Characteristic mTOR activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease – a combined tissue microarray, in vitro and in vivo study

The number of patients diagnosed with lymphoid malignancies has increased to 18,000 per year in Europe [1]. Hodgkin-lymphomas (HL) with characteristic histopathological subtypes comprise about 11% of all lymphomas [1, 2]. Tumor cells [Hodgkin-/Reed-Sternberg (HRS) cells] usually represent only a small fraction of diagnostic histology, while differences in microenvironment (reactive lymphocytes, extracellular matrix) allow subclassification of HL [3, 4]. The prognosis of HL patients is relatively good, however, some patients may relapse in spite of first line chemotherapy and radiation protocols, and can be further treated, sometimes cured by intensified chemotherapy and/or peripheral stem cell transplantation [5]. Unfortunately, these treatments still fail in 15-20% of HL patients [6]. Considering that the majority of HL patients are young and the survivors have a high risk of acute or late toxicity associated with therapy [7], more efficient and less toxic therapeutic strategies are needed. Targeting signaling pathways offers an attractive approach.

The PI3K/Akt/mTOR pathway is activated in a number of human neoplasms, accompanied by lower overall and disease free survival [8]. This pathway plays a key role in the regulation of cellular functions such as survival, proliferation, cell death and metabolic activities [9]. mTOR (mammalian target of rapamycin) – an important component of this network – is a serine-threonine kinase, which exists in two distinct multiprotein complexes (mTORC1 and mTORC2 – containing characteristic elements: Raptor and Rictor, respectively) [10]. The best known targets of mTORC1 are eukaryotic initiating factor-4E binding proteins (4EBP) and S6 kinase (S6K). mTORC2 can regulate Akt dependent antiapoptotic and survival mechanisms by phosphorylating Akt [11].

The PI3K pathway can be activated by several upstream receptors (IGF-R, Flt3, c-Kit, Notch, TCR, BCR) or intracellular proteins (Ras, BCR/ABL) in various hematological diseases [12]. Information about mTOR activity is very limited; however, transforming direct genetic modifications of PI3K, Akt, mTOR or PTEN are rare – such mutations occur in 5% of lymphoid malignancies [13]. mTOR has indeed been proven an important element in tumorigenesis in mantle cell lymphoma (MCL): its role was confirmed in MCL cell proliferation, mainly by influencing cyclin D1 expression [14]. This suggests that the mTOR pathway may play an important role in the development or progression of other lymphoma types as well, and can be considered as a useful therapeutic target.

Rapamycin (and its analogs: rapalogs) interacts with the FKBP12 protein, an element of the mTOR complex, and preferentially disrupts mTORC1 activity [15]. The response of mTORC2 to rapalogs remains conflicting [16]. Rapalogs have been used as immunosuppressive agents in organ transplantation since 1999, and they have been introduced into clinical oncology as a treatement option in renal cell carcinoma and recently in MCL as well [14]. Several trials using mTOR inhibitors in tumors with high mTOR activity are currently underway [17‐19].

The aim of our study was to investigate mTOR activity in different lymphomas, with a focus on HL. We found that the majority of HL cases (93%) displays high mTOR activity. Therefore we suggest that mTOR inhibition (e.g. by rapalogs) may be considered as a therapeutic option in HL, especially in patients with poor prognosis/relapse.

The introduction of new drugs has to be based on convincing evidence in malignancies where clinical response rate (or even cure rate) is rather high. A typical example is Hodgkin-lymphoma (HL); in fact, no new drugs have been approved by the FDA for HL in the last 30 years [22]. However, treatment failures in patients with advanced disease, insufficient response (recurrences and resistance) as well as late toxicity of the currently used chemotherapy – including second malignancies, cardiovascular toxicity and infertility – requires improvement in standard options for treating HL [23]. Targeted therapy is an innovative research field in oncology, where the defects of major regulatory steps fine-tuning critical cell functions such as survival, proliferation and apoptosis serve as molecular targets.

There is substantial evidence highlighting the importance of changes in the activity of different PI3K pathway members, including mTOR complexes. Here we show that mTOR activity is a characteristic feature in the majority (>50%) of MCL, BL, DLBCL, ALCL and HL cases. High mTOR activity of HRS cells is further supported by our second TMA study focusing on HLs.

Previous publications reported only small numbers of cases without considering subclassification of HL [24, 25]. Based on the evaluation of different downstream mTOR target proteins in 83 HL cases, increased mTOR activity was confirmed in more than 90% of HLs in our work, which was independent of HL subtype and clinical parameters. Low mTOR activity cases had no relapse, and these patients had more than 5 year disease free survival, with complete remission. However, high mTOR activity was observed in the case of both favorable and unfavorable clinical response, therefore it cannot be considered as a prognostic indicator. We are aware that the 83 HL patients included in our study comprise a heterogeneous patient group in respect of age, gender, stage, histological type and prognosis. Therefore, it is difficult to reach significant conclusions; nevertheless, our study offers a comprehensive overview of this heterogeneous group, which is obviously characterized by high mTOR activity in general.

At a molecular level, mTOR activity is known to play a role in cyclin D1 overexpression and cell cycle dysregulation in MCL [14]. Through the regulation of translation or by directly influencing the activity of p70S6K, mTOR can induce the antiapoptotic functions of mitochondrial proteins, e.g. by BAD phosphorylation, supporting the survival and proliferation of tumor cells [26]. The malfunction of apoptotic pathways and the overexpression of several cyclins (cyclin A, B1 and E) are also known in HL [27]. The overexpression of antiapoptotic signals (Bcl-xL) showed correlation with high mTOR activity in our study.

Each time a protein known to be a member of regulatory signaling pathways, participating in the development and/or progression of malignancies is brought into focus, the question arises: can we turn our knowledge to therapeutic advantage? In the case of mTOR, inhibitors already exist (rapamycin and its analogs: rapalogs), which are well tolerated [28], and rapamycin has also been shown to synergize with anticancer agents in several tumors [12, 29‐31]. Rapalogs/rapamycin inhibited proliferation and induced apoptosis, moreover, they increased the apoptotic effect of chemotherapeutic agents (doxorubicin, vincristine and etoposide) in HL cells in our xenograft and in vitro experiments. These results – along with others [32‐35] – suggest that mTOR inhibition is an option in tumors with increased mTOR activity. In this respect HL could be a good candidate, as high mTOR activity and mTORC1 expression could be detected in a high percentage of cases, and mTORC1 inhibition also had an antiproliferative and apoptotic effect in vitro and in vivo.

The efficiency of mTOR inhibitors may be dependent on the ratio of mTOR complexes [36]. While mTORC1 is sensitive to currently used mTOR inhibitors, the rapalog sensitivity of mTORC2 is still conflicting, and may vary in different cell types [37, 38]. New dual inhibitors – inhibiting both mTOR complexes, or mTORC1 and upstream elements of the PI3K/Akt/mTOR pathway – are being developed [39]. The inclusion of upstream proteins is quite logical, because the inhibition of mTORC1 may be able to activate them. The immunohistochemical detection of the phosphorylated forms of Akt (specifically, Ser473, which is connected to mTORC2) is very difficult. We tested different antibodies but we could not detect realiably specific staining in our lymphoid tissues. Baker et al. investigated the stability of phosphorylated Akt and they established that postoperative surgical samples may be of limited value for measuring phospho-Akt levels because Akt can be dephosphorylated quickly during tumor removal and fixation [40]. Considering this, we chose to investigate the expression of Rictor, one essential component of functioning mTORC2. We concluded that mTORC2 was not a characteristic feature when Rictor expression was not detected in the samples. Several solid and lymphoid malignancies such as non-GC DLBCLs overexpress Rictor (a characteristic protein in mTORC2), which potentially indicates increased mTORC2 activity [21, 41, 42]. Rictor was not overexpressed in our HL cell lines and cases, which can explain the sensitivity to rapamycin/rapalogs.

Taken together, Hodgkin-lymphoma is characterized by high mTOR activity, and this high mTOR activity does not exclude good prognosis. Moreover, mTORC1 may be a potential therapeutic target in HL, especially when commonly used protocols prove ineffective, and may also allow dose reduction of chemotherapeutic drugs in order to decrease late toxicity without diminishing treatment efficacy. The combination of mTOR inhibitors with other agents targeting critical molecular sites will likely be crucial for achieving the best clinical response.

Based on our results, mTOR activity may be a potential therapeutic tool in different lymphoma types. In particular, the majority of Hodgkin-lymphomas have high mTOR activity (with no mTORC2/Rictor expression). These data, along with our in vitro and in vivo results with mTOR inhibitors suggest that the inhibition of mTORC1 may be feasible in the therapy, especially in Hodgkin-lymphomas when standard protocols prove ineffective. The combination of mTOR inhibitors with other agents will probably offer the highest efficiency for achieving the best clinical response, and may also allow dose reduction in order to decrease late treatment toxicity in these cases.