TRAF6-Dominant Negative Peptides Show Potent Inhibitory Effects On Multiple Myeloma, Osteoclast Formation and Bone Resorption.

Abstract 611

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating NF-κB and JNK signal transduction pathway resulting in inhibition of tumor cell proliferation and osteoclast formation. The unique biological function of TRAF6 is largely determined within its TRAF-C domain which does not interact with peptide motifs that are recognized by other TRAFs including 1, 2, 3 or 5. We have recently reported inhibition of cell proliferation and increased apoptosis of multiple myeloma (MM) cells through regulation of the NF-κB and JNK pathways through silencing the TRAF6 C-domain mRNA. In this study, we determined the effects of TRAF6 dominant negative peptides on MM cells, osteoclast formation and bone resorption. We cloned a 167 amino acid (in residues 333 to 508) fragment to produce a TRAF6 negative dominant (TRAF6dn) construct and synthesized an inhibitory decoy peptide of the TRAF6 interaction domain with CD40 and another peptide interacting with the TRAF6-RANK binding domain as well as a control peptide. All peptides were synthesized with a 16 amino acid permeable peptide. Using the MM1s, RPMI8226, and U266, we evaluated the effects of these peptides on MM tumor cell growth using an MTS assay and apoptosis with an Annexin V assay. We found that TRAF6dn peptides significantly inhibited MM cell proliferation maximally at 72 hours whereas effects on induction of apoptosis in MM cells were most prominent at 48 hours. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration-dependent fashion. We found that TRAF6dn also markedly inhibited osteoclast cell formation from freshly derived human monocytes induced by RANKL and M-CSF in a concentration-dependent fashion comparing with cells exposed to control peptide. We further examined the effects on MM cell apoptosis of the TRAF6 decoy or CD40 decoy peptides alone and in cells exposed to the combination of both peptides. The results showed either decoy peptide alone slightly induced apoptosis of MM tumor cells whereas the combination of both peptides demonstrated marked apoptosis of MM cells. We also showed that although melphalan alone induced apoptotic cell death, this effect was markedly enhanced when this alkylating agent was combined with the TRAF6 decoy peptide. Although the CD40 peptide alone did not inhibit osteoclast formation, TRAF6 decoy peptide alone and the combination of both decoy peptides markedly inhibited formation of these bone resorbing cells. We also examined the effects of TRAF6dn on NF-κB and JNK by measuring JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-κB protein levels and phosphorylation of JNKK are both markedly reduced when MM cells are exposed to TRAF6dn fragment or TRAF6 decoy peptide. These studies suggest that TRAF6dn or the combination of TRAF6 decoy and CD40 decoy peptides may be excellent targets to block both myeloma cell and osteoclast cell formation. The study has been extended to assess the effects of these peptides in vivo using our SCID-hu murine model of human myeloma.