p62 as a Therapeutic Target for Myeloma Cell Growth and Osteoclast Formation.

Abstract 2857

Poster Board II-833

We reported that sequestosome 1 (p62) plays a critical role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with multiple myeloma (MM), and that p62 is a potential therapeutic target for MM. In contrast to treating patients with inhibitors of each of the multiple signaling pathways activated in marrow stromal cells by MM cells (e.g. NF-kB or p38 MAPK), blocking the function of p62 should inhibit the activation of the multiple pathways mediated by p62 and have a broader effect on the bone marrow microenvironment. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling in marrow stromal cells when they interact with myeloma cells, and develop inhibitory peptides as potential therapeutic agents that interfere with p62's role in these signaling complexes. To pursue this objective, we transfected p62^−/− stromal cells with p62 deletion constructs and assessed their effects on NF-kB and p38 MAPK signaling induced by MM cells or TNF-a. p62^−/− stromal cells support of MM growth or OCL formation was significantly decreased compared to WT stromal cells. We made a series of 5' deletion constructs of p62 that lacked specific p62 domains: ΔPB1 (Δ1) lacks homodimerization domain and binding to PKCz, ΔPB1, ZZ (Δ2) lacks PB1 and RIP1 binding domains, and ΔPB1, ZZ, TBS, (Δ3) the PB1, RIP1, p38 and TRAF6 binding domains have been deleted. These constructs were tested for their capacity to restore p62 function in p62^−/−stromal cells and support MM cell growth and OCL formation. GFP-labeled MM1.S myeloma cells were cocultured with p62^−/− and WT marrow stromal cells transduced with the different p62 deletion constructs. Transduction of p62^−/− stromal cells with the full-length p62 construct restored the capacity of p62^−/− stromal cells to enhance the growth of MM cells to levels induced by WT stromal cells. Transduction of p62^−/− stromal cells with the Δ1 construct also restored stromal cell support of MM growth. Therefore, the PB1 domain is not important for this function. Transduction of p62^−/− stromal cells with the Δ2 construct, resulted in an inability of the stromal cells to support MM cell growth. Additional loss of the p38 and TRAF6 binding domains did not further impair p62^−/− stromal cells support of MM cell growth. These results suggest that the RIP1 binding domain plays a critical role in supporting the growth of MM cells by marrow stromal cells. We then examined the capacity of p62^−/− stromal cells transduced with various p62 deletion constructs to support OCL formation. Normal CFU-GM, a source of OCL precursors, were cocultured with p62^−/− stromal cells transfected with the different p62 cDNA deletion constructs. The Δ1 construct completely rescued p62^−/− support of OCL formation. However, Δ2 construct transduced p62^−/− stromal cells only partly restored stromal cell support of OCL formation. Transduction of the Δ3 construct did not restore the capacity of the p62^−/− stromal cells to support OCL formation. Similarly, transduction of the Δ2 and Δ3 construction decreased WT stromal cell support of MM cell growth. We then tested the feasibility of using transduction domain (PTD) fusion peptides as a potential means of delivering dominant negative p62 constructs into stromal cells in vitro and in vivo to block MM cell growth and VCAM-1 expression induced by marrow stromal cells. PTD binding domain fusion peptides containing NEMO binding protein that blocks NF-kB activity was used as a model system to determine the feasibility of transducing marrow stromal cells with p62 constructs. Addition of PTD-NEMO fusion peptides to stromal cells significantly inhibited WT stromal cell to enhance MM cell growth and VCAM-1 expression on stromal cells, which is the capacity of dependent, in part, on NF-kB signaling. These results demonstrate that the ZZ, p38 MAPK and TRAF-6 domains of p62 together are required for stromal cell support of MM cell growth and OCL formation and suggest that PTD constructs containing dominant negatives for p62 may be a feasible method for blocking p62 function in the MM marrow microenvironment.