Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition

mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC) cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs) and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.     

Inhibition of autophagy enhances apoptosis induced by the PI3K/AKT/mTor inhibitor NVP‐BEZ235 in renal cell carcinoma cells

The PI3K/AKT/mTOR pathway plays a key role in the development of the hypervascular tumor renal cell carcinoma (RCC). NVP‐BEZ235 (NVP), a novel dual PI3K/mTOR inhibitor, showed great antitumor benefit and provided a treatment strategy in RCC. In this study, we test the effect of NVP on survival rate, apoptosis and autophagy in the RCC cell line, 786‐0. We also explore the hypothesis that NVP, in combination with autophagy inhibitors, leads to apoptosis enhancement in 786‐0 cells. The results showed that the PI3K/AKT/mTOR pathway proteins p‐AKT and p‐P70S6K were highly expressed in RCC tissue. We also showed that NVP inhibited cell growth and induced apoptosis and autophagy in RCC cells. The combination treatment of NVP with autophagy inhibitors enhanced the effect of NVP on suppressing 786‐0 growth and induction of apoptosis. This study proposes a novel treatment paradigm where combining PI3K/AKT/mTOR pathway inhibitors and autophagy inhibitors lead to enhanced RCC cell apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.     

The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.     

Allosteric and ATP-competitive kinase inhibitors of mTOR for cancer treatment

Over the past few years a number of components of the PI3K/mTOR pathway have been the subject of intense drug discovery activities both in pharmaceutical companies and in academia. This review article summarizes progress made in the identification and development of allosteric and ATP-competitive kinase inhibitors of mTOR and their potential therapeutic use in oncology.     

mTOR Inhibition Induces Compensatory,Therapeutically Targetable MEK Activation in Renal Cell Carcinoma

Rapamycin derivatives allosterically targeting mTOR are currently FDA approved to treat advanced renal cell carcinoma (RCC), and catalytic inhibitors of mTOR/PI3K are now in clinical trials for treating various solid tumors. We sought to investigate the relative efficacy of allosteric versus catalytic mTOR inhibition, evaluate the crosstalk between the mTOR and MEK/ERK pathways, as well as the therapeutic potential of dual mTOR and MEK inhibition in RCC. Pharmacologic (rapamycin and BEZ235) and genetic manipulation of the mTOR pathway were evaluated by in vitro assays as monotherapy as well as in combination with MEK inhibition (GSK1120212). Catalytic mTOR inhibition with BEZ235 decreased proliferation and increased apoptosis better than allosteric mTOR inhibition with rapamycin. While mTOR inhibition upregulated MEK/ERK signaling, concurrent inhibition of both pathways had enhanced therapeutic efficacy. Finally, primary RCC tumors could be classified into subgroups [(I) MEK activated, (II) Dual MEK and mTOR activated, (III) Not activated, and (IV) mTOR activated] based on their relative activation of the PI3K/mTOR and MEK pathways. Patients with mTOR only activated tumors had the worst prognosis. In summary, dual targeting of the mTOR and MEK pathways in RCC can enhance therapeutic efficacy and primary RCC can be subclassified based on their relative levels of mTOR and MEK activation with potential therapeutic implications.     

Targeting mTOR globally in cancer: Thinking beyond rapamycin

The mammalian target of rapamycin (mTOR) is centrally involved in growth, survival and metabolism. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for drug development. Until recently, we have relied largely on the use of rapamycin to study mTOR function and its anticancer potential. Recent insights now indicate that rapamycin is a partial inhibitor of mTOR through allosteric inhibition of mTOR complex-1 (mTORC1) but not mTOR complex-2 (mTORC2). Both the mechanism of action and the cellular response to mTORC1 inhibition by rapamycin and related drugs may limit the effectiveness of these compounds as antitumor agents. We and others have recently reported the discovery of second-generation ATP-competitive mTOR kinase inhibitors (TKIs) that bind to the active sites of mTORC1 and mTORC2, thereby targeting mTOR signaling function globally (see refs. 1-4). The discovery of specific, active-site mTOR inhibitors has opened a new chapter in the 40-plus year old odyssey that began with the discovery of rapamycin from a soil sample collected on Easter Island (see Vézina C, et al. J Antibiot 1975). Here, we discuss recent studies that highlight the emergence of rapamycin-resistant mTOR function in protein synthesis, cell growth, survival and metabolism. It is shown that these rapamycin-resistant mTOR functions are profoundly inhibited by TKIs. A more complete suppression of mTOR global signaling network by the new inhibitors is expected to yield a deeper and broader antitumor response in the clinic.     

Recent progress towards clinically relevant ATP-competitive Akt inhibitors

The frequency of PI3K/Akt/mTOR (PAM) Pathway mutations in human cancers sparked interest to determine if the pathway is druggable. The modest clinical benefit observed with mTOR rapalogs (temsirolimus and everolimus) provided further motivation to identify additional nodes of pathway inhibition that lead to improved clinical benefit. Akt is a central signaling node of the PAM pathway and could be an ideal target for improved pathway inhibition. Furthermore, inhibitors of Akt may be especially beneficial in tumors with Akt1 mutations. Recently, multiple ATP-competitive Akt inhibitors have been identified and are currently in clinical development. This review details the medicinal chemistry efforts towards identification of these molecules, highlights relevant preclinical data supporting clinical evaluation, and summarizes current clinical development plans.     

Recent advancements in the treatment of renal cell carcinoma--focus on international guidelines

Recent advances in understanding the fundamental biology underlying clear-cell RCC have opened the door to a series of targeted agents, such as tyrosine kinase inhibitors (TKIs) or mTOR inhibitors. These new agents have become the standard of care in managing advanced clear-cell RCC. Choice of initial medical management in patients with metastatic clear-cell RCC should be guided by randomised studies. On the evidence available, the first-line therapy in patients with good- or intermediate-risk mRCC should be either sunitinib or pazopanib, or bevacizumab plus interferon. In selected patients sorafenib is an option, as is high-dose interleukin-2 if performance status is good. In patients with poor prognosis, temsirolimus is recommended. In cytokine refractory patients, sorafenib, when patients have progressed on a tyrosine kinase inhibitor everolimus is the agent of choice. Biró K, Küronya Z. Recent advancements in the treatment of renal cell carcinoma - focus on international guidelines.     

New potential inhibitors of mTOR: a computational investigation integrating molecular docking,virtual screening and molecular dynamics simulation

The mTOR (mammalian or mechanistic Target Of Rapamycin), a complex metabolic pathway that involves multiple steps and regulators, is a major human metabolic pathway responsible for cell growth control in response to multiple factors and that is dysregulated in various types of cancer. The classical inhibition of the mTOR pathway is performed by rapamycin and its analogs (rapalogs). Considering that rapamycin binds to an allosteric site and performs a crucial role in the inhibition of the mTOR complex without causing the deleterious side effects common to ATP-competitive inhibitors, we employ ligand-based drug design strategies, such as virtual screening methodology, computational determination of ADME/Tox properties of selected molecules, and molecular dynamics in order to select molecules with the potential to become non-ATP-competitive inhibitors of the mTOR enzymatic complex. Our findings suggest five novel potential mTOR inhibitors, with similar or better properties than the classic inhibitor complex, rapamycin.     

Discovery and optimization of potent and selective imidazopyridine and imidazopyridazine mTOR inhibitors

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.     

Differential Effects of Selective Inhibitors Targeting the PI3K/AKT/mTOR Pathway in Acute Lymphoblastic Leukemia

Purpose

Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established.

Experimental Design

We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative.

Results

Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2.

Conclusions

Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.     

Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ～20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.     

Discovery and optimization of 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)

We discovered 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR). Since phenolic OH groups pose metabolic liability, one of the two hydroxyl groups was selectively removed. The SAR data showed the structural features necessary for subnanomolar inhibitory activity against mTOR kinase as well as selectivity over PI3Kα. An X-ray co-crystal structure of one inhibitor with the mTOR-related PI3Kγ revealed the key hydrogen bonding interactions.     

Specific Inhibition of the Nuclear Exporter Exportin-1 Attenuates Kidney Cancer Growth

Purpose

Despite the advent of FDA-approved therapeutics to a limited number of available targets (kinases and mTOR), PFS of kidney cancer (RCC) has been extended only one to two years due to the development of drug resistance. Here, we evaluate a novel therapeutic for RCC which targets the exportin-1 (XPO1) inhibitor.

Materials and Methods

RCC cells were treated with the orally available XPO1 inhibitor, KPT-330, and cell viability and Annexin V (apoptosis) assays, and cell cycle analyses were performed to evaluate the efficacy of KPT-330 in two RCC cell lines. Immunoblotting and immunofluorescence analysis were performed to validate mechanisms of XPO1 inhibition. The efficacy and on-target effects of KPT-330 were further analyzed in vivo in RCC xenograft mice, and KPT-330-resistant cells were established to evaluate potential mechanisms of KPT-330 resistance.

Results

KPT-330 attenuated RCC viability through growth inhibition and apoptosis induction both in vitro and in vivo, a process in which increased nuclear localization of p21 by XPO1 inhibition played a major role. In addition, KPT-330 resistant cells remained sensitive to the currently approved for RCC multi-kinase inhibitors (sunitinib, sorafenib) and mTOR inhibitors (everolimus, temsirolimus), suggesting that these targeted therapeutics would remain useful as second line therapeutics following KPT-330 treatment.

Conclusion

The orally-available XPO1 inhibitor, KPT-330, represents a novel target for RCC whose in vivo efficacy approaches that of sunitinib. In addition, cells resistant to KPT-330 retain their ability to respond to available RCC therapeutics suggesting a novel approach for treatment in KPT-330-naïve as well as -resistant RCC patients.     

Ligand-based 3-D pharmacophore generation and molecular docking of mTOR kinase inhibitors

The 3-D structure of the human mTOR kinase domain was modeled based on the crystal structure of PI3Kγ using comparative modeling methods, and the ATP-binding site of mTOR was characterized. The mTOR kinase 3-D model structure is similar to the structure of the PI3Kγ kinase domain, and exhibits great similarity to PI3Kγ at the active site of the kinase. Pharmacophore generation, the docking of mTOR inhibitors, and molecular dynamics (MD) simulations of mTOR–inhibitor docked complexes were carried out in this work. The best pharmacophore model generated from 27 ATP-competitive mTOR inhibitors comprised two hydrogen-bond acceptors, one aromatic ring, and one hydrophobic feature. These 27 inhibitors were docked into the ATP-binding site comprising the DFG motif, and the interactions in each protein–inhibitor complex were characterized. Mapping the pharmacophore model onto the docked inhibitors explained the specificity of the features of the pharmacophore and how they were arranged inside the active site of mTOR kinase. MD studies revealed important structural features, such as the large hydrophobic pocket “HP” and hydrophilic pocket “A1,” and the solvent-exposed hydrophilic pocket “A2” at the active site of mTOR. Our results provide structural models of mTOR–inhibitor complexes and clues that should aid in the design of better mTOR kinase inhibitors.     

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.     

Design,synthesis and SAR of new-di-substituted pyridopyrimidines as ATP-competitive dual PI3Kα/mTOR inhibitors

PI3Kα/mTOR ATP-competitive inhibitors are considered as one of the promising molecularly targeted cancer therapeutics. Based on lead compound A from the literature, two similar series of 2-substituted-4-morpholino-pyrido[3,2-d]pyrimidine and pyrido[2,3-d]pyrimidine analogs were designed and synthesized as PI3Kα/mTOR dual inhibitors. Interestingly, most of the series gave excellent inhibition for both enzymes with IC₅₀ values ranging from single to double digit nM. Unlike many PI3Kα/mTOR dual inhibitors, our compounds displayed selectivity for PI3Kα. Based on its potent enzyme inhibitory activity, selectivity for PI3Kα and good therapeutic index in 2D cell culture viability assays, compound 4h was chosen to be evaluated in 3D culture for its IC₅₀ against MCF7 breast cancer cells as well as for docking studies with both enzymes.     

mTOR-independent 4E-BP1 phosphorylation is associated with cancer resistance to mTOR kinase inhibitors

ATP-competitive mTOR kinase inhibitors (mTorKIs) are a new generation of mTOR-targeted agents with more potent anticancer activity than rapamycin in several tumor models. However, the sensitivity and resistance of cancer cells to mTorKIs remain poorly understood. In this study, we tested mTorKIs against a large panel of colorectal cancer (CRC) cell lines, and found that mTorKIs displayed broader anti-CRC activity than rapamycin, including CRC cells with K-Ras or B-Raf mutations, suggesting that these mTorKIs are particularly useful for CRCs resistant to EGFR inhibitors. Unexpectedly, we found that 40% CRC cell lines were intrinsically drug resistant. Moreover, we discovered an mTOR-independent 4E-BP1 phosphorylation that was correlated with mTorKI resistance. Altogether, our findings provide compelling preclinical support for testing mTorKIs in human CRC clinical trials. They further reveal the existence of significant intrinsic mTorKI drug resistance in cancer cells and suggest that 4E-BP1 phosphorylation is a predictive biomarker for mTorKI sensitivity and resistance.Key words: mTOR, kinase, colorectal cancer, drug resistance, 4E-BP1, phosphorylation     

8-Chloroadenosine Sensitivity in Renal Cell Carcinoma Is Associated with AMPK Activation and mTOR Pathway Inhibition

The adenosine analog 8-chloroadenosine has been shown to deplete ATP and inhibit tumor growth in hematological malignancies as well as in lung and breast cancer cell lines. We investigated effects of 8-chloroadenosine on clear cell (cc) renal cell carcinoma (RCC) cell lines. 8-chloroadenosine was effective against ccRCC cell viability in vitro, with IC₅₀ ranging from 2 μM in the most sensitive CAKI-1 to 36 μM in the most resistant RXF-393. Proteomic analysis by reverse-phase protein array revealed that 8-chloroadenosine treatment leads to inhibition of the mTOR pathway. In time-course experiments, 8-chloroadenosine treatment rapidly activated AMPK, measured by AMPK and ACC phosphorylation, and subsequently caused dephosphorylation of p70S6K and ribosomal protein RPS6 in the sensitive cell lines. However, in the resistant cell lines, AMPK activity and the mTOR pathway were unaffected by the treatment. We also noted that the resistant cell lines had elevated basal levels of phospho RPS6 and AKT. Inhibition of PI3K pathway enhanced the efficacy of 8-chloroadenosine across all cell lines. Our observations indicate that 8-chloroadenosine activity is associated with inhibition of the mTOR pathway, and that phospho RPS6 and PI3K pathway activation status may determine resistance. Among solid tumors, RCC is one of the few susceptible to mTOR inhibition. We thus infer that 8-chloroadenosine may be effective in RCC by activating AMPK and inhibiting the mTOR pathway.     

Crosstalks of GSK3 signaling with the mTOR network and effects on targeted therapy of cancer

The introduction of therapeutics targeting specific tumor-promoting oncogenic or non-oncogenic signaling pathways has revolutionized cancer treatment. Mechanistic (previously mammalian) target of rapamycin (mTOR), a highly conserved Ser/Thr kinase, is a central hub of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR network, one of the most frequently deregulated signaling pathways in cancer, that makes it an attractive target for therapy. Numerous mTOR inhibitors have progressed to clinical trials and two of them have been officially approved as anticancer therapeutics. However, mTOR-targeting drugs have met with a very limited success in cancer patients. Frequently, the primary impediment to a successful targeted therapy in cancer is drug-resistance, either from the very beginning of the therapy (innate resistance) or after an initial response and upon repeated drug treatment (evasive or acquired resistance). Drug-resistance leads to treatment failure and relapse/progression of the disease. Resistance to mTOR inhibitors depends, among other reasons, on activation/deactivation of several signaling pathways, included those regulated by glycogen synthase kinase-3 (GSK3), a protein that targets a vast number of substrates in its repertoire, thereby orchestrating many processes that include cell proliferation and survival, metabolism, differentiation, and stemness. A detailed knowledge of the rewiring of signaling pathways triggered by exposure to mTOR inhibitors is critical to our understanding of the consequences such perturbations cause in tumors, including the emergence of drug-resistant cells.Here, we provide the reader with an updated overview of intricate circuitries that connect mTOR and GSK3 and we relate them to the efficacy (or lack of efficacy) of mTOR inhibitors in cancer cells.