Preclinical Evaluation of Small Molecule p53 Activating Agent Prima-1met in Waldenstrom Macroglobulinemia
Maujendra N Saha, Sukhoon Koh, Hong Chang. University Health Network, Toronto, Canada
Background: Waldenstrom macroglobulinemia (WM) is an indolent, lymphoplasmacytic lymphoma. WM remains incurable given current therapy, with a 5-year survival rate of 50%. Targeted therapies based on small molecules including nutlin (a cis-imidazoline analog), RITA (a furan derivative), and prima-1met (a methylated derivative and more potent form of primer-1, an octan derivative) are attractive strategies for the human malignancies. Nutlin and RITA induce apoptosis in tumor cells harboring wild type p53, whereas prima-1met can restore wild type conformation of mutant p53 and is currently tested in phase I/II clinical trials in various of solid tumors. To date, the activity of these small molecules has not been explored in WM.
Design: A WM cell line MWCL-1, derived from a patient with WM, was used as a representative model for evaluating in vitro anti-tumor activity of nutlin, RITA, and prima-1met. MWCL-1 cells were treated with each molecule individually, at different concentrations for different time period. Cells treated with di-methyl sulfoxide (DMSO) were used as controls. The cytotoxicity of the drugs was evaluated by measuring the viability of the cells by MTT assay. The induction of apoptosis was analysed by flow cytometry for Annexin V binding. The expression of apoptotic targets was examined by Western blot analysis of the total cell lysates collected from the cells treated with the drugs or DMSO control.
Results: Treatment of the MWCL-1 cells (bearing a missense mutation at exon 5 of p53) with prima-1met resulted in a dose- and time-dependent killing of the cells. At 48 hrs after treatment about 76% MWCL-1 cells were killed by 20 µM prima-1met as measured by MTT cell viability assay. Flow cytometry for quantitative analysis of apoptosis induction showed at least 25% Annexin V-positive cells compared to the cells treated with DMSO control. In contrast, nutlin or RITA did not have any significant cytotoxic/apoptotic effect on MWCL-1 cells. Western blotting of the whole cell lysates collected after 24 hrs treatment of MWCL-1 cells with prima-1met showed the activation of caspase-3 and PARP, confirming that prima-1met induced apoptosis in WM cells. Furthermore, the apoptosis induced by prima-1met was associated with up-regulation of a pro-apoptotic protein puma.
Conclusions: Our data demonstrated potent anti-tumor activity of prima-1met and dissected its pro-apoptotic pathways in WM cells. Thus, this study provides the preclinical framework for further evaluation of prima-1met as a novel therapeutic approach for the treatment of WM patients, especially with p53 mutations.
Monday, March 19, 2012 1:00 PM
Poster Session II # 219, Monday Afternoon