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

Abstract

The ubiquitin-proteasome pathway has been validated as a target in non-Hodgkin's lymphoma through demonstration of the activity of the proteasome inhibitor bortezomib. 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. 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 micromol/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. These findings support the hypothesis that inhibition of the HDM-2/p53 interaction may be a promising approach both by itself and in combination with currently used chemotherapeutics against lymphoid malignancies.