In this review we highlight recent progress in the study of Rheb family GTPases. Structural studies using X-ray crystallography and NMR have given us insight into unique features of this GTPase. Combined with mutagenesis studies, these works have expanded our understanding of residues that affect Rheb GTP/GDP bound ratios, effector protein interactions, and stimulation of mTORC1 signaling. Analysis of cancer genome databases has revealed that several human carcinomas contain activating mutations of the protein. Rheb’s role in activating mTORC1 signaling at the lysosome in response to stimuli has been further elucidated. Rheb has also been suggested to play roles in other cellular pathways including mitophagy and peroxisomal ROS response. A number of studies in mice have demonstrated the importance of Rheb in development, as well as in a variety of functions including cardiac protection and myelination. We conclude with a discussion of future prospects in the study of Rheb family GTPases.

Background: Rheb activates mTORC1 to stimulate mRNA translation. Results: Rheb binds to CAD and activates CPSase activity. Conclusion: Rheb affects intracellular pyrimidine nucleotide pools. Significance: Our results provide a new mechanism that TSC/Rheb signaling regulates cell growth.

BackgroundRHEB is a unique member of the RAS superfamily of small GTPases expressed in all tissues and conserved from yeast to humans. Early studies on RHEB indicated a possible RHEB-RAF interaction, but this has not been fully explored. Recent work on cancer genome databases has revealed a reoccurring mutation in RHEB at the Tyr35 position, and a recent study points to the oncogenic potential of this mutant that involves activation of RAF/MEK/ERK signaling. These developments prompted us to reassess the significance of RHEB effect on RAF, and to compare mutant and wild type RHEB.MethodsTo study RHEB-RAF interaction, and the effect of the Y35N mutation on this interaction, we used transfection, immunoprecipitation, and Western blotting techniques. We generated cell lines stably expressing RHEB WT, RHEB Y35N, and KRAS G12V, and monitored cellular transforming properties through cell proliferation, anchorage independent growth, cell cycle analysis, and foci formation assays.ResultsWe observe a strong interaction between RHEB and BRAF, but not with CRAF. This interaction is dependent on an intact RHEB effector domain and RHEB-GTP loading status. RHEB overexpression decreases RAF activation of the RAF/MEK/ERK pathway and RHEB knockdown results in an increase in RAF/MEK/ERK activation. RHEB Y35N mutation has decreased interaction with BRAF, and RHEB Y35N cells exhibit greater BRAF/CRAF heterodimerization resulting in increased RAF/MEK/ERK signaling. This leads to cancer transformation of RHEB Y35N stably expressing cell lines, similar to KRAS G12 V expressing cell lines.ConclusionsRHEB interaction with BRAF is crucial for inhibiting RAF/MEK/ERK signaling. The RHEB Y35N mutant sustains RAF/MEK/ERK signaling due to a decreased interaction with BRAF, leading to increased BRAF/CRAF heterodimerization. RHEB Y35N expressing cells undergo cancer transformation due to decreased interaction between RHEB and BRAF resulting in overactive RAF/MEK/ERK signaling. Taken together with the previously established function of RHEB to activate mTORC1 signaling, it appears that RHEB performs a dual function; one is to suppress the RAF/MEK/ERK signaling and the other is to activate mTORC1 signaling.Electronic supplementary materialThe online version of this article (10.1186/s12885-017-3938-5) contains supplementary material, which is available to authorized users.

Tuberous sclerosis complex (TSC) is a multisystem disorder that affects multiple organ systems, including tumors of the brain, heart, kidney, skin and lung. TSC is caused by germline loss-of-function gene mutations in TSC1 or TSC2, which inhibit the mammalian target of rapamycin (mTOR) signaling pathway. Rapalogs are effective cytostatic agents for the treatment of TSC, but continual lifelong therapy is needed. Therapies for TSC that induce a selective cytocidal response in TSC-deficient cells are not currently available, and could have a major clinical impact for TSC patients. We performed a quantitative high-throughput screen (qHTS) of the NCATS Pharmaceutical Collection (NPC) of 2816 compounds, many of which are drugs already approved for other diseases, for their effect on the proliferation of TSC2-deficient ELT3 cells vs. ELT3 cells in which TSC2 was re-expressed. Agents were tested at fifteen concentrations, from 0.6 nM to 46 µM. Six agents selectively inhibited the growth of the TSC2-deficient cells relative to the TSC2-reexpressing cells. One of top inhibitory agents, methotrexate (MTX), a dihydrofolate reductase inhibitor, is FDA-approved, making it a candidate for “repurposing”. In our studies, we confirmed the selective inhibitory effect of MTX on patient-derived TSC2-deficient cells. Flow cytometry analysis demonstrated that MTX (5 µM, 24 h) arrested 60.8% of the TSC2-deficient cells in S-phase, vs. 26.6% of the TSC2-expressing cells. The accumulation of S-phase arrested cells ultimately leads to apoptosis selectively in the TSC2-deficient cells, which was detected by flow cytometry of Propidium Iodide/Annexin V stained cells (52.3% of apoptosis in TSC2-deficient cells vs. 11% in TSC2-reexpressing cells) and immunoblotting for cleaved Caspase3 and cleaved PARP. Co-treatment with the mTOR inhibitor Rapamycin and MTX prevented MTX-induced apoptosis (52.3% apoptotic cells with MTX alone vs. 11.8% with MTX plus Rapamycin). In vivo, mice bearing TSC2-deficient ELT3 cell xenograft tumors were treated with MTX (25mg/kg or 50mg/kg) or rapamycin (1mg/kg) by intraperitoneal injection every other day over 3 weeks. We found that 50mg/kg of MTX significantly decreased the volume of tumors within two weeks of treatment, to a similar extent as Rapamycin treatment did. We conclude that Methotrexate acts as a selective cytotoxic agent in TSC2-deficient cells. This is consistent with recent discoveries that mTOR regulates the de novo purine and pyrimidine synthesis pathways. As an FDA-approved agent, Methotrexate has potential for clinical “repurposing” for the treatment of TSC-deficient tumors. Citation Format: Amine Belaid, Harilaos Filippakis, Hilaire Lam, Alexander Valvezan, Ruili Huang, Menghang Xia, Tianmu Wen, Christian Baglini, Srilatha Sakamuru, John Asara, Nguyen Truong Sinh, Jeffrey J. Heard, Fuyuhiko Tamanoi, Christopher Austin, Brendan Manning, Elizabeth P. Henske. Therapeutic targeting of TSC2-deficient cells with Methotrexate: Results of a drug repurposing screen. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A09.

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