**6. mTOR inhibitors in cancer therapy**

#### **6.1. Rapamycin and its derivatives**

As discussed previously, mTOR is involved in many cell signaling pathways, and clinical trials for cancer treatment showed that tumor cells with mutations in p53 or PTEN are susceptible to mTOR inhibitors [115]. mTOR inhibitors are categorized in first- and second-generation presenting a wide variety of target and mechanism. The first-generation mTOR inhibitors include rapamycin and its analogs that employ allosteric mechanism to block, whereas the second-generation mTOR inhibitors (AZD8055, Torin1, PP242, and PP30) have as target ATP binding site to impede kinase activity of both mTORC1 and mTORC2 [116].

Rapamycin, discovered in 1975, is a macrocyclic lactone isolated from the soil bacterium *Streptomyces hygroscopicus*, and it has clinical applications including antifungal, immunosup‐ pressant, and anticancer proprieties [117,118]. FDA approved this drug in 1997 for prevention of host-rejection during kidney transplants [119]. Preclinical studies have shown that rapa‐ mycin presents strong antiangiogenic and antiproliferative properties against a variety of human cancers such as the phase II study, which showed rapamycin potentiates the effect of paclitaxel in endometrial cancer cell lines [120].

Three different mechanisms of action have been proposed: first, the binding of the FKBP-12– rapamycin complex to mTOR that could lead dephosphorylation of downstream effector molecules such as S6K-1 and 4EBP1 [121]; second, the FKBP-12–rapamycin complex competes with phosphatidic acid to bind to the FRB domain of mTOR, blocking mTOR kinase function [122]; and third, the FKBP-12–rapamycin complex bounds to mTOR and destabilizes the mTOR–raptor–4EBP1/S6K-1 scaffold complex, leading to dephosphorylation of S6K-1 and 4EBP1 [123,124].

osteosarcoma, ovarian, multiple myeloma, leukemia, and glioma [100,101]. Additional *in vitro* data demonstrate synergistic effects of perifosine and traditional chemotherapeutic agents such as paclitaxel and cisplatin in ovarian cancer [102,103], etoposide in leukemia cells [104], doxorubicin in multiple myeloma cells [105], and gemcitabine in pancreatic cells [106].

Despite these encouraging preclinical studies, results from phase I/II clinical trials of perifosine as single agent in a various tumor types (metastatic breast cancer, metastatic head and neck

In behalf of the poor efficacy of perifosine as a single agent observed in most tumor types evaluated thus far, efforts have been made to combine this drug with target agents and chemotherapy. Phase I studies have now confirmed the safety of these combinations with different agents, including sorafenib in patients with Hodgkin lymphoma and taxanes in highgrade epithelial ovarian cancer [112,113]. Currently, one clinical trial with perifosine is recruiting patients, a phase II study with perifosine and temsirolimus in patients with

GSK-690693 is a potent ATP-competitive AKT inhibitor selective for all three AKT isoforms versus the majority of kinases assessed by biochemical tests [114]. GSK690693 displayed antiproliferative activity *in vitro* and *in vivo* models of ovarian, breast, and prostate cancer [114]. The compound has entered phase I trials for refractory hematologic malignancies but

As discussed previously, mTOR is involved in many cell signaling pathways, and clinical trials for cancer treatment showed that tumor cells with mutations in p53 or PTEN are susceptible to mTOR inhibitors [115]. mTOR inhibitors are categorized in first- and second-generation presenting a wide variety of target and mechanism. The first-generation mTOR inhibitors include rapamycin and its analogs that employ allosteric mechanism to block, whereas the second-generation mTOR inhibitors (AZD8055, Torin1, PP242, and PP30) have as target ATP

Rapamycin, discovered in 1975, is a macrocyclic lactone isolated from the soil bacterium *Streptomyces hygroscopicus*, and it has clinical applications including antifungal, immunosup‐ pressant, and anticancer proprieties [117,118]. FDA approved this drug in 1997 for prevention of host-rejection during kidney transplants [119]. Preclinical studies have shown that rapa‐ mycin presents strong antiangiogenic and antiproliferative properties against a variety of human cancers such as the phase II study, which showed rapamycin potentiates the effect of

Three different mechanisms of action have been proposed: first, the binding of the FKBP-12– rapamycin complex to mTOR that could lead dephosphorylation of downstream effector

binding site to impede kinase activity of both mTORC1 and mTORC2 [116].

cancer, locally advanced soft tissue sarcoma, prostate cancer, and metastatic

malignant gliomas (NCT02238496).

138 Updates on Cancer Treatment

**6.1. Rapamycin and its derivatives**

was withdrawn prior to enrolment (NCT00666081).

**6. mTOR inhibitors in cancer therapy**

paclitaxel in endometrial cancer cell lines [120].

This inhibitor has limited bioavailability due to its poor aqueous solubility. In an effort to improve its pharmacokinetics, several rapamycin analogs, named rapalogs, have been developed, such as temsirolimus (CCI-779), everolimus (RAD001), and ridaforolimus (MK-8669/AP23573) [125-127].

Some studies have shown that these compounds are able to disrupt the mTORC2 complex in a dose-, time-, and cell type-dependent manner [24,128,129]. A possible mechanism by which rapamycin and rapalogs could inhibit mTORC2 relies on the interaction of newly synthesized mTOR molecules and rapamycin/rapalogs-FKBP12 complexes. In turn, this interaction would prevent mTOR from the interaction with RICTOR, thus inhibiting mTORC2. Indeed, it has been shown that prolonged exposure of cancer cells to rapamycin can promote its binding to mTOR before the assembly of the mTORC2 complex, with subsequent inhibition of the AKTmediated signaling [24].

Rapamycin and its derivates exhibit a safe toxicity profile, being the side effects of skin rashes and mucositis dose dependent [130]. Other symptoms commonly described are fatigue, nausea, anemia, hypertriglyceridemia, hypercholesterolemia, and neutropenia [131]. Further‐ more, temsirolimus and sirolimus are associated with significant rate of pulmonary toxicity [130,131]. Rare side effects of the aforesaid drugs include interstitial lung disease, risk of secondary lymphoma, and reactivation of latent infections [35].

Everolimus (Afinitor®), the oral mTOR inhibitor, has been approved by the FDA in 2009 for advanced renal cell cancer. Everolimus exhibit strong antiangiogenic and antiproliferative activity against various human cancer such as metastatic or unresectable pancreatic neuroen‐ docrine tumors, subependymal giant cell astrocytoma [132], metastatic renal cell carcinoma, and advanced estrogen receptor (ER)-positive [133] and human epidermal growth factor receptor-2 (HER2)-negative breast cancer [134].

Several studies have been conducted to analyze the effectiveness of rapamycin and rapalogs alone and in combination with standard chemotherapy, hormonal therapy such as anti-VEGF inhibitors in the treatment of several types of cancers such breast, ovarian, cervical, and endometrial. Phase II studies are ongoing in order to test everolimus in combination with chemotherapy (cisplatin and gemcitabine) in patients with metastatic triple negative breast cancer (NCT01939418 and NCT01931163). In addition, a recent study of breast cancer (BO‐ LERO-3) demonstrated that the combination of everolimus with trastuzumab and vinorelbine significantly prolonged progression-free survival (PFS) in patients with trastuzumab-refrac‐ tory and taxane-pretreated, HER2-positive advanced breast cancer [135]. Moreover, another breast cancer study, BOLERO-1, evaluated patients treated with paclitaxel and trastuzumab with or without everolimus as first-line therapy [136]. Furthermore, clinical studies have evaluated the aromatase inhibitor letrozole in combination with everolimus in patients with metastatic endometrial carcinoma (NCT01068249) and breast cancer (NCT00107016).

Temsirolimus (Torisel®), the first rapamycin analog to be FDA approved as an anticancer drug, is an intravenous injection drug and gets converted into rapamycin *in vivo* [137]. This drug was valued with bevacizumab or in combination with chemotherapeutic agents in endometrial cancer cell lines, and results showed the increase progesterone mRNA expression and inhibition of ER mRNA expression [138,139]. Also, preliminary phase II study using temsiro‐ limus in patients with metastatic cervical cancer showed positives results [140]. Another phase II clinical study (NCT01196429) evaluates additional effects of the temsirolimus combined with paclitaxel/carboplatin therapy have been conducted in patients with stages III/IV clear cell adenocarcinoma [141]. However, some studies failed to show the efficiency of temsirolimus in patients with persistent/recurrent epithelial ovarian cancer/primary peritoneal cancer showing a modest activity of this mTOR inhibitor, and the results were insufficient to justify further study in a phase III [142].

Ridaforolimus (MK-8669/AP23573), a non-rapamycin prodrug, is available in both oral and intravenous formulations. This mTOR inhibitor is actively being evaluated as either mono‐ therapy or in combination with other therapies for treatment of various cancers, including sarcomas, endometrial, prostate, breast, and non-small cell lung cancer [143]. Studies had been conducted in patients with advanced endometrial cancer and clinical benefit response was reported in 33% of the patients [144]. Another phase II study using oral ridaforolimus in patients with advanced or recurrent endometrial cancer also showed partial response in 7.7% patients [145].

Although clinically promising, the efficacy of rapalogs is partially limited by the negative feedback loops in the mTOR pathway. With this regard, the exclusive inhibition of the mTORC1 complex by the rapalogs compromises the S6K-1-mediated feedback loop towards IRS-1, resulting in the activation of both the PI3K/AKT and the mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) pathways, hence promoting compensatory cell survival, and the acquisition of chemoresistant phenotype [127,146,147]. Efforts have been made to overcome the previously mentioned clinical limitation by means of developing new generation mTOR inhibitors, which inhibit the catalytic activity of both mTORC1 and mTORC2 complexes.
