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

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

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

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

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

Cell migration after zoledronic acid treatment in melanoma cells.

<p>(<b>A-C</b>) Migrated distance as a function of time and (<b>D</b>) average migrated distance after zoledronic acid (ZA) treatment in melanoma cells measured by videomicroscopy. A profound and significant increase in migrated distance was found in all of the BRAF mutant cells. A modest but significant increase in migration was found in VM-47 triple wild-type and VM-15 NRAS mutant cells. Colors blue, red and green indicate BRAF, NRAS mutation and wild-type for these genes, respectively. Data shown as average ± SEM are from at least three independent measurements. Asterisks indicate significant difference of p < 0.05 from the respective control with unpaired two-tailed T test.</p

<i>In vivo</i> effects of zoledronic acid treatment.

<p>Effect of zoledronic acid (ZA) treatment using <i>in vivo</i> subcutaneous xenograft model of human melanoma cells in SCID mice (<b>A, C, E</b>). ZA treatment failed to show effects in the subcutaneous growth of melanoma cells with either mutation. (<b>B, D, F</b>) Effect of ZA treatment using <i>in vivo</i> spleen liver colonization model of human melanoma cells in SCID mice. ZA did not inhibit the primary tumor or metastatic growth of melanoma cells. Data shown as average ± SEM.</p

Cell viability, clonogenic growth and apoptosis in melanoma cells after zoledronic acid treatment.

<p>(<b>A</b>) Dose-response analysis of cell viability of human melanoma cell lines with different mutations after 72hs treatment with ZA measured by SRB assay; Most pronounced reduction in cell viability was observed in two NRAS mutant lines (<b>B</b>) Long-term effect of 10 days of 5 μM ZA treatment on clonogenic growth. Resistance was found in BRAF mutant and PTEN wild-type cells. Of note, NRAS and BRAF double mutant cells demonstrated intermediate sensitivity (<b>C</b>) Apoptosis induction after 25μM ZA treatment demonstrated by the proportion of TUNEL positive cells and (<b>D</b>) apoptosis induction of 72hs ZA treatment evaluated by the immunoblot of cleaved PARP. Limited apoptosis induction was found in BRAF mutant and PTEN wild-type cells and in the MDM2 over expressing WM239 cells. Colors green, red, blue, and dark-blue indicate triple wild-type, NRAS, BRAF, and BRAF mutant/PTEN-null mutational status of the cells, respectively. Data shown as average ± SEM are from at least 5 repeats. Asterisks indicate the lowest concentration of ZA treatment resulting in a significant difference with p < 0.05 from control by ANOVA and Dunnett’s post hoc test in the cell viability assay. Data shown as average ± SEM are from at least 3 measurements. Asterisks indicate significant difference of p < 0.05 between given mutational group and the BRAF mutant PTEN wild-type group by Kruskal-Wallis and Dunn’s post hoc test in the clonogenic assay, and p < 0.05 difference from the respective control by unpaired two tailed T test in the apoptosis assay. (C = control; Z = zoledronic acid).</p

Activation of downstream elements of the RAS/RAF pathway in melanoma cells after zoledronic acid treatment.

<p>(<b>A</b>) Representative blots of the effect of 48hs zoledronic acid (ZA) treatment on the activation of Erk1/2 and S6. (<b>B</b>) Quantification of the effect of ZA treatment on the activation of Erk1/2. Treatment with ZA resulted in robust increase in the phosphorylation of Erk1/2 in MEWO and M24met cells. (<b>C</b>) Quantification of the effect of ZA treatment on the activation of S6. After the treatment with ZA, decreased activation of S6 proteins was found only in NRAS mutant M24met and VM-15 cells. Colors blue, red and green indicate BRAF, NRAS mutation and wild-type for these genes, respectively. (C = control; ZA = zoledronic acid).</p