A dominant-negative F-box deleted mutant of E3 ubiquitin ligase, β-TrCP1/FWD1, markedly reduces myeloma cell growth and survival in mice

// Ramaswamy Sharma 1,* , Paul J. Williams 1,* , Anjana Gupta 1 , Brandon McCluskey 1 , Shylesh Bhaskaran 1,2 , Steve Munoz 1,3 and Babatunde O. Oyajobi 1,4 1 Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 2 Current address: Oklahoma Medical Research Foundation, Oklahoma City, OK, USA 3 Current address: Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, Nashville, TN, USA 4 Cancer Therapy and Research Center, San Antonio, TX, USA * These authors contributed equally to this work Correspondence to: Babatunde O. Oyajobi, email: // Keywords : FWD1/β-TrCP1, 5TGM1, myeloma, plasmacytoma, proteasome Received : December 23, 2014 Accepted : April 30, 2015 Published : May 12, 2015 Abstract Treatment of multiple myeloma with bortezomib can result in severe adverse effects, necessitating the development of targeted inhibitors of the proteasome. We show that stable expression of a dominant-negative F-box deleted (∆F) mutant of the E3 ubiquitin ligase, SCF β-TrCP/FWD1 , in murine 5TGM1 myeloma cells dramatically attenuated their skeletal engraftment and survival when inoculated into immunocompetent C57BL/KaLwRij mice. Similar results were obtained in immunodeficient bg-nu-xid mice, suggesting that the observed effects were independent of host recipient immune status. Bone marrow stroma offered no protection for 5TGM1-∆F cells in cocultures treated with tumor necrosis factor (TNF), indicating a cell-autonomous anti-myeloma effect. Levels of p100, IκBα, Mcl-1, ATF4, total and cleaved caspase-3, and phospho-β-catenin were elevated in 5TGM1-∆F cells whereas cIAP was down-regulated. TNF also activated caspase-3 and downregulated Bcl-2, correlating with the enhanced susceptibility of 5TGM1-∆F cells to apoptosis. Treatment of 5TGM1 tumor-bearing mice with a β-TrCP1/FWD1 inhibitor, pyrrolidine dithiocarbamate (PDTC), significantly reduced tumor burden in bone. PDTC also increased levels of cleaved Mcl-1 and caspase-3 in U266 human myeloma cells, correlating with our murine data and validating the development of specific β-TrCP inhibitors as an alternative therapy to nonspecific proteasome inhibitors for myeloma patients.