Multiple Myeloma Cell Induction of GFI-1 in Stromal Cells Suppresses Osteoblast Differentiation in Patients with Myeloma.

Abstract 742

Multiple Myeloma (MM) is a plasma cell malignancy characterized by formation of lytic bone lesions in approximately 90% of the patients which do not heal even after prolonged complete remission. The basis for this selective and protracted suppression of osteoblast (OBL) differentiation from immature bone marrow stromal cells (MSC) is unknown. Although factors that inhibit OBL differentiation in MM have been identified, such as DKK-1, sFRP2, IL-3, IL-7, and TNF-a, none of these factors have been shown to be responsible for the protracted suppression of OBL differentiation in MM. Further, inhibition of Runx2 activity, a critical transcription factor required for OBL differentiation has been reported in MM, but the mechanisms responsible are still unclear. To address the basis for the protracted inhibition of OBL differentiation in MM, we have developed a murine model of MM-induced OBL suppression using a genetically modified murine myeloma cell line that expresses GFP and thymidine kinase (5TGM1-GFP-TK MM cells). Injection of these 5TGM1-GFP-TK MM cells into SCID mice resulted in persistent inhibition of OBL differentiation even when the MM cells were totally depleted by ganciclovir treatment. The MSC from these mice had selective inhibition of OBL differentiation, but not adipogenesis, and minimally differentiated to OBL even when treated with BMP2. These MSC expressed elevated levels of the SNAG family Zn-finger containing transcriptional repressor, Gfi-1, which we found can cause both acute and protracted suppression of RUNX2. In support of these results, decreased RUNX2 expression and elevated GFI-1 levels were also protracted in MSC from 7 MM patients with impaired OBL differentiation compared to normals. Further, 5TGM1 inhibition of OBL differentiation in vitro was dependent on TNF-a and IL-7, and neutralizing antibodies to TNF-a and IL-7 blocked MM-induced Runx2 suppression. In addition, TNF-a and IL-7 increased Gfi-1 in a murine OBL precursor cell line (MC4). Deletion analysis of the Runx2 P1 promoter revealed that a 943-bp region containing 27 putative Gfi-1 binding sites (AA(T/G)C core) was responsible for MM repression of Runx2 expression. Importantly, siRNA knockdown of GFI-1 expression restored RUNX2, OCN, BSP and OSX expression in both MM exposed MC4 cells and in MSC from MM patients. These results support an important role for GFI-1 in repressing RUNX2 expression in MSC exposed to MM cells, thereby inhibiting osteoblastogenesis in MM.