Overcoming resistance to mTOR inhibition for enhanced strategies in clinical trials

Keywords::

• angiogenesis
• mTOR
• Phase I
• resistance

1. Introduction

In this issue, the authors provided a comprehensive review of mTORC1 inhibitors (sirolimus (rapamycin), temsirolimus, everolimus and ridaforolimus) and dual kinase inhibitors (OSI-027, AZD8055, AZD2014 and INK128). The mammalian target of rapamycin (mTOR) pathway is a crucial target in new drug development. Since first being described in the 1990s, the mTOR pathway has been greatly researched [1]. In many different cancers, activations of PI3K/AKT/mTOR signalling through mutations of pathway components play a crucial role in deregulation of proliferation, in angiogenesis and in resistance to apoptosis resulting in cell survival and resistance to chemotherapy and radiotherapy [2-6].

Activation of the mTOR complex, which is mainly located in the nuclear fraction of both normal and neoplastic cells, prompts resting cells to increase translation of a subset of mRNA [7]. These translated proteins escalate cell cycle progression from G1 to S phase. In response to mitogenic signals, the mTOR complex is phosphorylated, which leads to mRNA translation in the presence of favourable nutrient environments [8,9]. In vitro, mTOR-inhibitor-treated cells expressed mTOR inactivation, downregulation of translation, G1 arrest, accumulation of glycogen stores and altered transcription patterns. Strikingly, these conditions were similar to those observed under starvation [9]. More recent studies have demonstrated that mTOR is involved in regulating many aspects of cell growth, including organisation of the actin cytoskeleton, membrane traffic, protein degradation, protein kinase C signalling, ribosome biogenesis and transcription [10].

2. FDA-approved mTOR inhibitors

Currently, there are four mTORC1 inhibitors: sirolimus, temsirolimus, everolimus and ridaforolimus [11-13]. Sirolimus is approved as an immunosuppressive agent for patients with renal transplants [14]. Temsirolimus is an ester of the macrocyclic immunosuppressive agent sirolimus. Temsirolimus has been shown to inhibit the growth of a wide range of histologically diverse tumours, including adrenocortical carcinoma [15], the Ewing's sarcoma family of tumours [16], central nervous system cancers [17], mantle cell lymphoma [18], breast cancer [19] and renal cell carcinoma [20]. Temsirolimus is Food and Drug Administration (FDA)-approved for renal cell carcinoma [12,20].

Everolimus in combination with exemestane has been FDA-approved for hormone receptor-positive metastatic breast cancer based on the BOLERO-2 trial [13,21]. Everolimus is also FDA-approved to treat patients with advanced renal cell carcinoma and neuroendocrine tumours [22-25]. Ridaforolimus has not yet been approved by the FDA, however its activity has been observed in sarcoma patients [26,27].

3. Toxicity associated with mTOR inhibitors

Some of the common toxicities associated with mTOR inhibitors include clinical toxicities such as asthenia, rash and mucositis and laboratory abnormalities such as anaemia, hyperglycaemia and hyperlipidaemia [12,15,16,28-30]. Pneumonitis, even though it is rare, is a clinically noteworthy complication of treatment with mTOR inhibitors [31]. In general, single-agent mTOR inhibitors are well tolerated; however, mTOR in combination with other agents may cause significant side effects depending on the mechanisms of the second drug. Mucositis and endocrine complications have been observed in patients who received temsirolimus and insulin-like-growth factor receptor (IGF-1R) inhibitor [15,16,29]. Even though mTOR combination therapy causes hyperlipidaemia and hyperglycaemia, patients with diabetes and high cholesterol should not be excluded from combination-therapy clinical trials, as long as their blood sugar and lipids are well controlled. In clinical trials combining MEK inhibitors and mTOR inhibitors, the most frequent toxicities were mucositis, thrombocytopenia and skin rash [32,33]. It is highly recommended to avoid strong CYP3A4 modifiers when using mTOR inhibitors since CYP3A4 is the primary enzyme responsible for metabolism of this class of drugs [34].

4. Resistance to mTOR inhibition and strategies for overcoming resistance

Understanding the mechanism of resistance to mTOR inhibition is critical. Various pathways have been delineated as responsible for resistance to mTOR inhibition. There are multiple strategies for overcoming this resistance.

4.1 Dual inhibition of mTOR complexes

Since mTOR exists in two complexes – mTOR complex 1 (mTORC1) containing Raptor, which is rapamycin sensitive, and mTOR complex 2 (mTORC2) containing Rictor, which is rapamycin insensitive – there is an opportunity for dual inhibition [35]. mTORC1 phosphorylates 4-EPB1 and p70S6 kinase, which results in cell cycle progression. mTORC2 has been shown to directly phosphorylate and activate the upstream kinase AKT [35,36]. Inhibition of mTORC1 impedes the negative feedback loop between S6 kinase and insulin receptor substrate 1 (IRS-1), resulting in increased PI3K (phosphatidylinositol 3-kinase) and AKT activity, thereby limiting the activity of mTORC1 inhibitors [35]. A dual kinase inhibits both mTORC1 and mTORC2 complexes (Figure 1). By controlling both S6K and AKT, dual kinase inhibitors provide a broader spectrum of activity than does mTORC1 inhibitor alone [35,37,38].

Figure 1. Crosstalk of multiple pathways, mechanisms of the resistance, and strategies for overcoming resistance to mTOR inhibitors.

4.2 Inhibition of the PI3K/AKT/mTOR and MAPK pathways

The PI3K pathway and the mitogen-activated protein kinase (MAPK) pathway share common upstream activators. Both activated by oncogenic RAS, these two pathways are significantly interconnected by feedback loops, and one pathway provides compensatory signalling when the other is inhibited [39,40]. The inactivation of either the MAPK or PI3K pathway can lead to incomplete tumour growth inhibition. Parallel inhibition of the PI3K/AKT/mTOR and MAPK pathways leads to synergistic increases in apoptosis in both phosphatase and tensin homolog (PTEN)-mutant and wild-type cells [41,42]. In vivo studies have shown marked synergies when PI3K/AKT/mTOR inhibitors are combined with MEK inhibitors in KRAS-mutant cancers [43]. Thus, combining an mTOR inhibitor and a MEK inhibitor is an appropriate strategy to enhance mTOR-targeted anti-cancer therapy (Figure 1).

4.3 Inhibition of the PI3K/AKT/mTOR and IGF pathways

AKT activation plays a crucial role in many cellular processes, including cancer cell survival, proliferation and growth [2]. Thus, an unfavourable consequence of mTOR inhibition is the induction of phosphorylated AKT, in turn causing resistance to mTOR inhibition [2]. Several studies have shown that mTOR-induced AKT activation is due to the release of feedback inhibition mediated by the IGF pathway. IGF-1R inhibitors can reduce AKT phosphorylation induced by mTOR inhibitors. AKT activation is related to the escape/resistance mechanism of mTOR inhibitors, but combination studies with mTOR inhibitors and IGF-1R inhibitors suggest additive anti-tumour effects compared with treatment with single agents alone (Figure 1). Furthermore, as mTOR is involved in signal transduction downstream of the IGF pathway, the combination potentially enhances the activity of IGF-1R inhibitors [29]. Therefore, simultaneous inhibition of both pathways could lead to a more effective anti-tumour strategy when compared with individual pathway blockade [15,16].

Moreover, AKT activation is pivotal in resistance to mTOR inhibition, there are several AKT inhibitors (MK-2206, RX-0201, PBI-05204, GSK2141795) currently being investigated in clinical trials as single agent or in combination with other mTOR inhibitors or chemotherapy [44].

4.4 Metformin in combination with mTOR inhibitors

The combination of rapamycin and metformin, an anti-diabetic drug, was shown to expand the therapeutic window of normal human cell types (RPE, NKE, WI-38t cells) exposed to paclitaxel and nocodazole, both of which are microtubule inhibitors [45]. The drug combination accomplished these results by selectively inducing G1 and G2 arrest in normal cells. Metformin and rapamycin protected normal cells in low-glucose conditions, but the combination was cytotoxic to cancer cells [45].

This combination is attractive because the mTOR inhibitor and metformin appear to inhibit the mTOR pathway by different mechanisms [46]. mTORC1 inhibitors inhibit mTOR activity by binding to FKBP12, which can result in the unintended upregulation of AKT through a positive feedback loop that might undesirably accelerate cell proliferation. Metformin inhibits the mTOR pathway through upstream activation of 5′-adenosine monophosphate-activated protein kinase (AMPK) [46]. In contrast to mTOR inhibitors, metformin decreases AKT activation. Via AMPK activation, metformin phosphorylates an inhibitory site of IRS-1, which is associated with decreased AKT activation, reduced mTOR activation and reduced feedback inhibition [46].

Metformin, independent of AMPK, also induces mTOR inhibition and cell cycle arrest through REDD1 (a negative regulator of mTOR) in LNCaP (androgen-sensitive human prostate adenocarcinoma) cell lines [47]. In fact, metformin upregulates REDD1, leading to an anti-proliferative effect. Metformin-induced cell cycle arrest and cyclin D1 decrease are not dependent on AMPK in LNCaP cell lines [47]. Therefore, in addition to improving resistance to mTOR inhibition, metformin augments further independent inhibition of the mTOR pathway [47].

In addition, AMPK activation counteracts the inhibitory effect of the Ras/Raf/ERK pathway on tuberous sclerosis complex 2 (TSC2) [48]. These results support the rationale that dual inhibition of the AMPK/TSC2/mTOR pathway through combining metformin and an mTOR inhibitor will enhance the anti-tumour activity [48]. Furthermore, metformin improved hyperglycaemia caused by mTOR inhibitor [29]. Both metformin and the mTOR inhibitor significantly influence intracellular energy homeostasis and interfere with autophagy mechanisms [49]. Thus, combining an mTOR inhibitor and metformin may be an appropriate strategy to enhance mTOR-targeted anti-cancer therapy (Figure 1).

4.5 Combination of histone deacetylase inhibitors and mTOR inhibitors

Histone deacetylases (HDACs) are enzymes that regulate chromatin structure and function through the catalysis of the removal of acetyl modifications from the lysine residues of histones [50]. Inhibition of HDACs reverses aberrant epigenetic changes associated with malignancy [51], and HDAC inhibitors possess anti-tumour activities against multiple cancers [52]. The HDAC inhibitor SAHA (suberoylanilide hydroxamic acid) directly diminished the kinase activity of PI3K in several cell lines, including PC3 (prostate cancer) and Jeko1 (mantle cell lymphoma) in vitro [53]. Inhibiting PI3K activity in this manner could counteract the paradoxical increase in phosphorylated-AKT in response to mTOR inhibition. Inhibiting HDACs with LBH589 diminished resistance to mTOR inhibition by decreasing AKT activation by two additional mechanisms: activating protein phosphatase PP1 and inhibiting the mTORC2 complex [54]. A combination of a novel HDAC inhibitor (FK228) and a PI3K/AKT pathway inhibitor (LY294002) showed synergy in cytotoxic activity in lung adenocarcinoma cell lines [55]. Adding an HDAC inhibitor to an mTOR inhibitor substantially suppressed survivin levels and generated better tumour growth inhibition than did the single-agent mTOR inhibitor (Figure 1) [56].

5. mTOR inhibition as a tool to overcome resistance to other drugs

Beyond their function of inhibiting mTOR complexes, mTOR inhibitors play an important role in overcoming resistance to other drugs, such as anti-VEGF therapies and anti-EGFR treatment.

5.1 Resistance to anti-angiogenesis treatment

mTOR inhibitors can improve the anti-angiogenic activity of anti-VEGF therapies by several mechanisms. First, mTOR inhibitors inhibit endothelial cell function in neoangiogenesis. Additionally, they inhibit VEGF production induced by hypoxia-stimulated hypoxia-inducible factor 1-α (HIF-1α) [57]. Increased HIF-1α is associated with increased expression of VEGF, aggressive tumour growth and poor prognosis. This phenomenon is commonly observed as a resistance mechanism in tumours treated with anti-VEGF therapy. HIF-1α inhibition in combination with anti-angiogenic therapy is a promising strategy for targeting tumour resistance [58]. This important property of mTOR inhibitors makes them ideal candidates for combination therapy with anti-angiogenic agents.

5.2 Resistance to anti-EGFR treatment

The combination of an EGFR inhibitor and an mTOR inhibitor has the reciprocal benefit of improving resistance to each other. Cancer cell lines exposed to mTOR inhibition have demonstrated increased EGFR/Ras/Raf/Erk/MEK pathway signalling [59]. EGFR possesses kinase-independent activity that induces cancer cell survival by preventing autophagic cell death and maintaining intracellular glucose levels through interaction and stabilisation of the sodium/glucose co-transporter 1 (SGLT1) [60]. Downregulation of EGFR led to a loss of SGLT1 expression, resulting in lower intracellular glucose concentration [61]. Therefore, adding an EGFR inhibitor to an mTOR inhibitor will provide a strategy to overcome resistance.

mTOR is a central player in the autophagy pathway, and downregulation of the PI3K pathway induces autophagy [62]. In glioma cell lines resistant to EGFR tyrosine kinase inhibitors, increased activities of the IGF pathway and of its downstream effectors, PI3K/AKT/mTOR, were observed [63]. Simultaneous inhibition of the EGFR and PI3K/AKT/mTOR pathways inhibited tumour cells that were resistant to small-molecule inhibitors of EGFR [63-65]. Therefore, adding mTOR inhibition to an anti-EGFR antibody/small molecule will provide a strategy for overcoming resistance.

6. Conclusion

In conclusion, there are several ways to improve efficacy of mTOR inhibition. Combination of mTOR inhibitors with other agents that decrease resistance to mTOR inhibition can improve the efficacy of mTOR inhibition. Additionally, mTOR inhibition can improve the therapeutic effect of anti-VEGF therapies and anti-EGFR therapies. With combination therapies, toxicities may be a major obstacle. Hence, it is of great importance to devise an optimal dosing schedule, maintain appropriate sequence of drug administration and optimize management of side effects. Further understanding of resistance and response mechanisms of mTOR inhibitors is critical. Biopsies at baseline before initiation of the treatment and at the time of progression are necessary and may present biological evidence of mutational changes in tumour specimens. Gene sequencing platforms may provide insight into resistance mechanisms, thereby providing more opportunities to improve the efficacy of this class of drugs.

Declaration of interest

The author states no conflict of interest and has received no payment in preparation of this manuscript.