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. 2016 Nov 8;7(45):72886-72897.
doi: 10.18632/oncotarget.12128.

Synergistic Activity of ALK and mTOR Inhibitors for the Treatment of NPM-ALK Positive Lymphoma

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Free PMC article

Synergistic Activity of ALK and mTOR Inhibitors for the Treatment of NPM-ALK Positive Lymphoma

Sara Redaelli et al. Oncotarget. .
Free PMC article

Abstract

ALK-positive Anaplastic Large Cell Lymphoma (ALCL) represents a subset of Non-Hodgkin Lymphoma whose treatment benefited from crizotinib development, a dual ALK/MET inhibitor. Crizotinib blocks ALK-triggered pathways such as PI3K/AKT/mTOR, indispensable for survival of ALK-driven tumors.Despite the positive impact of targeted treatment in ALCL, resistant clones are often selected during therapy. Strategies to overcome resistance include the design of second generation drugs and the use of combined therapies that simultaneously target multiple nodes essential for cells survival. We investigated the effects of combined ALK/mTOR inhibition. We observed a specific synergistic effect of combining ALK inhibitors with an mTOR inhibitor (temsirolimus), in ALK+ lymphoma cells. The positive cooperation resulted in an increased inhibition of mTOR effectors, compared to single treatments, a block in G0/G1 phase and induction of apoptosis. The combination was able to prevent the selection of resistant clones, while long-term exposure to single agents led to the establishment of resistant cell lines, with either ALK inhibitor or temsirolimus. In vivo, mice injected with Karpas 299 cells and treated with low dose combination showed complete regression of tumors, while only partial inhibition was obtained in single agents-treated mice. Upon treatment stop the combination was able to significantly delay tumor relapses. Re-challenge of relapsed tumors at a higher dose led to full regression of xenografts in the combination group, but not in mice treated with lorlatinib alone. In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for ALK+ ALCL patients.

Keywords: ALK/ALCL; TKI; resistance; synergy; targeted therapy.

Conflict of interest statement

CONFLICTS OF INTEREST

CGP has research funding from Pfizer.

Figures

Figure 1
Figure 1. Evaluation of combined treatment effect on cellular proliferation
Karpas 299 cells or normal lymphocytes were treated for 72 hours with selected nanomolar concentrations of ALK inhibitors and temsirolimus as single agents (ALK inhibitor alone, blue circles; Temsirolimus alone, red squares) or in combination (combo, green triangles). After 72 hours, tritiated thymidine incorporation was measured. Drug concentrations are indicated below the graphs: selected ratios, corresponding to those reported in Table 1, are shown. Each data point is normalized over the DMSO-treated control. Results are the average of at least 3 independent experiments performed in quadruplicate. Cri = Crizotinib, CH = Alectinib, PF = Lorlatinib, Tems = Temsirolimus.
Figure 2
Figure 2. Immunoblot analysis of ALK/mTOR downstream pathways
Two million Karpas 299 cells were treated for 4 hours with different concentrations of temsirolimus (T) and alectinib (CH) (A) or lorlatinib (PF) (B) as single agents or in combination. Whole cell lysates were loaded on a gel and probed with the indicated antibodies in western blot. Densitometry analysis of phospho-p70S6K and phospho-STAT3, were calculated relative to the control sample, normalized over the actin signal. The data are representative of two independent experiments. Lanes of interest derived from a single western blot image were juxtaposed.
Figure 3
Figure 3. Effect of combined treatment on cell cycle
Karpas 299 were treated up 96 hours with the indicated concentrations of temsirolimus, alectinib or lorlatinib either as single agents or in combination. Cell cycle analysis was evaluated after 72 hours with propidium iodide staining: (A) shows results for alectinib treated cells, (B) for lorlatinib treated cells. Results are the average of three independent experiments. (C, D) PI-Annexin V-FITC double staining on Karpas 299 samples performed in parallel to cell cycle analysis, after 72 hours of incubation with the drugs. Data presented refer to three independent experiments. (E) Karpas 299 viability was indirectly assessed by MTS assay after 96 hours of treatment. Percentage of viability normalized over the control sample is shown in the graph as average of 3 independent experiments ± SD For all the experiments t-test was performed to assess the statistical significance of the differences observed (*p-values < 0.05, **p-values < 0.01).
Figure 4
Figure 4. Selection and characterization of resistant cell lines
(A) proliferation rate expressed as population doublings for Karpas 299 cells exposed to increasing concentrations of alectinib, temsirolimus or combination. Colored arrows indicate a dose escalation: red arrows correspond to temsirolimus, blue arrows to alectinib, green to the combination. The corresponding doses are indicated by a dot in the curves (B) dose-response curve of Karpas 299 cells resistant to alectinib (K299-CHR80) treated with alectinib. Parental Karpas 299 cells are used as control. Proliferation was assessed by tritiated thymidine incorporation assay after 72 hours of treatment. (C) western blot characterization of K299-CHR80 cell line treated with increasing concentrations of alectinib; comparison with parental Karpas 299. (D) dose-response curve of Karpas 299 cells resistant to temsirolimus (K299-TemR200) treated with temsirolimus. Parental Karpas 299 cells are used as control. Proliferation was assessed by tritiated thymidine incorporation assay after 72 hours of treatment (E) western blot characterization of K299-TemR200 treated with increasing concentrations of temsirolimus, compared to parental cells.
Figure 5
Figure 5. Effect of combined treatment on soft agar assay
To explore the effects of long-term exposure to single or combined drugs on anchorage-independent growth, we performed a soft-agar colony assay on Karpas 299. Five thousands cells were seeded in 6-well plates, embedded in 0.3% low-melting agarose with or without different inhibitors concentrations as indicated on a 0.5% bottom agar layer, as previously described. Three weeks after seeding, colony formation was virtually suppressed by the simultaneous treatment with alectinib and temsirolimus.
Figure 6
Figure 6. In vivo evaluation of the effect of combined treatment
(A) summary of treatment schedule. For each group the doses and time of treatment are indicated. For lorlatinib and treatment combination group the two time scales (from treatment start and from dose increase) are reported. (B) relative tumor volumes in mice injected with Karpas 299 and treated with single agents, combination or vehicle only. For the graphical representation the tumor volume of each mouse was normalized over its volume at day 1. Shaded area indicates treatment period. Mean ± SEM is plotted. Mice receiving combination treatment showed a statistically significant reduction in the normalized tumor volumes compared to lorlatinib alone, both at day 7 (median = 0.53 vs 1.19 ***p-value = 0.003) and at day 15 (median = 0.15 vs 2.38,***p-value = 0.003. (C) Event-free survival analysis. The tumor growth for each mouse was monitored and normalized to its size at day 1. A two fold tumor increase was considered as an event. (D) Relative mice weight measurements. For the graphical representation the weight of each mouse was normalized over its weight at day 1. Mean ± SEM is plotted (E) relative tumor volumes during treatment increase. Lorlatinib was administered twice a day at 0.25 mg/kg either alone (8 mice) or in combination (8 mice) with temsirolimus [2 mg/kg]. The curves refer to the average normalized tumor volume.

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