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

Authors' Affiliations: Departments of ¹ Lymphoma and Myeloma and ² Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, Texas; ³ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; ⁴ University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin; and ⁵ Ascenta Therapeutics, Inc., San Diego, California

Requests for reprints: Robert Z. Orlowski, Department of Lymphoma and Myeloma, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 429, Houston, TX 77030-4009. Phone: 713-792-2860; Fax: 713-563-5067; E-mail: rorlowsk{at}mdanderson.org.

Purpose: 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.

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 IC₅₀ 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 G₁-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 may be a promising approach both by itself and in combination with currently used chemotherapeutics against lymphoid malignancies.