RITA Induces Cytotoxicity and Apoptosis in Multiple Myeloma Cells Harbouring Wild Type p53: Evidence for Synergistic Cytotoxic Responses with Conventional Chemotherapeutic Drugs.
Manujendra N Saha, Hua Jiang, Donald R Branch, Hong Chang. University Health Network, Toronto, ON, Canada
Background: MDM2 is a key negative regulator of p53 that binds to and targets p53 for proteasomal degradation. RITA (reactivation of p53 and induction of tumor cell apoptosis) binds p53 and prevents its interaction with MDM2, resulting in stabilization and activation of wild type p53. Since p53 mutation is relatively rare in multiple myeloma (MM), activation of the p53 pathway by RITA may offer an attractive therapeutic strategy.
Design: Human MM cell lines harbouring wild type, mutant or null p53 and primary MM samples were treated with RITA alone or in combination with other chemotherapeutic drugs such as doxorubicin (doxo) and dexamethasone (dxm). Cells were assayed for cell viability, apoptosis induction and activation of the p53 pathway.
Results: RITA induces a time and dose-dependent cytotoxic response in MM cell lines harbouring wild type p53 (MM.1S and H929) but not with mutant (LP1 and U266) or null (8226R5) p53. Cytotoxic response of RITA was also observed in 4 of 5 primary MM samples tested. Importantly, RITA in combination with doxo and dxm displays p53-dependent synergistic responses in killing of MM cells: 75% of H929 cells were killed by the combination of 2 µM RITA and 0.5 µM doxo, whereas 41% and 25% cells were killed when treated with RITA or doxo alone at these doses. Similar synergistic responses were observed with the same combination of RITA and dxm. The RITA-induced apoptotic response was also further enhanced by doxo or dxm treatment as evidenced by an increase in Annexin-V positive cells. The RITA-induced apoptotic response was due to activation of the p53 pathway as observed by significant up-regulation of p53 and its pro-apoptotic target, Noxa and down-regulation of an anti-apoptotic target, Mcl1. p53-dependent apoptosis induced by RITA was mediated through an extrinsic pathway as shown by activation of caspase-8 and -3 but not -9. This was confirmed by inhibition of apoptosis induction by a caspase-8 specific inhibitor. In addition, global gene expression profiling identified induction of several ER-stress response markers such as JUN, ATF3, and CHOP, which was further validated by qRT-PCR. Also, apoptosis induction by RITA was significantly inhibited by two inhibitors of the ER-stress response. Thus, RITA-induced apoptosis in MM cells is associated with the ER-stress response.
Conclusions: Our study provides the rationale for further clinical evaluation of RITA either as a single agent or in combination with chemotherapeutic drugs as a novel strategy for the treatment of MM.
Tuesday, March 1, 2011 9:30 AM
Poster Session III # 248, Tuesday Morning