Inhibition of the p53 E3 ligase HDM-2 induces apoptosis and DNA damage - Independent p53 phosphorylation in mantle cell lymphoma

Purpose: The ubiquitin-proteasome pathwayhas been validated as a target in non - Hodgkin's lymphoma through demonstration of the activity of the proteasome inhibitor bortezomib. Experimental Design: Another potentially attractive target is the human homologue of the murine double minute-2 protein, HDM-2, which serves as the major p53 E3 ubiquitin ligase; we therefore evaluated the activity of a novel agent, MI-63, which disrupts the HDM-2/p53 interaction. Results: Treatment of wild-type p53 mantle cell lymphoma (MCL) cell lines with MI-63 resulted in a dose-and time-dependent inhibition of proliferation, with an IC ₅₀ in the 0.5 to 5.0 μmol/L range. MI-63 induced p53 and HDM-2 accumulation, as well as other downstream p53 targets such as p53 up-regulated modulator of apoptosis and p21 ^Cip1. This was associated with cell cycle arrest at G ₁-S; activation of caspase-3, caspase-8, and caspase-9; cleavage of poly-(ADP-ribose) polymerase; and loss of E2F1. HDM-2 inhibition caused phosphorylation of p53 at multiple serine residues, including 15, 37, and 392, which coincided with low levels of DNA strand breaks. DNA damage occurred in a small percentage of cells and did not induce phosphorylation of the DNA damage marker H2A.X ^Ser139. Combinations of MI-63 with the molecularly targeted agents bortezomib and rapamycin showed synergistic, sequence-dependent antiproliferative effects. Treatment of primary MCL patient samples resulted in apoptosis and induction of p53 and p21, which was not seen in normal controls. Conclusions: These findings support the hypothesis that inhibition of the HDM-2/p53 interaction maybe a promising approach both by itself and in combination with currently used chemotherapeutics against lymphoid malignancies.