Small Molecule RITA Induces Apoptosis In Multiple Myeloma Cells through Activation of the p53 Pathway and Induction of ER-Stress response

Abstract 790

TP53 mutations or deletions are relatively rare in multiple myeloma (MM). Thus, restoration of p53 tumor suppressor function in MM by blocking the inhibitory role of MDM2 is a promising and applicable therapeutic strategy. RITA (Reactivation of p53 and Induction of Tumor-cell Apoptosis), binds to p53, prevents the p53-MDM2 interaction and induces accumulation of p53 in solid tumors and leukemic cells. However, whether RITA exerts anti-myeloma activity is not known. Therefore, we examined the apoptotic effect of RITA and molecular mechanisms of RITA-induced apoptosis in MM cells. Treatment of MM cells with RITA caused a dose-dependent inhibition of survival and an increase in apoptosis of cells harboring wild type (MM.1S, H929) but not mutant (U266, LP1) or null p53 (8226R5) suggesting that RITA-induced apoptosis in MM cells is p53-dependent. MM cells were sensitive to low doses of RITA, killing about 80% of MM.1S or H929 cells at 2 or 6 μM, respectively. In addition, RITA elicited a dose-dependent inhibition of myeloma cell growth in 4 of the 5 primary MM samples tested. Importantly, RITA did not have a significant inhibitory effect on the survival of bone marrow or peripheral blood mononuclear cells obtained from 5 healthy donors at the concentrations that induced apoptosis of MM cells, indicating a preferential killing of myeloma cells by this drug. Furthermore, the combination of RITA with an MDM2 antagonist, nutlin displayed a strong synergism on the killing of H929 or MM.1S cells (CI=0.56-0.82). Western blot analysis showed that RITA induced an increase in the expression of p53 only in cells harboring wild type p53 but not mutant p53. The activation of p53 induced by RITA was mediated through modulation of multiple apoptotic regulatory proteins, including up-regulation of a pro-apoptotic protein Noxa, down-regulation of an anti-apoptotic protein Mcl-1 and activation of caspase-8 and -3. RITA-induced caspase-mediated apoptosis was followed by extrinsic pathway as shown by decrease in the percentage of annexin V-positive cells (p<0.05) as well as inhibition of cleavage of PARP and caspase-3 and/or caspase-8 activation in MM.1S or H929 cells when treated with either pan-caspase inhibitor, Z-VAD-FMK or caspase-8 specific inhibitor, Z-IETD-FMK. To further delineate the mechanisms of apoptosis, we performed gene expression profiling and identified up-regulation of transcription factors including JUN, DDIT3, ATF3 and ATF4, and down-regulation of survival factors, MYC and IGF1R in RITA-induced MM.1S cells, which were validated by qRT-pCR. Up-regulations of DDIT3, ATF3 and ATF4 are thought to be associated with the induction of endoplasmic reticulum (ER)-stress response. To examine whether apoptosis induction by RITA in MM cells is mediated through ER-stress response pathway, cells were treated with either an inhibitor of ER-stress-induced apoptosis, salubrinal, or a specific inhibitor of c-Jun N-terminal kinase (JNK), SP600125. Treatment of MM.1S or H929 cells with these inhibitors resulted in a significant inhibition of apoptosis induced by RITA as evidenced by inhibition of cleavage of caspase-3 and PARP, and decrease in the percentage of annexin V-positive cells (p<0.05). However, the above phenomenon was not observed in MM cells harboring mutant or null p53. Taken together, our results identify RITA as a promising agent in treatment of MM with two potential mechanisms of action: 1) activation of the p53 apoptotic pathway through up-regulation of Noxa and down-regulation of Mcl-1; and 2) p53-mediated induction of the ER-stress response pathways that may inhibit MM proliferation or survival. Our data provide the rationale for the clinical evaluation of RITA as a potential novel therapeutic intervention in MM.