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Can mTOR kinase inhibitors beat rapalogues in fighting against cancer?

The mammalian target of rapamycin (mTOR) has emerged as a promising cancer therapeutic target. Some rapamycin analogues (rapalogues) as mTOR allosteric inhibitors are FDA-approved drugs for treatment of certain types of cancers. However, the modest clinical anticancer activity of rapalogues, which preferentially inhibit mTOR complex 1, in most types of cancer, has spurred the development of ATP competitive mTOR kinase inhibitors (TORKinibs) that inhibit both mTOR complex 1 and complex 2, in the hope of developing a novel generation of mTOR inhibitors with better therapeutic efficacy than rapalogues. So far, several TORKinibs have been developed and some are under clinical testing. With a strong rationale, we expect great success in the treatment of cancer with TORKinibs.

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family and plays a central role in positively regulating cell growth, survival and other cellular functions. It is generally thought that mTOR exerts these biological functions through interacting with multiple proteins and forming two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1, which is composed of mTOR and other four associated proteins, raptor, mLST8, PRAS40 and DEPTOR, controls cell growth primarily by enhancing translation of multiple oncogenic proteins such as cyclin D1, c-Myc, hypoxia-inducible factor 1 (HIF1) and vascular endothelial growth factor (VEGF) through activating S6 kinase (S6K) and suppressing eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). mTORC2, which contains mTOR, rictor, mLST8, DEPTOR, mSin1 and protor, positively regulates cell survival and proliferation primarily by phosphorylating Akt and other proteins such as serum and glucocorticoid-inducible kinase (SGK) and PKCα1-4. Hence both mTORC1 and mTORC2 are involved in the positive regulation of cancer cell survival and proliferation. Due to a hyperactive mTOR axis in many types of human cancers, mTOR and mTORCs have emerged as promising cancer therapeutic targets3,5,6.

Accordingly, there are two types of inhibitors that exert their anticancer activity through targeting the mTOR axis: rapamycin (sirolimus) and its analogues (rapalogues) and ATP-competitive mTOR kinase inhibitors (TORKinibs). This short review will highlight recent advances in the development of TORKinibs and discuss their potential as cancer therapeutic drugs.

Rapalogues

Rapalogues are conventional mTOR inhibitors that were developed before TORKinibs. These agents are specific allosteric inhibitors of mTOR that inhibit mTOR kinase activity through binding and recruiting an accessory protein named FKBP12 to the FRB domain of mTOR. It is generally believed that rapalogues function though inhibition of mTORC1 with weak activity against mTORC27. Some rapalogues such as CCI-779 (temsirolimus) and RAD001 (everolimus) have been actively tested in various phases of oncology clinical trials8,9 and have demonstrated encouraging clinical efficacy in treatment of patients with metastatic renal cell carcinoma10-12, pancreatic neuroendocrine tumours13, and postmenopausal hormone receptor-positive advanced breast cancer14. As a result, both CCI779 and RAD001 were approved by the FDA for the treatment of advanced renal cancer and RAD001 was also approved for the treatment of pancreatic neuroendocrine tumours and postmenopausal hormone receptor-positive advanced breast cancer (Table 1).

Despite these advances, the single agent activity of rapalogues in most other cancer types has been modest at best6,15,16. This may be largely due to the fact that rapalogues lack effective activity against mTORC2, exert incomplete inhibitory effects on mTORC1 (e.g., weak activity in suppression of 4EBP1 phosphorylation), and induce feedback activation of Akt, Mnk/eIF4E and ERK/RSK2 survival pathways that attenuate their anticancer efficacy7,17-20.

TORKinibs

The limitations of rapalogues as discussed above and the discovery of mTORC2 as an Akt S473 kinase21 and the subsequent demonstration of its involvement in cancer development22,23 have encouraged great efforts in developing a novel generation of mTOR inhibitors that are able to inhibit both mTORC1 and mTORC2 activity. Consequently, several relatively specific ATP-competitive inhibitors of mTOR (i.e., TORKinibs) such as Ku-0063794, PP244, INK126, OSI-027, Torin 1, AZD8055, AZD2014 and WYE-354 have been developed and some of these have been tested in clinical trials8,24-27.

In the preclinical setting, TORKinibs have been shown to inhibit mTORC1 signalling (particularly 4E-BP1 phosphorylation) and protein synthesis, suppress Akt S473 phosphorylation and induce G1 arrest and/or apoptosis in some cancer cells more dramatically than rapamycin28–31. A robust in vivo anticancer activity of these inhibitors against certain types of cancers was also observed29,32,33.

To date, only a few TORKinibs (e.g., INK128, OSI-027, AZD8055 and AZD2014) have been tested in clinical trials (Table 1), most in patients with advanced solid malignancies7,27,34. Except for AZD8055, the results of clinical trials with TORKinibs have not been completed and reported. Although the drug may have an acceptable toxicity profile, no substantial response has been observed from the AZD8055 studies35,36.

Beyond mTOR, the PIKK family includes several other cancer-related protein kinases such as PI3K, ataxia–telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK)37. The kinase domains of mTOR and these proteins are closely related or similar to each other. Some compounds that inhibit catalytic activity of both mTOR and one or two other kinases have been developed as well. This includes several mTOR/PI3K dual inhibitors (e.g., NVP-BEZ235, NVP-BGT226, GSK2126458, XL765, PF-04691502 and GDC0980), which are under clinical testing (Table 1)7,34. In addition, the mTOR/DNA-PK dual inhibitor, CC-115, is currently being tested in Phase I clinical trials (Table 1; http://clinicaltrials.gov/show/NCT01353625). Torin 2 was optimised from Torin 1 and initially reported to be a TORKinib38. However, a recent study from the same group has shown that Torin 2 also exhibits potent biochemical and cellular activity against ATM, ATR and DNA-PK in addition to inhibiting mTOR39.

Challenges and opportunities

Rapalogues are macrocycle compounds with complex chemical structures. The synthesis of rapalogues, including both biosynthesis and total synthesis, involves complicated procedures40 and thus is cost-ineffective. In comparison, the chemical structures of currently identified TORKinibs are relative simple and can easily be synthesised. Hence this should be a great advantage for TORKinibs over rapalogues in regard to reducing manufacturing costs of the drugs.

Rapalogues act through an allosteric inhibition mechanism to very specifically inhibit mTOR without any inhibitory effects on many known protein kinases even at a concentration of 1 μM, which is 10–20 fold higher than that required to completely inhibit mTORC1 activity in cell-based assays41. Like other ATP-competitive kinase inhibitors, selectivity and potential off-target effects of TORKinibs are always an unavoidable issue, particularly when used at high therapeutic concentration ranges27. This concern should be kept in mind when we interpret results, particularly when using TORKinibs as a research tool to demonstrate the involvement of mTORCs. The concentration is a critical factor when considering the selectivity and potency of a given drug. In general, selectivity will be reduced as the concentration increases although the potency will be increased. Many studies have claimed that TORKinibs are more potent than rapalogues (e.g., rapamycin) in suppressing growth and inducing apoptosis of cancer cells. However these studies used much higher concentrations (e.g., µM ranges) of TORKinibs compared with lower concentrations of rapalogues (e.g., nM ranges) as we have discussed previously27.

From a therapeutic point of review, less specific TORKinibs such as mTOR/PI3K dual inhibitors and mTOR/DNA-PK dual inhibitors may be beneficial and may have better therapeutic outcomes against cancer, considering the fact that more than one PIKK kinase pathway (e.g., PI3K and mTOR) is activated in many types of cancers and TORKinibs induce feedback activation of other survival and proliferative signalling molecules (e.g., Akt and ERK1/2)27. This may be an opportunity for us to develop novel and effective cancer therapeutic drugs.

One rationale in favour for TORKinibs as better cancer therapeutic agents is that these agents will be more potent than rapalogues in inhibiting both mTORC1 and mTORC2 activities. However, some studies have suggested that TORKinibs exert their growth inhibitory effects or anti-cancer effects through strong inhibition of mTORC1 signalling, particularly p-4E-BP1 T37/46, rather than through inhibition of mTORC228,31. This notion is further supported by a recent study showing that incomplete inhibition of 4E-BP1 phosphorylation is a mechanism of primary resistance of cancer cells to TORKinibs42. Moreover, suppression of mTORC1 or mTORC2 signalling does not predict tumour sensitivity as suggested in a recent study43. Hence, it is urgent to further elucidate the involvement of mTORC2 in the regulation of cancer cell survival and growth.

Another rationale in favour for TORKinibs is that these agents, unlike rapalogues, will not induce activation of PI3K/Akt signalling while suppressing mTOR signalling. However, recent studies have suggested that some TORKinibs, as rapalogues do, can also initiate feedback activation of certain cell survival signaling pathways (e.g., Akt and ERK1/2), which in turn results in attenuation of TORKinibs’ therapeutic efficacies and likely development of resistance to TORKinibs albeit through a different mechanism44,45. Thus, rational combinations that will interrupt or block these feedback activations may still be needed to enhance TORKinib-based cancer therapy or prevent development of eventual resistance to TORKinibs.

Certain genetic alterations such as frequent mutations in PIK3CA and/or Ras, loss of expression of PTEN and/or over-expression of receptor tyrosine kinases can result in hyper-activation of the PI3K/mTOR axis and may impact cell sensitivity to agents that target this axis. A study with a panel of 31 breast cancer cell lines suggests that breast cancer cells harbouring PIK3CA mutations are selectively sensitive to PP242. However, cells with PTEN loss of function were not sensitive to these drugs46. Another recent study that screened a panel of over 600 human cancer cell lines suggests that Ras and PIK3CA mutations are the most significant genetic markers for resistance and sensitivity to PP242, respectively. Specifically, colon cancer cell lines with K-Ras mutations are most resistant to PP242, whereas those without K-Ras mutations are most sensitive42. Hence it is critical to develop various predictive biomarkers that are able to help us to identify patient populations or cancer types that may indeed benefit from TORKinib-based therapy.

Concluding remarks

With strong scientific rationale, considerable effort has been made thus far in the development of TORKinibs. The availability of various TORKinibs will undoubtedly provide us with valuable research tools to fully understand or dissect the biological functions of mTORCs essential for regulation of cell proliferation and survival in the preclinical setting and is highly appreciated. The development of TORKinibs as cancer therapeutic drugs faces some challenges as discussed above, but has great potential based on their strong scientific rationale. Several rapalogues are FDA-approved drugs albeit limited to only a few rare cancer types. Although most clinical trials with TORKinibs have not been completed, the Phase I results with AZD8055 have not demonstrated major responses thus far35,36. Hence, it is still too early to tell whether TORKinibs are indeed superior to rapalogues.

Acknowledgements

Research in the author’s laboratory is supported by NIH/NCI R01 CA118450 and R01 CA160522. The author is grateful to Dr. A. Hammond for editing the manuscript.

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Biography

Shi-Yong Sun earned his PhD in cancer pharmacology from Peking Union Medical College / Chinese Academy of Medical Sciences in Beijing, China, in 1990. He received his first postdoctoral training in cancer biology at Peking University Health Science Center in Beijing, China, from 1990 to 1992 and second postdoctoral training in cancer and retinoid biology at the University of Texas M.D. Anderson Cancer Center in Houston, Texas, USA, from 1994 to 1996. In 2003, he joined Emory University School of Medicine and Winship Cancer Institute in Atlanta, Georgia, USA, as an Assistant Professor and was soon promoted to a tenured Associate Professor in 2007. Since 2011, he has been a tenured Professor in the Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute. His research primarily focuses on studying regulation of the extrinsic apoptotic pathway in cancer and its implications in cancer therapy. In addition, his lab is interested in understanding mTOR signalling in cancer and targeting the mTOR axis for cancer therapy.

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