular stimuli. Its activity is essential for cell survival and adaptation under various stress conditions. A recent study identified the TOR complex 1 (TORC1), a central regulator in cell growth and metabolism, as a direct target of the small GTPase. This novel crosstalk extends the signaling network of Rho1 into many TORC1-dependent processes and sheds light on how yeast cells coordinate polarized spatial expansion with mass increase.

A MAP kinase dependent feedback mechanism controls Rho1 GTPase and actin distribution in yeast.

In the yeast Saccharomyces cerevisiae the guanosine triphosphatase (GTPase) Rho1 controls actin polarization and cell wall expansion. When cells are exposed to various environmental stresses that perturb the cell wall, Rho1 activates Pkc1, a mammalian Protein Kinase C homologue, and Mpk1, a mitogen activated protein kinase (MAPK), resulting in actin depolarization and cell wall remodeling. In this study, we demonstrate a novel feedback loop in this Rho1-mediated Pkc1-MAPK pathway that involves regulation of Rom2, the guanine nucleotide exchange factor of Rho1, by Mpk1, the end kinase of the pathway. This previously unrecognized Mpk1-dependent feedback is a critical step in regulating Rho1 function. Activation of this feedback mechanism is responsible for redistribution of Rom2 and cell wall synthesis activity from the bud to cell periphery under stress conditions. It is also required for terminating Rho1 activity toward the Pkc1-MAPK pathway and for repolarizing actin cytoskeleton and restoring growth after the stressed cells become adapted.

Rapamycin activates Tap42-associated phosphatases by abrogating their association with Tor complex 1.

In Saccharomyces cerevisiae, the Tap42-phosphatase complexes are major targets of the Tor kinases in the rapamycin-sensitive signaling pathway. The immunosuppressive agent, rapamycin, induces a prompt activation of the Tap42-associated phosphatases, which is vitally important in Tor-mediated transcriptional regulation. However, the mechanism for the rapid phosphatase activation is poorly understood. In this study, we show that the Tap42-phosphatase complexes exist mainly on membrane structures through their association with Tor complex 1 (TORC1). Rapamycin abrogates this association and releases the Tap42-phosphatase complexes into the cytosol. Disassembly of the Tap42-phosphatase complexes occurs subsequently, following the release but at a much slower rate, presumably caused by Tap42 dephosphorylation. Release of the Tap42-phosphatase complexes from membrane structures also occurs when cells are deprived of nutrient. These findings suggest that the association of the Tap42-phosphatase complexes with TORC1 represents an important mechanism by which nutrient controls Tor signaling activity. In addition, our data support a model in which rapamycin acts not by inhibiting the kinase activity of Tor but by disrupting its interaction with downstream targets.

A single residual replacement improves the folding and stability of recombinant cassava hydroxynitrile lyase in E. coil.

Substitution of Ser113 for Gly113 in the cap domain of hydroxynitrile lyase from Manihot esculenta (MeHNL) was performed by site-directed mutagenesis to improve its self-generated folding and stability under denaturation conditions. The yield of the recombinant mutant HNL1 (mut-HNL1), which had higher specific activity than the wild type HNL0 (wt-HNL0), was increased by 2 to 3-fold. Thermostability of MeHNL was also enhanced, probably due to an increase in content of the beta-strand secondary structure according to CD analysis. Our data in this report suggest that Ser113 significantly contributes to the in vivo folding and stability of MeHNL and demonstrates an economic advantage of mut-HNL1 over the wt-HNL0.