Plitidepsin (Aplidin) is a potent inhibitor of diffuse large cell and Burkitt lymphoma and is synergistic with rituximab

Abstract

Plitidepsin (Aplidin), an antitumor agent of marine origin, presently is undergoing phase II/III clinical trials, and has shown promise for the treatment of lymphoma. Here, we describe the antitumor effects of plitidepsin alone and in combination with rituximab and investigated the effects of each drug and the combination on the cell cycle and mechanism of cell death. Several Diffuse Large Cell Lymphoma (DLCL) lines and Burkitt cell lines were tested for sensitivity to plitidepsin and rituximab. All DLCL and Burkitt lymphoma cell lines were inhibited by plitidepsin in nanomolar concentrations, while rituximab sensitivity varied among different cell lines. Ramos and the RL cell lines proved sensitive to rituximab and were used to test the effects of each of the two drugs. The two agents exhibited synergism at all tested concentrations. For in vivo studies, irradiated athymic nude mice were engrafted with the Ramos lymphoma. Treatment was initiated when the tumors were ~0.5 cm in diameter, and toxic and therapeutic effects were monitored. In the in vivo study, additive effects of the combined two drugs, was demonstrated without an increase in host toxicity. The in vitro synergy and the in vivo additive antitumor effects without an increase in host toxicity with two relatively non-marrow suppressive agents encourages further development of this combination for treatment of aggressive B-cell lymphomas.

Key words:

• lymphoma
• rituximab
• plitidepsin
• synergy
• combination therapy

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

N.B. was supported by a T32 grant CA108455 from the NIH

Figures and Tables

Figure 1 Relative expression of CD20 on lymphoma B-cell lines. B-cell lines were tested for CD20 expression and analyzed by flow cytometry. Histograms of CD20 fluorescence intensity are shown compared with the IgG1 isotype. The results presented are representative of at least three independent experiments.

Figure 2 Combination of plitidepsin and rituximab synergistically inhibit B cells. Dose-response curves (log scale) of cells cultured in RPMI plus 10% non-heat inactivated FBS exposed to plitidepsin, rituximab and their combination (A), compared with doxorubicin, rituximab and their combination (B), after 96 h of treatment. (C) Sequence of drug concentrations at a fixed plitidepsin:rituximab dose ratio with the IC₅₀ at 1.25 nM: 2.17 nM.

Figure 3 Plitidepsin blocks cells in the G₁/S phase of the cell cycle. Ramos (A), and RL cells (B), were incubated with rituximab, plitidepsin, or their combination at 37°C for 24 h. Cells (1 á 10⁵) were stained with 0.5 ml PI/RNase staining buffer and analyzed by flow cytometry with CellQuest software (Becton-Dickinson). Results analyzed with the Modfit software are presented as the mean and SD from three independent experiments. In Ramos cells p < 0.005 only for the plitidepsin treatments in comparison with the control. In RL cells p < 0.005 for all treatments in comparison with the control. (C) Histograms of the cell cycle fluorescence intensity after 24 h treatment with each drug alone and in combination.

Figure 4 Apoptosis induced by plitidepsin, rituximab or their combination in rituximab-sensitive B cells. Ramos and RL cells were incubated with rituximab, plitidepsin, or their combination at 37°C for 48 h. Cells (1 × 10⁵) were stained with Annexin V-FITC and 7AAD and analyzed by flow cytometry. Data are representative of three independent experiments. (A) Y axis represents the apoptotic cells. In Ramos and RL cells p < 0.001 for the rituximab treatments alone and in combination with plitidepsin in comparison with the control. (B) In the histograms, FL-1 (Annexin V-FITC) thresholds were determined by using cells stained without Annexin buffer per manufacturer's protocol. (C) Expression of caspase-3 and caspase-7 was analyzed by western blotting in Ramos cells treated with plitidepsin (C), plitidepsin plus rituximab (D) and rituximab (E) for the indicated times.

Figure 5 Western blot analysis of caspase activation in B cells expressing different levels of CD20. B cells with different levels of resistance to rituximab were treated for the indicated times with plitidepsin, plitidepsin plus rituximab and rituximab. Expression of caspase-7 (A) and caspase-3 (B) was analyzed by western blotting.

Figure 6 The effect of plitidepsin, rituximab and the combination of these drugs in Ramos tumor xenografts in mice. (A) Ramos cells (10⁷) were inoculated subcutaneously in the flanks of irradiated athymic nude female Ncr mice. When the tumor diameter reached 0.5 cm, mice were simultaneously treated with rituximab (200 µg/kg), plitidepsin (0.2 or 0.4 mg/kg), or the combination of these drugs at four doses 3 d apart beginning on day 15 after tumor cell inoculation. Tumor measurements were taken at 2 d intervals (B) Survival of the mice treated with plitidepsin or rituximab alone, and in combination. Survival curves between control and each drug and control and combination treatment was significant (p = 0.03).

Table 1 Effect of rituximab and plitidepsin on lymphoma B-cell lines