ALBERT

Description: Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

Description: Protracted inhibition of osteoblast (OB) differentiation characterizes multiple myeloma (MM) bone disease and persists even when patients are in long-term remission. However, the underlying pathophysiology for this prolonged OB suppression is unknown. Therefore, we developed a mouse MM model in which the bone marrow stromal cells (BMSCs) remained unresponsive to OB differentiation signals after removal of MM cells. We found that BMSCs from both MM-bearing mice and MM patients had increased levels of the transcriptional repressor Gfi1 compared with controls and that Gfi1 was a novel transcriptional repressor of the critical OB transcription factor Runx2. Trichostatin-A blocked the effects of Gfi1, suggesting that it induces epigenetic changes in the Runx2 promoter. MM-BMSC cell-cell contact was not required for MM cells to increase Gfi1 and repress Runx2 levels in MC-4 before OBs or naive primary BMSCs, and Gfi1 induction was blocked by anti–TNF-α and anti–IL-7 antibodies. Importantly, BMSCs isolated from Gfi1−/− mice were significantly resistant to MM-induced OB suppression. Strikingly, siRNA knockdown of Gfi1 in BMSCs from MM patients significantly restored expression of Runx2 and OB differentiation markers. Thus, Gfi1 may have an important role in prolonged MM-induced OB suppression and provide a new therapeutic target for MM bone disease.

Description: Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

Description: Introduction Multiple myeloma (MM) is a clonal malignancy of plasma cells that frequently causes skeleton destruction. The osteolytic lesions rarely heal even in those patients who are in long-term remission. In MM bone disease, differentiation of osteoblasts is suppressed due to the down-regulation of the Runx2 gene, a master osteoblast differentiation transcription factor. Growth independent factor 1 (Gfi1) is a 55 kDa protein that contains an N-terminal SNAG domain and 6 zinc finger domains at the C-terminus. It is a transcription factor that can recruit chromatin modifiers to genes. We reported that MM cells induce expression of Gfi1 in bone marrow stromal cells, and that Gfi1 directly represses the Runx2 gene. Our previous data indicated that a histone deacetylases (HDAC) inhibitor, Trichostatin A, could block Gfi1-mediated Runx2 promoter repression. However, how Gfi1 activity is modulated by HDAC inhibitors and whether Gfi1 acetylation is involved in the HDAC inhibitor prevention of Runx2 repression are unknown. Therefore, we investigated if Gfi1 is acetylated and if the activity of Gfi1 is regulated by its acetylation status. We also examined the MM-induced changes in Gfi1 and HDACs occupancy on the Runx2 gene. Methods Full length or truncated Gfi1 cDNA constructs were used to express Gfi1 protein by transient transfections. After immunoprecipitation of Gfi1, the acetylation status of Gfi1 was determined by anti-acetylated lysine antibody using western blotting. Overexpression or knockdown of lysine acetyltransferase p300 was done by co-transfection of p300 cDNA or shRNA plasmids. Trichostatin A (5 µM) and nicotinamide (10 mM) were used to inhibit the activity of HDACs. Site-directed mutagenesis was used to change Gfi1 residue lysine 292 to arginine (Gfi1-K292R). For luciferase reporter assays, the pGL4.10 vector constructed with a mouse Runx2 promoter region (-976 ∼ +111) was transfected into mouse pre-osteoblast cell line MC-4. A biotinylated double-stranded oligonucleotide spanning the Gfi1 binding region (-40 ∼ -1) in the mouse Runx2 promoter was utilized for oligonucleotide pull-down assays. Chromatin immunoprecipitation (ChIP) was used to characterize the MM-induced changes in the Runx2 gene chromatin. Results We found that Gfi1 was a target of acetylation and that p300 was co-localized and co-immunoprecipitated with Gfi1. Interestingly, the deacetylation of Gfi1 was controlled by both Zn2+ and NAD+ dependent deacetylases. Using different truncated Gfi1 cDNA constructs, we identified lysine 292 in Gfi1 zinc finger 2 as a critical site for acetylation. Mutant Gfi1-K292R displayed significant resistance to acetylation in the presence of HDAC inhibitors, confirming that Gfi1-K292 is the major acetylation site. Importantly, oligonucleotide pull down assays showed that Gfi1-K292R exhibited stronger DNA binding activity than Gfi1-WT to the Gfi1 binding site in the Runx2 promoter. In luciferase assays, increasing or decreasing lysine acetyltransferase activity by overexpression or knockdown of p300 resulted in the attenuation or enhancement in Gfi1 repression of the Runx2 promoter, respectively. Further, the acetylation-resistant Gfi1-K292R more strongly repressed the Runx2 promoter, which was not rescued by HDAC inhibitors, indicating that the contribution of HDAC inhibitors to increased acetylated Gfi1 is a major mechanism for prevention of Gfi1-mediated Runx2 repression. Lastly, ChIP analysis of the Runx2 gene in MC-4 cells before and after co-culture with myeloma cells revealed that increased Gfi1 occupancy at the Runx2 gene correlated with increased HDAC1 and decreased Pol-II and H3K9ac, confirming that the interaction between Gfi1 and HDACs is involved in the repression of the Runx2 gene. Conclusions Taken together, our results indicate that Gfi1 is an acetylated protein and suggest that increasing acetylation of Gfi1represents a potential therapeutic target for reversing Gfi1-mediated Runx2 repression and promoting osteoblast differentiation in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.