Data from Inhibition of the p53 E3 Ligase HDM-2 Induces Apoptosis and DNA Damage–Independent p53 Phosphorylation in Mantle Cell Lymphoma

Abstract

<div>Abstract<p><b>Purpose:</b> 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.</p><p><b>Experimental Design:</b> 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.</p><p><b>Results:</b> 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<sub>50</sub> 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<sup>Cip1</sup>. This was associated with cell cycle arrest at G<sub>1</sub>-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<sup>Ser139</sup>. 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.</p><p><b>Conclusions:</b> 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.</p></div>