Inhibition of the Human Double Minute (HDM)-2 E3 Ubiquitin Ligase Activates Different Programmed Cell Death (PCD) Pathways in Models of Non-Hodgkin Lymphoma (NHL) with Wild Type (wt) and Mutant (mut) p53

Background: The ubiquitin-proteasome pathway has been validated as a target for NHL with the recent approval of bortezomib for mantle cell lymphoma (MCL). In addition to anti-tumor activity, however, proteasome inhibitors have pleiotropic effects, including activation of anti-apoptotic heat shock proteins, and their use clinically is complicated by toxicities such as peripheral neuropathy. By targeting E3 ubiquitin ligases, which are involved in ubiquitination of only a small subset of cellular proteins, it may be possible to achieve more specific anti-tumor effects with a better therapeutic index. One attractive target is HDM-2, which is responsible for ubiquitination of the p53 tumor suppressor.

Methods: To evaluate the therapeutic potential of agents targeting HDM-2, we studied the impact of the small molecule JNJ-26854165, an inhibitor of HDM-2-function, in both p53 wt and mut cell line models.

Results: Treatment of wt p53 NHL cell lines with JNJ-26854165 induced a dose- and time-dependent inhibition of proliferation, with an IC₅₀ in the 0.02-0.3 μM range. Cell death, which was typically seen within 48 hours of HDM-2 inhibition, occurred through induction of type I PCD, as judged by the appearance of increased staining with Annexin V and activation of caspase 3. While cell lines with mut p53 were generally less sensitive than their wt p53 counterparts, JNJ-26854165 remained potent, with an IC₅₀ in the 0.05-0.6 μM range. The latter cell lines showed a longer kinetics of death, with PCD being seen within 72 hours of drug exposure. Notably, in these mut p53 cell lines, very little Annexin V staining or caspase 3 activation was seen, consistent with a minor role for type I PCD. Instead, mut p53 cell lines demonstrated an increased content of acidic vacuoles by acridine orange staining, increased expression of Beclin 1 and Sequestosome 1/p62, and conversion of microtubule-associated protein 1 light chain 3 form I to form II, consistent with activation of type II PCD, or autophagy. Also, electron microscopy showed an increased presence of autophagosomes and autolysosomes, further supporting the activation of this pathway. Combinations of JNJ-26854165 with other agents, including rapamycin, doxorubicin, and an inhibitor of Bcl 2, showed enhanced anti-proliferative effects in a sequence-dependent fashion, which were greatest when the chemotherapeutic preceded the HDM-2 inhibitor. Combination index analysis revealed that these interactions met criteria for synergy.

Conclusions: Inhibition of the function of HDM-2 using JNJ-26854165 is a promising approach that is effective against both wt and mut p53 models by activating type I and type II PCD, respectively. The effectiveness of JNJ-26854165 was enhanced in combination with currently used chemotherapeutics in a sequence specific manner, providing a rationale for translation of this novel approach into the clinic.