TRAIL as the ATO-Target Gene Uniquely Activated In the Hyperdiploid Subtype of Myeloma with Prognostic Relevance, Resulting In Better Prognosis

Abstract 1904

Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying mechanism remains elusive. In this study, the molecular mechanisms of ATO-induced myeloma apoptosis were explored on four myeloma cell lines OPM2, U266, RPMI8226, and KMS28PE of wild type or mutant p53 status and six primary myeloma cells. Gene expression profiling (GEP) of CD138⁺ bone marrow plasma cells from 22 healthy individuals (NPC), 44 patients with monoclonal gammopathy of undetermined significance (MGUS), and 351 newly diagnosed MM patients were published previously (Zhan et al. Blood. 2006;108:2020-8. Shaughnessy et al. Blood. 2007;109:2276-84.); and GEP from 9 myeloma cell lines were used in this study from the unpublished data of the University of Utah. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. GEP was performed by using the Affymetrix U133Plus2.0 microarray. ATO induced potent inhibition of myeloma cell growth and myeloma cell apoptosis, compared with controls. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb while up-regulating p53, p21Cip1, and p27Kip1 proteins, resulting in G0/G1 or G2/M cell cycle arrest. ATO treatment increased mRNA levels of interferon regulatory factor-1 and TRAIL, as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway in the mutated p53 myeloma cells. ATO also activated caspases 3 and 9, indicating involvement of the intrinsic apoptotic pathway in the wild type p53 myeloma cells. The usage of ATO and TRAIL agonist together has a synergistic effect, indicated by a combination index of less than 1. More importantly, these molecular changes induced by ATO-treated myeloma cells are very similar to the baseline expression pattern of hyperdiploid myeloma, which has a relative good prognosis with high expression of TRAIL and interferon related genes. Together, our data suggest that ATO induces apoptosis in MM through either extrinsic or intrinsic signaling pathway depending on the p53 genetic background. These observations may be employed as prognostic tools and lead to novel therapies in primary myelomas.