- Open Access
An MDM2 antagonist (MI-319) restores p53 functions and increases the life span of orally treated follicular lymphoma bearing animals
- Ramzi M Mohammad1Email author,
- Jack Wu†1,
- Asfar S Azmi1,
- Amro Aboukameel†1,
- Angela Sosin1,
- Sherwin Wu2,
- Dajun Yang4,
- Shaomeng Wang3 and
- Ayad M Al-Katib1
© Mohammad et al; licensee BioMed Central Ltd. 2009
Received: 3 February 2009
Accepted: 3 December 2009
Published: 3 December 2009
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.
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 μM, 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.
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.
Follicular lymphoma is a slow growing B-cell lymphoma and is the second most common type of non-Hodgkin's lymphoma (NHL), which is expected to have more than 66,000 new cases in the USA in 2008 . Despite improvement of survival rates in recent years [2, 3], follicular lymphoma remains incurable due mainly to limitations of the current first-line standard of treatment, which usually involves concomitant administration of humanized anti-CD20 monoclonal antibody rituximab and a chemotherapy regimen . In the pivotal clinical trial that led to the approval of rituximab for clinical use in the USA, only 48% of patients with relapsed follicular lymphoma responded . Therefore, better therapeutics is needed to further improve the outcome of afflicted patients.
A growing number of recent reports suggest that small molecule inhibitors targeting the MDM2-p53 interaction may represent very promising, specific and novel therapeutics against various types of cancers [6–9]. The p53 gene is an important tumor suppressor. It can promote cell cycle arrest by up-regulating the expression of genes involved in cell cycle control, such as p21WAF1[10, 11]; and can also promote apoptosis, possibly by the up-regulation of pro-apoptotic genes, such as Bax and PUMA [12–14]. Among all the cancer patients, approximately half of them have mutated or deleted p53 gene, which leads to defective p53 protein or complete missing of functional p53 protein [15, 16]. Among the remaining patients with wild-type p53 gene, functional p53 protein is quickly degraded after protein translation, primarily through direct interaction with the MDM2 protein . Thus, using small molecules to block the MDM2-p53 interaction is an attractive approach to stabilize functional p53 protein and restore its anti-tumor activity in tumors with wild-type p53 gene.
Unlike in many solid tumors, alterations of the p53 gene are far less common in hematological malignancies (generally < 15%) . Therefore, small-molecule inhibitors that interrupt the MDM2-p53 interaction might represent a new therapeutic strategy for the treatment of most patients with this kind of disease. Previous studies demonstrated that a different inhibitor of MDM2, Nutlin-3, is indeed able to efficiently induce apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) [19–24]. To our knowledge, however, there are no reports so far on the studies of this kind of small-molecule inhibitors against follicular lymphoma. In the present study, we report on the evaluation of a new inhibitor of the MDM2-p53 interaction, named MI-319, against a follicular small cleaved B-cell lymphoma line (FSCCL) in vitro by using cultured cells and in vivo by using a systemic model in mice with severe combined immunodeficiency (SCID). MI-319 is closely related to MDM2 antagonists MI-219  and Nutlin-3  in terms of the expected main working mechanisms. For comparison purpose, we also assessed these three compounds side by side against FSCCL and three other B-cell hematological tumor cell lines in growth inhibition and gene expression profiling experiments.
MI-319 binds to MDM2 protein with high affinity
FSCCL cell growth can be effectively inhibited by MI-319, MI-219 and Nutlin-3
Summary of cell P53 status
p53 mutation status
FSCCL cells exhibited increased protein levels of p53, MDM2, p21 and cleaved PARP after treatment with MI-319, MI-219 or Nutlin-3
MI-319 induced differential cell cycle arrest responses among FSCCL, WM, RL and DLCL2 cells
MI-319 induces apoptotic cell death only in FSCCL cells
MI-319 has significant anti-lymphoma activity in FSCCL systemic SCID mouse model
Follicular lymphoma is the second most common type of NHL, which has increased incidence over the past three decades and is now the fifth most common cancer in the United States . Current therapeutic tools for follicular lymphoma, such as monoclonal antibodies, radio-immunotherapy, vaccines and chemotherapeutic agents, all have limitations . In an attempt to search for a targeted and less toxic agent that can be administered orally, we evaluated the anti-lymphoma activity of MI-319 in a follicular small cleaved cell lymphoma cell line established in our laboratory. Data obtained in our studies is encouraging and is consistent with the following statements: i) MI-319 is able to bind to MDM2 protein with a high affinity that is over 500-fold more potent than a natural p53 peptide; ii) MI-319 effectively inhibited proliferation of FSCCL cell (p53 wild-type) in vitro, with IC50 value of 2.5 μM for 48-hour treatment; iii) Inhibition of FSCCL cell proliferation by MI-319 involves induction of both cell cycle arrest and apoptotic death; iv) MI-319 displayed potent anti-tumor efficacy in the FSCCL-SCID mouse model.
MI-319 was designed to stabilize p53 protein in cells by blocking the MDM2-p53 interaction. Although many genes in addition to p53 are usually altered in tumors, recent studies suggest that restoring p53 function alone is sufficient to cause regression of established sarcomas, lymphomas, and liver tumors in mice [28–30]. Therefore, restoring functional p53 activity by using small molecules, such as MI-319, to block MDM2-p53 interaction and stabilize p53 protein is an attractive pharmacological approach. Since the discovery of the Nutlins , there has been a great deal of interest in the evaluation of small-molecule inhibitors of the MDM2-p53 interaction against various types of cancer . Currently there are two major classes of such small-molecule inhibitors. One class is represented by Nutlin-3 [6, 31, 32]; the other one is represented by MI-219 . MI-319 is a very close analogue of MI-219. In our fluorescence polarization-based competitive binding assay, MI-319 exhibited a binding affinity to human MDM2 protein that is slightly higher than that of MI-219 and Nutlin-3. Nevertheless, the three compounds have similar potency against the cells tested in this report in terms of growth inhibition and regulation of expression of p53 target genes, such as MDM2, p21, Bax and PUMA. Therefore, we believe that MI-319, MI-219 and Nutlin-3 are probably equal as an MDM2 antagonist. In our remaining cell cycle analysis, apoptotic cell death assays and animal model studies, we assessed only MI-319 simply due to availability issues.
In our studies, we assessed FSCCL side by side with three other cells that have mutant p53-WSU-WM (R213Q), RL (A138P) and WSU-DLCL2 (R248Q). Interestingly, the cells expressing the three mutants behaved differently in terms of cell proliferation, cell cycle arrest and expression of some of p53's target genes, such as MDM2 and p21. WM responded the strongest among the three and RL and DLCL2 responded much weaker. Previous reports have documented that these three p53 mutants still retain some of wild-type p53 protein's regulatory functions. When A138P and R248Q mutants were expressed in p53 null cells, it was found that both of them still retain a little (< 5%) of p53's regulatory activities [36, 37]; according to the studies by Pan et al., the R213Q mutant is still partially functional  and therefore probably retains much more of wild-type p53's regulatory activities. Compared with wild-type p53, R213Q mutant p53 protein has a weaker transactivating activity for p21 gene . Our gene expression profiling data obtained by Western blotting agreed quite well with this result. Treatment with MI-319, MI-219 or Nutlin-3 all led to dose-dependent up-regulation of p21 protein in WM cells, but appeared less robust than that in FSCCL. Overall, it appeared that the different responses of the three cells with p53 mutants correlated very well with the level of wild-type p53's regulatory activities retained by the corresponding p53 mutant proteins.
The main goal of our study is to find a novel agent that holds promises to make its way into clinical trials for the treatment of follicular lymphoma. Thus, we tested the anti-lymphoma activity of MI-319 in vivo by using a systemic FSCCL SCID mouse model. MI-319 given orally for one week showed no major toxicity, such as > 15% weight loss in treated animals, whereas the treatment showed a significant therapeutic impact (p < 0.0001); conferring a more than 28% (14.4 days) increase in life span (ILS).
Our studies showed that MI-319, MI-219 and Nutlin-3 have similar potency as an MDM2 antagonist. MI-319 has potent anti-lymphoma activities against FSCCL cells. It stabilizes p53 protein and induces cell cycle arrest and apoptosis in follicular lymphoma cells that retain wild-type p53. When administered orally to the animals, MI-319 showed significant anti-lymphoma activity. Our results provide confidence towards the development of MDM2 inhibitors for lymphoma patients in the clinic.
Chemical synthesis and competitive binding assay
MI-319 and MI-219 were synthesized by using methods published previously . Nutlin-3 was purchased from Sigma-Aldrich. For cell culture experiments, MI-319, MI-219 and Nutlin-3 were dissolved in 100% DMSO to make 10 mM stock solutions which were kept at -70°C. Fluorescence polarization-based competitive binding assays were performed to determine the binding affinity of MI-319 and MI-219 with a recombinant His-tagged MDM2 protein. The assays were carried out as described previously .
P53 genomic DNA and full-length cDNA sequencing
Genomic DNAs were extracted by adapting a procedure described previously . The amount of genomic DNA was determined by UV absorption at 260 nm. 200 ng was used in each reaction of PCR amplification. Primers to amplify exons 5/6, 7, and 8/9 of human p53 and adjacent intronic sequences were adopted from the literature  with modification of the p53-E5/6-F primer sequence as 5'-ggaggtgcttacgcatgtttg-3'. Amplified PCR products were analyzed by agarose gel electrophoresis, cleaned with Wizard SV Gel/PCR Cleanup kit (Promega, Madison, WI), and sequenced directly. Sequencing was done with the Applied Biosystems ABI Prism 3700 sequencer (Applied Biosystems, CA). Sequencing reactions were performed using the ABI BigDye®Terminator v3.1.
In order to sequence full-length p53 cDNA, total RNAs were isolated from cells using the RNeasy® Isolation Kit (Qiagen, Valencia, CA). The amount of total RNA was estimated by UV absorption at 260 nm. The extracted RNAs (2 μg of each sample) were reverse-transcribed with the ImProm-II(TM) Reverse Transcription System, following the manufacturer's instructions (Promega, Madison, WI). PCR reactions were performed subsequently to amply p53 cDNA with an Eppendorf AG Mastercycler (Hamburg, Germany). Two pairs of primers were used to amplify the full-length p53 coding cDNA sequence. The sequence of the primers is as follows: p53-F1/p53-R1 (5'-aagtctagagccaccgtcca-3'/5'-catagggcaccaccacacta-3'), p53-F2/p53-R2 (5'-gtggaaggaaatttgcgtgt-3'/5'-gtggggaacaagaagtggag-3'). The PCR products were analyzed by agarose gel electrophoresis, cleaned with Wizard SV Gel/PCR Cleanup kit (Promega, Madison, WI), and sequenced directly.
Cell lines FSCCL, WM and DLCL2 were established in our laboratory [25, 42, 43]. The cell line RL was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Mononuclear cells were isolated from four lymphoma patients - BP071708 is diffuse large B-cell lymphoma (DLCL) intermediate grade; RM072307 is marginal zone B-cell lymphoma (MZL) low grade; JC012706 is another marginal zone B-cell lymphoma (MZL) low grade; and CH012306 is small lymphocytic lymphoma (SLL) low grade. All patients had stage IV disease. Lymphoma cells were isolated by Ficoll gradient centrifugation (GE Healthcare, Little Chalfont, United Kingdom), seeded into growth medium right away or aliquoted into fetal bovine serum with 10% DMSO and cryopreserved in liquid nitrogen. Studies involving human tissues were done according to IRB-approved protocol and all patients had signed informed consent prior to tissue procurement. Cells were usually seeded at a density of 2 × 105 viable cells per ml in 24-well or 6-well culture plates (Costar, Cambridge, MA), or 10-cm cell culture dishes (Corning Inc., Corning, NY). All cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, Utah) and 1% Penicillin-Streptomycin (Invitrogen, Carlsbad, CA), at 37°C in a humidified incubator with 5% CO2. The number of viable cells was determined by trypan blue exclusion test with trypan blue (0.4%) purchased from Sigma Chemical Co. (St. Louis, MO) and MTT assay . Statistical analysis was done using the t test (two tailed) with 95% confidence intervals between treated and untreated samples. P < 0.05 was used to indicate statistical significance.
Cells were collected by centrifugation, washed twice with cold PBS, and lysed at 4°C in lysis buffer containing protease inhibitors as described previously . Total protein content in lysates was estimated by the Bradford method . The primary antibodies used in the experiments included p53 (Cell Signaling, Danvers, MA), MDM2 (R&D System, Minneapolis, MN), p21 (Cell Signaling, Danvers, MA), Bax (Sigma, St. Louis, MO), PUMA (Sigma, St. Louis, MO), PARP (Cell Signaling), and β-actin (Sigma).
Cell cycle analysis
Cells were collected by centrifugation and washed twice with cold PBS. Cell pellets were resuspended in 0.5 ml of cold PBS and fixed in 4.5 ml of 70% ethanol and stored at 4°C. On the day of analysis, cells were collected by centrifugation and each pellet was resuspended in 1 ml of staining buffer, which contains 50 μg/mL of propidium iodide, 100 μg/mL of RNase A, and 0.1% of Triton X-100. The cell suspensions were incubated in the dark for 30 minutes at room temperature and subsequently analyzed on a Coulter EPICS 753 flow cytometer for DNA content. The percentage of cells in different phases of the cell cycle was determined using a ModFit 5.2 computer program.
Apoptotic cell death was determined with two methods: Annexin V-FITC staining and Tunnel assay. Annexin V-FITC staining kit and ApoDIRECT In Situ DNA Fragmentation Assay (Tunnel assay) kit were purchased from BioVision (Mountain View, CA). Experiments were performed by following the manufacturer's instructions. Quantifications of the percentage of apoptotic cells were done with a Coulter EPICS 753 flow cytometer.
FSCCL systemic xenograft model
All animal studies were conducted according to Animal Investigation Committee (AIC)-approved protocol of Wayne Sate University. This systemic model was initiated by injecting 20 × 106 FSCCL cells via the tail vein (iv) of acclimated 3-4 week old female severe combined immune deficient mice (ICR-SCID) (Taconic Farms, Germantown, NY). Animals were monitored daily for changes in weight, side effects of the treatment or signs of any sickness. 8-10 weeks post inoculation, symptoms such as diarrhea, dehydration, ascites, lethargy, paralysis and/or general weakness became evident, thus animals were euthanized, tissues such as liver, spleen, bone marrow, lymph nodes, blood and brain were harvested and subjected to H&E staining to evaluate pattern of dissemination, involvement and confirmation of engraftment. Engraftment rate for this model is 100%.
Animal preclinical efficacy trial design
The in vivo anti-tumor activity of MI-319 was assessed against our FSCCL xenograft model. To ensure randomness, 14 animals were combined in a single cage and inoculated with FSCCL. Seventy-two hours later, mice were pooled and 2 groups of seven animals each were randomly and unselectively assigned to two interventions; control and MI-319-treated group. MI-319 was administered orally at 300 mg/kg BIDx7.
Percent Increase in Host life Span (%ILS)
%ILS was calculated by subtracting the median day of death of the treated tumor-bearing mice from median day of death of the tumor-bearing control divided by the median day of death of the tumor-bearing control animals. Statistical analysis of data was carried out with GraphPad Prism software. Survival distribution of the treated (T) and control (C) groups was compared using the log-rank test. In this report, survival was characterized as the duration of the animal's life span 24-hours after the initiation of the xenograft until an observed event (euthanasia or death).
Pathological evaluation of mouse tissues
Necropsy was carried out to determine extent of macroscopic lymphoma. Major organs including the brain, femur (for bone marrow), liver, and spleen were harvested for microscopic examination. In addition, peripheral blood smears were examined for evidence of circulating lymphoma cells.
This work was supported by the following grants: Leukemia and Lymphoma Society grant 6028-8 (RMM); National Institutes of Health grant R01 CA109389 (RMM); Department of Defense Breast Cancer Program grant BC0009140 (SW); and National Institute of Health grant P30 CA22453-20 (SW).
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