An MDM2 antagonist (MI-319) restores p53 functions and increases the life span of orally treated follicular lymphoma bearing animals

Abstract

Background: MI-319 is a synthetic small molecule designed to target the MDM2-P53 interaction. It is closely related to MDM2 antagonists MI-219 and Nutlin-3 in terms of the expected working mechanisms. The purpose of this study was to evaluate anti-lymphoma activity of MI-319 in WSU-FSCCL, a B-cell follicular lymphoma line. For comparison purpose, MI-319, MI-219 and Nutlin-3 were assessed side by side against FSCCL and three other B-cell hematological tumor cell lines in growth inhibition and gene expression profiling experiments.

Results: MI-319 was shown to bind to MDM2 protein with an affinity slightly higher than that of MI-219 and Nutlin-3. Nevertheless, cell growth inhibition and gene expression profiling experiments revealed that the three compounds have quite similar potency against the tumor cell lines tested in this study. In vitro, MI-319 exhibited the strongest anti-proliferation activity against FSCCL and four patient cells, which all have wild-type p53. Data obtained from Western blotting, cell cycle and apoptosis analysis experiments indicated that FSCCL exhibited strong cell cycle arrest and significant apoptotic cell death; cells with mutant p53 did not show significant apoptotic cell death with drug concentrations up to 10 muM, but displayed weaker and differential cell cycle responses. In our systemic mouse model for FSCCL, MI-319 was tolerated well by the animals, displayed effectiveness against FSCCL-lymphoma cells in blood, brain and bone marrow, and achieved significant therapeutic impact (p < 0.0001) by conferring the treatment group a > 28% (%ILS, 14.4 days) increase in median survival days.

Conclusion: Overall, MI-319 probably has an anti-lymphoma potency equal to that of MI-219 and Nutlin-3. It is a potent agent against FSCCL in vitro and in vivo and holds the promises to be developed further for the treatment of follicular lymphoma that retains wild-type p53.

Figure 1

Chemical structure of MI-319 and MI-219 and MDM2 protein binding assay. (A), Chemical structure of MI-319 and MI-219. (B), Fluorescence polarization-based MDM2 binding assay. The binding affinities (Ki values) were determined by a competitive fluorescence polarization-based binding assay using recombinant His-tagged MDM2 (amino acids 1-118) and PMDM6-F (5-FAM-βAla-βAla-Phe-Met-Aib-pTyr-(6-Cl-_L-Trp)-Glu-Ac3c-Leu-Asn-NH2), a fluorescently labeled high-affinity p53-based peptide.

Figure 2

Effect of MI-319, MI-219 and Nutlin-3 on cell proliferation in vitro. Established tumor cell lines. Cells were grown for 48 hours. The number of viable cells was determined by MTT assay.

Figure 3

Effect of MI-319, MI-219 and Nutlin-3 on cell proliferation in vitro. Mononuclear cells isolated from lymphoma patients - BP071708 is diffuse large B-cell lymphoma (DLCL), RM072307 is marginal zone B-cell lymphoma (MZL), JC012706 is another marginal zone B-cell lymphoma (MZL), and CH012306 is small lymphocytic lymphoma (SLL). The number of viable cells was determined by trypan blue exclusion test. Data represents mean of three independent experiments. * represents p < 0.05 ** represents p < 0.01.

Figure 4

Gene expression profiling by Western blotting. Cells were treated with indicated chemical or equal volume of 100% DMSO for 12 hours.

Figure 5

Cell cycle analysis. Cells were treated with MI-319 or equal volume of 100% DMSO for 24 hours. Numbers plotted here are the average of at least three independent experiments. * represents p < 0.05 and ** represents p < 0.01.

Figure 6

MI-319 induces cell apoptosis in FSCCL cells only. (A) Cells were stained with Annexin V-FITC and quantification of the percentage of apoptotic cells was done with a Coulter EPICS 753 flow cytometer. (B) Tunnel assay. TdT+ cells (apoptotic cells) were assessed with a Coulter EPICS 753 flow cytometer. Numbers plotted here are the average of three independent experiments. * represents p < 0.05 and ** represents p < 0.01.

Figure 7

Survival of FSCCL-SCID mice is prolonged with the treatment of MI-319. (A) Treatment with MI-319 did not result in significant body weight changes (> 15%). Wn=mouse body weight at day (n); W4 = mouse body weight at day (4) when the administration of MI-319 was started. (B) H&E staining of mouse blood smear and sections of mouse brain, bone marrow, liver, and spleen. Arrows in the upper blood panel (untreated) point to lymphocytes, indicating lymphoma involvement. Lower brain panel (MI-319-treated) is an example of no involvement by lymphoma. Note the thin meningeal lining of the brain (between opposing arrows). In comparison, the upper panel (untreated) shows examples of involvement of the meninges by lymphoma. Note the expanded space between the opposing arrows indicative of lymphoma cell infiltration. For the bone marrow (femurs) sections, lower panel (MI-319-treated) shows examples of normal marrow and upper panel (untreated) shows examples of involvement by WSU-FSCCL (note the cavities filled with lymphocytes). (C) The survival percentage of untreated and MI-319-treated mice is plotted against days post FSCCL inoculation. The control animals have a median survival day of 51.6, whereas the treatment animals have a median survival day of over 66.0. n = 7 for control group, n = 6 for treatment group, p < 0.0001.

Figure 8

MI-319-induced FSCCL apoptotic cell death might be p53 transcription-independent. (A) FSCCL cells were treated for 12 hours and Western blotting experiments were performed to examine the protein levels of p53, MDM2, p21, Bax, PUMA and PARP. (B) FSCCL cells were grown for 24 hours after drug treatment and apoptotic cell death was assessed with Tunnel assay.