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

BACKGROUND: Targeting signaling pathways is an attractive approach in many malignancies. The PI3K/Akt/mTOR pathway is activated in a number of human neoplasms, accompanied by lower overall and/or disease free survival. mTOR kinase inhibitors have been introduced in the therapy of renal cell carcinoma and mantle cell lymphoma, and several trials are currently underway. However, the pathological characterization of mTOR activity in lymphomas is still incomplete. METHODS: mTOR activity and the elements of mTOR complexes were investigated by immunohistochemistry on tissue microarrays representing different human non-Hodgkin-lymphomas (81 cases) and Hodgkin-lymphomas (87 cases). The expression of phospho-mTOR, phospho-4EBP1, phospho-p70S6K, phospho-S6, Rictor, Raptor and Bcl-2, Bcl-xL, Survivin and NF-kappaB-p50 were evaluated, and mTOR activity was statistically analyzed along with 5-year survival data. The in vitro and in vivo effect of the mTOR inhibitor rapamycin was also examined in human Hodgkin-lymphoma cell lines. RESULTS: The majority (>50%) of mantle cell lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma and Hodgkin-lymphoma cases showed higher mTOR activity compared to normal lymphoid tissues. Hodgkin-lymphoma was characterized by high mTOR activity in 93% of the cases, and Bcl-xL and NF-kappaB expression correlated with this mTOR activity. High mTOR activity was observed in the case of both favorable and unfavorable clinical response. Low mTOR activity was accompanied by complete remission and at least 5-year disease free survival in Hodgkin-lymphoma patients. However, statistical analysis did not identify correlation beetween mTOR activity and different clinical data of HL patients, such as survival. We also found that Rictor (mTORC2) was not overexpressed in Hodgkin-lymphoma biopsies and cell lines. Rapamycin inhibited proliferation and induced apoptosis in Hodgkin-lymphoma cells both in vitro and in vivo, moreover, it increased the apoptotic effect of chemotherapeutic agents. CONCLUSIONS: Targeting mTOR activity may be a potential therapeutic tool in lymphomas. The presence of mTOR activity probably indicates that the inclusion of mTOR inhibition in the therapy of Hodgkin-lymphomas may be feasible and beneficial, especially when standard protocols are ineffective, and it may also allow dose reduction in order to decrease late treatment toxicity. Most likely, the combination of mTOR inhibitors with other agents will offer the highest efficiency for achieving the best clinical response

Mammalian Target of Rapamycin (mTOR) Activity Dependent Phospho-Protein Expression in Childhood Acute Lymphoblastic Leukemia (ALL)

Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25–30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments

The Effects of Different mTOR Inhibitors in EGFR Inhibitor Resistant Colon Carcinoma Cells

Several monoclonal antibodies and inhibitors targeting signalling pathways are being used in personalised medicine. Anti-EGFR antibodies seem to be effective, however, therapy resistance often occurs in colon carcinoma cases. mTOR inhibitors (mTORIs) could have a potential role in the breakthrough of therapy resistance. The mTOR activity related protein expression patterns and the in vitro effects of EGFR inhibitors (EGFRIs), mTORIs and their combinations were studied in different colon carcinoma cell lines (with different genetic backgrounds). Alamar Blue test and flow cytometry were used to analyse the in vitro proliferation and apoptotic effects of cetuximab, gefitinib, cisplatin, rapamycin, PP242 and NVP-BEZ235. The expressions of mTOR activity related proteins (p-70S6K, p-S6, Rictor, p-mTOR, Raptor) were studied by Western blot, immunocytochemistry and Duolink staining. The EGFRI resistance of the studied colon carcinoma cell lines related to their known mutations were confirmed, neither gefitinib nor cetuximab inhibited the proliferation or induced apoptosis in vitro. Individual differences in Rictor and Raptor expressions were detected by Western blot and immunocytochemistry beside elevated mTOR activity of these different colon carcinoma cell lines. These expression patterns correlated to the mTORIs sensitivity differences, moreover, mTORIs could enhance the effects of EGFRIs and other in vitro treatments. Our results suggest that mTORI combinations could be helpful in both EGFRI and platinum-based therapy of colon carcinomas. Moreover, we suggest determining both mTOR complex activity and mutations in Akt/mTOR signalling pathways for selecting the appropriate mTORIs and patients in potential future combination treatments

Summary of clinical data and mTOR activity related p-4EBP1 in 49 primary ALL patients.

§<p>cutoff equals 1.1 (OD p-4EBP1 ELISA).</p>*<p>based on cytogenetic and molecular genetic analysis; the hyperdiploid group also includes two cases with ETV6/RUNX1 fusion (good prognosis). High risk genetic lesions are: BCR/ABL fusion (two cases), ABL amplification (one case) and RUNX1 amplification (one case).</p>#<p>based on steroid response and current status; see methods: SR - standard, IR - intermediate, HR - high risk arm of the therapy; TX – transplantation, TRM - transplantation related mortality.</p>φ<p>– Fisher’s exact probability and,</p>χ<p>- chi² tests.</p>π<p>- significant correlation with mTOR activity level.</p

Multivariate analysis of various standard independent prognostic factors in ALL cases.

*<p>Cutoff value is 1.1 p-4EBP1 OD.</p><p>RR – relative risk, 95% CI –95% confidence interval.</p><p>na – not applicable.</p

mTOR activity related p-4EBP1 expression in samples of ALL patients with different prognoses (ELISA) and detection of p-4EBP1 and p-S6 in ALL cells by flow cytometry.

<p>(a) P-4EBP1 OD values in 49 ALL samples (n = 37 for patients with good prognosis; n = 12 for poor prognosis, criteria for prognostic groups are described in the Methods section). P-4EBP1 OD levels were significantly different between patients with good and poor prognosis (p<0.05). The cutoff value between p-4EBP1 OD values representing high and low mTOR activity was determined to be 1.1 by ROC curve analysis. (b) p-S6 and p-4EBP1 overexpression is confirmed by flow cytometry in ALL cells. Representative histograms for p-4EBP1 and p-S6 flow cytometric analysis in normal PMNC and in ALL samples. Expression was calculated as the difference between MFIs of p-4EBP1 or p-S6 stained (p-4EBP1 and p-S6, respectively) and parallel unstained controls (-co). Differences between MFIs (ΔMFI) was 2.35 (pS6) and 1.56 (p-4EBP1) for normal peripheral mononuclear cells. However, ΔMFI was 42.6 and 32.4 for p-S6 and p-4EBP1, respectively, in the representative ALL case shown here.</p

Survival analysis (Kaplan-Meier curves).

<p>Kaplan-Meier curves show overall and relapse free survivals for patients (n = 49) stratified by mTOR activity. Cutoff value between low and high mTOR activity groups (n = 37 and n = 12, respectively) was determined to be 1.1 for p-4EBP1 ODs (ELISA). We found that patients with low mTOR activity had significantly longer overall and relapse free survival (P<<0.05 with log-rank test; o: complete event, +: censored cases).</p

Changes in mTOR related p-4EBP1 expression in samples of ALL patients after <i>in vivo</i> chemotherapy and in cultured ALL cells treated with rapamycin <i>in vitro</i>.

<p>(a) P-4EBP1 OD is reduced by chemotherapy, but re-increases during relapse in ALL patient samples. Data is shown from 2 representative patients with good prognosis and 2 patients with poor prognosis at day 0, day 33, after three months of treatment, and at relapse (ELISA). (b) <i>In vitro</i> rapamycin sensitivity is variable in different human ALL samples (n = 4). P-4EBP1 levels variably decrease or increase after <i>in vitro</i> rapamycin treatment (50 ng/ml, 24 h) in isolated ALL cells (ELISA). A decrease in p-4EBP1 was associated with concomitant induction of apoptosis by rapamycin (>50% apoptotic cells in the first two samples; data not shown). (The first three samples were cultured from cells isolated from primary ALL cases and one at relapse.) *- P<0.05.</p

The amount of mTOR activity dependent phospho-proteins (p-4EBP1 and p-S6) in lymphoma/leukemia cells (ELISA).

<p>(a.) P-4EBP1 ODs are shown for normal control peripheral blood mononuclear cells (N1, N2, N3), isolated peripheral normal B- and T-cells (B, T), isolated peripheral blood mononuclear cells and isolated bone marrow mononuclear cells from non-leukemic patients (NL) and isolated primary childhood ALL cells from representative patient samples, and from leukemia (Jurkat, CEM, Mn60, Nalm6) and lymphoma cell lines (KMH2). (b.) Relative p-4EBP1 and p-S6 expression was determined in samples from ALL patients (n = 10) and in leukemia/lymphoma cell lines (Jurkat, CEM, Mn60, Nalm6, KMH2); expression of normal lymphoid cells is considered to be 1. (OD: optical density; MNC: mononuclear cells. Equal cell numbers and equal protein concentrations were confirmed for comparisons.).</p