TRAF6 Dominant Negative Peptides Block Intracellular Signaling Resulting in Anti-Myeloma and Anti-Bone Resorptive Effects in Multiple Myeloma
Authors: Li Mingjie , Dreyer Marissa P , Ahles Cameryn P , Ramirez David , Nichols Cydney M , Lee Tiffany , Song More , Wang Cathy S , Sanchez Eric , Chen Haiming , Bonavida Benjamin , Berenson James R. 
Published: October 2019

Abstract
Abstract 2919
Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. We have previously reported inhibition of cell proliferation and increase of apoptosis in multiple myeloma (MM) cells through regulation of these intracellular pathways through silencing of TRAF6 C-domain mRNA. To determine TRAF6 protein expression in fresh MM tumor cells, we performed an immunofluorescence assay (IFA). The results showed that expression of this factor in tumor cells from bone marrow (BM) from MM patients with progressive disease is higher than in cells from patients with monoclonal gammopathies without disease progression or normal controls. We further examined the effects of TRAF6 negative dominant peptides on intracellular signaling pathways. Briefly, cells from the RPMI8226 or MM1s MM cell lines or primary MM BM samples were treated with or without TRAF6 inhibition peptide for 24 hours and then stimulated with either IGF1 (30ng/ml) or IL1 β (20ng/ml) for 30 minutes. The cells were lysed and Western blot analysis performed to determine protein phosphorylation and RT-PCR for gene expression. TRAF6 has been found to be an E3 ligase for Akt ubiquitination. We found that IGF1 increased the phosphorylation of AKT and treatment with TRAF6 inhibition peptide markedly decreased its phosphorylation compared to treatment with a control peptide in RPMI8226 and primary MM tumor cells. Downstream of AKT, C-Raf phosphorylation was also significantly reduced with treatment with TRAF6 inhibition peptide. Notably, cyclin D gene expression in MM tumor cells treated with TRAF6 inhibition peptide was reduced as determined with an RT-PCR. In contrast, the gene expression of mTOR was increased in RPMI8226 cells treated with TRAF6 inhibition peptide whereas there was no change in its expression in MM1s and primary MM tumor cells. It is quite possible that the increase in mTOR expression in RPMI8226 cells may act as a negative feedback which results from blockage of the ubiquitination of TRAF6. We further examined the effect of the TRAF6 inhibition peptide on NF-kB and JNK signaling as determined through evaluation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-kB protein was reduced and phosphorylation of JNKK was clearly decreased with exposure to the TRAF6 inhibition peptide. We examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. Total endogenous c-Jun is reduced following exposure of RPMI8226 cells to the TRAF6 inhibition peptide. Consistent with our past findings, TRAF6 inhibition peptide significantly inhibited osteoclast formation from CD14+ induced by RANKL and M-CSF with in a concentration dependent fashion whereas control peptides showed no effects on osteoclast formation. In addition, inhibition of the TRAF6 signaling blocked not only myeloma cell proliferation induced by AKT and NF-kB activation but also osteoclast cell formation mediated through transcription at TRE/AP-1 elements. The study has been extended to our SCID-hu murine model of human myeloma.

Disclosures:
No relevant conflicts of interest to declare.