In unstressed cells, p53 is tightly regulated by HDM2 to ensure that its anti-proliferative and anti-apoptotic activity will not harm normal cells. HDM2 and p53 exist in a finely tuned balanced auto regulatory feedback loop by which the two proteins mutually control their cellular levels . In many cancers, this balance is skewed when overexpression of HDM2 leads to constant suppression of p53 posttranslational modifications (such as phosphorylation, acetylation, methylation) which are necessary for the p53 response to stress. Although the majority of lymphoma tumors retain wt-p53 status, mechanisms to compromise p53 signaling, such as HDM2 overexpression, deletions in p14ARF, and viral oncogenes exist to prevent p53 activation in response to stress or initiate programmed cell death [28–30]. Because of the importance of HDM2 in regulating p53 function, attempts are being made to test the effects of its inhibition in cancer. The discovery of small molecular inhibitors of HDM2-p53 interaction is considered a significant development in the treatment of all types of cancers, particularly those in which the activation of wt- p53 is suppressed. These small molecules are designed to physically sit in the p53 binding pocket of HDM2, thereby preventing HDM2-p53 interaction . HDM2 inhibitors R7112 (Nutlin-3 analog) and JNJ-26854165 (a tryptamine derivative) are currently under clinical evaluation in Phase I studies (NCT00623870, NCT00559533, NCT00676910) and the first results from Phase 1 pharmacokinetic and pharmacodynamic study for JNJ-26854165 or Serdemetan in patients with advanced solid tumors has recently been published . These first results indicate that this agent showed clinical efficacy but at elevated doses some toxic effect were of concern. The authors concluded newer derivatives would provide more optimal results.
We had previously investigated MI-319 in preclinical studies using lymphoma cell lines with encouraging results and found that MI-219, MI-319 and Nutlin-3 exhibited similar activity against the cell lines at the concentrations used in this study . Here, we investigated selected effects of MI-219, a clinical grade spiro-oxindole  against lymphoma cell lines and patient-derived primary indolent non-Hodgkin’s SLL/CLL lymphoma samples. Using the cis-imidazoline inhibitor, Nutlin-3 as a benchmark for comparison, we noted significant differences between the two classes which may have clinical implications. Compared to Nutlin-3, differences could be attributed to enhanced MI-219-induced HDM2 autoubiquitination and degradation which resulted in higher levels of p53 expression and p53 stabilization at equivalent concentrations. Our results indicate that the enhanced MI-219-induced increase in p53 is the result of disruption of the HDM2-p53 complex and released p53 rather than de novo transcribed or translated p53. Further in-depth studies with the WSU-FSCCL cell line demonstrated that MI-219 enhanced efficacy was associated with significantly increased p53-induced p53AIP1 mRNA at the expense of p53-induced HDM2 mRNA at 24 h which was not seen with Nutlin-3. Nutlin-3 induce a greater increase in p53-induced p21 mRNA than MI-219 at 48 h. These results provide evidence that MI-219 can induce cell death pathways earlier and more robustly than Nutlin-3 in cell lines and in patient samples. The enhanced effects of MI-219 could be attributed to several factors including higher binding affinity reported for MI-219 to HDM2 (7-fold higher than Nutlin-3) , differences in chemical structure or differences in solubility between the two agents. Although in our previous study, we found the binding affinity of MI-219 to be only ~3 times higher than that for Nutlin-3, it is highly unlikely that a 3-fold difference is crucial as both HDM2 inhibitors are over 500 times more potent than the natural p53 peptide . Polar groups added to MI-319 to produce the structure of MI-219, increased its solubility for better dispersal in aqueous media and resulted in increased biological activity . The relationship between aqueous solubility and activity has also been demonstrated in the development of another HDM2 SMI, HLI373, which was found to be more potent than its insoluble parent compound HLI 98 s . Three p53 amino acids (Phe19, Trp23 and Leu26) have been found to be essential for binding between the two proteins and are inserted into a deep pocket on the surface of HDM2. Nutlin-3 mimics the p53 binding pocket at these three amino acids. In contrast, MI-219 mimics 4 key binding residues in p53 (Phe 19, Leu22, Trp23 and Leu26) resulting in more optimal hydrogen binding and hydrophobic interaction with HDM2 and this alone may account for its increased efficacy.
While it is known that p53 is induced in response to a number of stimuli including DNA damage, if p53 is bound to HDM2, that response is suppressed. During DNA damage, in normal cells, HDM2 can be phosphorylated at multiple sites which leads to HDM2 ubiquitination, degradation and p53 stabilization [35, 36]. Once again, in cells in which HDM2 is overexpressed, more HDM2 protein can quickly bind to and suppress p53 activation. The focus of our study here was to demonstrate that HDM2 inhibition using HDM2 SMIs would allow free p53 to be activated, if and when necessary, in response to internal as well as external stimuli or stress. Because HDM2 inhibition with these HDM2 SMI does not involve a response to DNA damage, the question remains as to what then initiates the activated p53-induced death pathways in lymphoma cells once p53 suppression is released. Are they the same factors encountered by normal cells and is the response the same for cancer and non-cancerous cells are question for future studies. However, p53 is not the only partner for HDM2. There are more than 30 known proteins that form interactions with HDM2 and include proteins such as NF-κB, p21,pRb, TGFβ-1 as well as others . Their function and significance are not fully understood, although they may have therapeutic implications and have been shown to play a role in cellular responses such as transcriptional regulation, apoptosis and the cell cycle [15, 38].
In addition to disrupting the HDM2-p53 interaction, there is evidence that HDM2 SMIs may induce posttranslational modification of p53. Poyurovsky et al. recently showed that Nutlin-3 induced the modification of the C-terminus of p53 (including acetylation) which may explain its lower efficiency in dissociating p53-HDM2 in vitro . In other studies, using solid tumors, MI-219 was found to target the class II histone deacetylase SIRT 1 (which deacetylates p53 allowing HDM2 to target p53 to ubiquitination) and the Bax-Ku70 interaction . Suppression of Ku70, in theory, would allow pro-apoptotic Bax localization to mitochondria and p53-mediated apoptosis.
Nutlin-3 was one of the first HDM2-SMIs to show significant efficacy in a number of models, thus, there are more published reports on this agent than on MI-219. Bixby et al. confirmed that p53 status was the most important predictor of response to HDM2 SMI in isolated CLL cells studied ex vivo . Results of other studies indicate CLL cells exposed to Nutlin-3 ex vivo in combination with cytotoxic chemotherapeutic agents have demonstrated synergy; while activity of Nutlin-3 as single agent, was modest . Ex vivo studies using isolated multiple myeloma cells indicate that Nutlin-3 was as effective as melphalan without the genotoxic effects . In treatment resistant acute myelogenous leukemia cell, Nutlin-induced apoptosis was mediated by transcriptional activation of pro-apoptotic Bcl-2 family proteins and transcriptional-independent mitochondrial permeabilization via mitochondrial p53 translocation . In Hodgkin lymphoma samples, Nutlin-3 was able to mediate p53 stabilization, cell cycle arrest and initiate the apoptotic death pathway, including Caspase-3 and PARP cleavage.
Differences in response to Nutlin-3 and MI-219 between the WSU-FSCCL and KM-H2 cell lines were seen in our study and may reflect biological differences between Hodgkin’s and non-Hodgkin’s lymphomas which they represent, including different time-frame of response to these two HDM2 SMIs.
This study is one of the first, if not the first, to compare the effects of Nutlin-3 and MI-219 in SLL/CLL samples ex vivo. These SLL/CLL and MZL samples represent slowly dividing tumor cells which have lost the ability to undergo programmed cell death or apoptosis and this leads to bone marrow replacement by lymphoma which impedes its function. Agents which affect mitotic or cell cycle pathways are generally not useful as a therapeutic regimen. Instead, optimal anti-cancer agents are sought to quickly initiate cell death processes for these lymphomas while sparing normal cells. The population studied here exhibits varied genetic characteristics and varied responses to standard treatment and thus, offered an excellent opportunity to test the effects of HDM2 SMIs for treatment of this disease. The results show that the response to MI-219-induced HDM2 autoubiquitination and degradation and associated increase in p53 varied among the studied samples and reflected the genetic diversity which has thwarted the development of a single cure for this group. In other studies, failure of CLL to respond upregulation of p53 have been attributed to polymorphism in the p21 gene , transactive defective spliced variants of the p53 gene , and altered p53-induced effectors .
For extended experimental research studies, we utilized cell lines derived from patients with aggressive forms of NHL. Since these cells do proliferate, effects of anti-cancer agent modifying cell cycle associated proteins (such as p21) can be demonstrated. Further studies in wt-p53 WSU-FSCCL NHL cell line confirmed that MI-219 induced enhance HDM2 autoubiquitination and degradation compared to that in Nutlin-3-treated cells. As this was a consistent finding observed in both wt-p53 cell lines and in patient samples, we demonstrated that none of the HDM2 SMIs (MI-219, MI-319 or Nutlin-3) inhibited recombinant HDM2 ubiquitination and degradation by themselves in a cell-free ubiquitination assay. This indicates that the E3 ligase function of HDM2 is not affected by the inhibitors. The exact mechanism for enhanced MI-219 induced HDM2 autoubiquitination will require further detailed investigation. Possible mechanisms include binding of MI-219, but not Nutlin-3, to HDM2 results in its interaction with one of the many proteins that are known to bind to, and modulate activity of HDM2. Some of these HDM2-interacting proteins have been shown to facilitate or induce HDM2 autoubiquitination and include L11, 14-3-3σ, SCL-BP1, FKBP25, ZNF668, TR3, and RASSF1A [48–54]. In addition, anticancer agents such as Parthenolide accelerate HDM2 autoubiquitination through mechanisms that required ATM and Berberine, which downregulates HDM2 at the posttranslational level through modulation of death domain-associated protein (DAXX) have recently been shown to induce apoptosis [55, 56].