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High-frequency relapse remains a clinical hurdle for complete remission of T-cell acute lymphoblastic leukemia (T-ALL) patients, with heterogeneous dysregulated signaling profiles—including of Raf-MEK-ERK and Akt-mTORC1-S6K signaling pathways—recently being implicated in disease outcomes. Here we report that GM-CSF/IL-3/IL-5 receptor common β-chain-associated protein (CBAP) is highly expressed in human T-ALL cell lines and many primary tumor tissues and is required to bolster leukemia cell proliferation in tissue culture and for in vivo leukemogenesis in a xenograft mouse model. Downregulation of CBAP markedly restrains expansion of leukemia cells and alleviates disease aggravation of leukemic mice. Transcriptomic profiling and molecular biological analyses suggest that CBAP acts upstream of Ras and Rac1, and functions as a modulator of both Raf-MEK–ERK and Akt-mTORC1 signaling pathways to control leukemia cell growth. Specifically, CBAP facilitated Akt-dependent TSC2 phosphorylation in cell-based assays and in vitro analysis, decreased lysosomal localization of TSC2, and elevated Rheb-GTP loading and subsequent activation of mTORC1 signaling. Taken together, our findings reveal a novel oncogenic contribution of CBAP in T-ALL leukemic cells, in addition to its original pro-apoptotic function in cytokine-dependent cell lines and primary hematopoietic cells, by demonstrating its functional role in the regulation of Akt-TSC2-mTORC1 signaling for leukemia cell proliferation. Thus, CBAP represents a novel therapeutic target for many types of cancers and metabolic diseases linked to PI3K-Akt-mTORC1 signaling.

Rheb is a Ras family GTPase, which binds to and activates mammalian target of rapamycin complex 1 (mTORC1) when GTP loaded. Recently, cancer genome sequencing efforts have identified recurrent Rheb Tyr35Asn mutations in kidney and endometrial carcinoma. Here we show that Rheb-Y35N causes not only constitutive mTORC1 activation, but sustained activation of the MEK-ERK pathway in a TSC1/TSC2/TBC1D7 protein complex and mTORC1-independent manner, contributing to intrinsic resistance to rapamycin. Rheb-Y35N transforms NIH3T3 cells, resulting in aggressive tumor formation in xenograft nude mice, which could be suppressed by combined treatment with rapamycin and an extracellular signal-regulated kinase (ERK) inhibitor. Furthermore, Rheb-Y35N inhibits AMPKα activation in response to nutrient depletion or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), leading to attenuated phosphorylation of BRAF-S729 and retained mitogen-activated protein kinase (MAPK) activation. Finally, we demonstrate that Rheb-WT can bind AMPK to facilitate AMPK activation, whereas Rheb-Y35N competitively binds AMPK, impairing AMPK phosphorylation. In summary, our findings indicate that Rheb-Y35N is a dominantly active tumor driver that activates both mTORC1 and MAPK to promote tumor growth, suggesting a combination of mTORC1 and MAPK inhibitors may be of therapeutic value in patients whose cancers sustain this mutation.

Tuberous sclerosis complex (TSC) is an autosomally inherited disorder that causes tumors to form in many organs. It is frequently caused by inactivating mutations in the TSC2 tumor-suppressor gene. TSC2 negatively regulates the activity of the GTPase Rheb and thereby inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. Activation of mTORC1 as a result of lack of TSC2 function is observed in TSC and sporadic lymphangioleiomyomatosis (LAM). TSC2 deficiency has recently been associated with elevated AMP-activated protein kinase (AMPK) activity, which in turn correlated with cytoplasmic localization of p27Kip1 (p27), a negative regulator of cyclin-dependent kinase 2 (Cdk2). How AMPK in the absence of TSC2 is stimulated is not fully understood. In this study, we demonstrate that Rheb activates AMPK and reduces p27 levels in Tsc2-null cells. Importantly, both effects occur largely independent of mTORC1. Furthermore, increased p27 levels following Rheb depletion correlated with reduced Cdk2 activity and cell proliferation in vitro, and with inhibition of tumor formation by Tsc2-null cells in vivo. Taken together, our data suggest that Rheb controls proliferation of TSC2-deficient cells by a mechanism that involves regulation of AMPK and p27, and that Rheb is a potential target for TSC/LAM therapy.

The Rheb1 and Rheb2 small GTPases and their effector mTOR are aberrantly activated in human cancer and are attractive targets for anti-cancer drug discovery. Rheb is targeted to endomembranes via its C-terminal CAAX (C=cysteine, A=aliphatic, X=terminal amino acid) motif, a substrate for posttranslational modification by a farnesyl isoprenoid. After farnesylation, Rheb undergoes two additional CAAX-signaled processing steps, Ras converting enzyme 1 (Rce1)-catalyzed cleavage of the AAX residues and isoprenylcysteine carboxyl methyltransferase (Icmt)-mediated carboxylmethylation of the farnesylated cysteine. However, whether these postprenylation processing steps are required for Rheb signaling through mTOR is not known. We found that Rheb1 and Rheb2 localize primarily to the endoplasmic reticulum and Golgi apparatus. We determined that Icmt and Rce1 processing is required for Rheb localization, but is dispensable for Rheb-induced activation of the mTOR substrate p70 S6 kinase (S6K). Finally, we evaluated whether farnesylthiosalicylic acid (FTS) blocks Rheb localization and function. Surprisingly, FTS prevented S6K activation induced by a constitutively active mTOR mutant, indicating that FTS inhibits mTOR at a level downstream of Rheb. We conclude that inhibitors of Icmt and Rce1 will not block Rheb function, but FTS could be a promising treatment for Rheb- and mTOR-dependent cancers.

Rheb (Ras homolog enriched in brain) is a component of the phosphatidylinositol 3-kinase (PI3K)–target of rapamycin (TOR) signaling pathway, functioning as a positive regulator of TOR. Constitutively active mutants of Rheb induce oncogenic transformation in cell culture. The transformed cells are larger and contain more protein than their normal counterparts. They show constitutive phosphorylation of the ribosomal protein S6 kinase (S6K) and the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), two downstream targets of TOR. The TOR-specific inhibitor rapamycin strongly interferes with transformation induced by constitutively active Rheb, suggesting that TOR activity is essential for the oncogenic effects of mutant Rheb. Rheb-induced transformation is also dependent on a C-terminal farnesylation signal that mediates localization to a cellular membrane. An engineered N-terminal myristylation signal can substitute for the farnesylation. Immunofluorescence localizes wild type (wt) and mutant Rheb to vesicular structures in the cytoplasm, overlapping with the endoplasmic reticulum (ER).

The mTOR/S6K/4E-BP1 pathway integrates extracellular signals derived from growth factors, and intracellular signals, determined by the availability of nutrients like amino acids and glucose. Activation of this pathway requires inhibition of the tumor suppressor complex TSC1/2. TSC2 is a GTPase-activating protein for the small GTPase Ras homologue enriched in brain (Rheb), GTP loading of which activates mTOR by a yet unidentified mechanism. The level at which this pathway senses the availability of amino acids is unknown but is suggested to be at the level of TSC2. Here, we show that amino-acid depletion completely blocks insulin- and TPA-induced Rheb activation. This indicates that amino-acid sensing occurs upstream of Rheb. Despite this, amino-acid depletion can still inhibit mTOR/S6 kinase signaling in TSC2-/- fibroblasts. Since under these conditions Rheb-GTP levels remain high, a second level of amino-acid sensing exists, affecting mTOR activity in a Rheb-independent fashion.

Cell growth (an increase in cell mass and size through macromolecular biosynthesis) and cell cycle progression are generally tightly coupled, allowing cells to proliferate continuously while maintaining their size. The target of rapamycin (TOR) is an evolutionarily conserved kinase that integrates signals from nutrients (amino acids and energy) and growth factors (in higher eukaryotes) to regulate cell growth and cell cycle progression coordinately. In mammals, TOR is best known to regulate translation through the ribosomal protein S6 kinases (S6Ks) and the eukaryotic translation initiation factor 4E-binding proteins. Consistent with the contribution of translation to growth, TOR regulates cell, organ, and organismal size. The identification of the tumor suppressor proteins tuberous sclerosis1 and 2 (TSC1 and 2) and Ras-homolog enriched in brain (Rheb) has biochemically linked the TOR and phosphatidylinositol 3-kinase (PI3K) pathways, providing a mechanism for the crosstalk that occurs between these pathways. TOR is emerging as a novel antitumor target, since the TOR inhibitor rapamycin appears to be effective against tumors resulting from aberrantly high PI3K signaling. Not only may inhibition of TOR be effective in cancer treatment, but rapamycin is an FDA-approved immunosuppressive and cardiology drug. We review here what is known (and not known) about the function of TOR in cellular and animal physiology.