Sp1 Transcription Factor as a Novel Therapeutic Target in Multiple Myeloma (MM)

Sp1 transcription factor has an important role in regulating expression of cell differentiation, cell cycle and apoptosis related genes. Increased Sp1 activity has been shown to augment growth and metastatic potential of tumor cells. In multiple myeloma (MM), modulation of autocrine IL-6 secretion by Sp1 has been described. Using genome expression profiling analysis we observed high expression of Sp1 in primary myeloma cells obtained from bone marrow aspirates of MM patients. This observation was confirmed by western blot analysis. Therefore, we evaluated Sp1 as a novel therapeutic target in MM, analyzing both in vitro and in vivo anti-MM activity of a Sp1 inhibitor (Terameprocol, EM-1421). Terameprocol significantly (≥80% inhibition, P≤0.001) inhibited DNA synthesis in 12 MM cells lines tested in a dose-dependent fashion at 24 hour with an IC₅₀ of 1–10 uM. Longer exposure of MM cells to Terameprocol resulted also in cytotoxity, as assessed by MTT assay, in all cell lines tested (IC₅₀ of 20–40 uM at 72 hours). Terameprocol induced apoptosis in a time- and dose-dependent manner in all MM cell lines examined, as assessed by Annexin V+/PI staining, and triggered caspase-9/3 and PARP cleavage. Since Sp1 modulates transcription of survivin and survivin is an anti-apoptotic protein overexpressed in most human cancers that inhibits caspases-3 and -9 activity, we next examined effect of Terameprocol on survivin protein expression. Consistent with induction of caspase-3 and apoptosis, Terameprocol significantly reduced survivin protein in a time-dependent manner. To identify additional potential downstream targets of Sp1 inhibition in MM, we evaluated change in expression profiling in MM cells following Terameprocol treatment (10 uM for 12 hours). We observed alteration (>2-fold changes) in 68 genes. In particular, we recognized significant down-regulation of c-Jun and up-regulation of p53 and p21 following exposure to Terameprocol. Changes in protein expression of these genes were also confirmed by western blot analysis. Since bone marrow stromal cells (BMSCs) trigger growth, survival, and drug resistance of MM cells we next confirmed the ability of Terameprocol to overcome protective effects of BMSCs on MM cell growth and survival. Finally, we have confirmed in vivo activity of Terameprocol on MM cell growth in a xenograft murine model of human MM. In conclusion, our results demonstrate that Sp1 may be an attractive target for MM therapy and provide the rationale for clinical evaluation of Terameprocol in MM.