Role of Gfi1 Transcription Factor in Myeloma Cells Growth and Survival

Abstract

RationalMultiple myeloma (MM) is a plasma cell malignancy that is the second most common hematologic malignancy and is currently incurable for the overwhelming majority of patients. We reported that MM cells induce expression of the transcriptional repressor, Growth independent factor 1 (Gfi1), in bone marrow stromal cells (BMSC) that results in prolonged suppression of osteoblast differentiation by repressing transcription of the Runx2 gene. Since Gfi1 is an anti‐apoptotic factor in other hematologic malignancies, we hypothesized that Gfi1 has an important pro‐survival role in MM cells by blocking apoptosis and regulating MM cell growth and survival.MethodsCD138+ cells isolated from MM patients and healthy donors and human MM cell lines (H929, JJN3, U266, RPMI‐1640 and MM1.S) were tested for Gfi1 expression levels and the effects of Gfi1 knock down (KD) on MM cell survival by transduction with pLKO.1‐puro lentivirus vectors encoding Gfi1 or non‐mammalian shRNAs. Since acetylation of Gfi1 and p53 can affect their activity and ability to bind each other, we also characterized HDAC inhibitor (HDACi)‐induced changes in p53 enrichment at the Noxa, PUMA and p21 gene promoters by ChIP assays and the effects on acetylation of Gfi1 on its p53 binding capacity in MM cells treated with trichostatin A (5 μM) and nicotinamide (10 mM). Gfi1 was immune‐precipitated followed by western blotting with anti‐acetylated lysine or anti‐p53.ResultsWe found that Gfi1 is highly expressed, at the mRNA and protein level, in CD138+ cells from MM patients and MM cell lines compared with CD138+ cells from normal donors. Gfi1 expression was further increased in MM cells by exogenous IL‐6 (5ng/ml) and sphingosine‐1‐phosphate (S1P) (0.1 μM), but not by TNFα (10 ng/ml). KD of Gfi1 inhibited the growth and induced apoptosis of MM cells, as measured by increased mRNA levels of Bax, PUMA, Noxa, increased cleaved caspase 3 protein levels and decreased protein levels of Mcl‐1 and c‐Myc. Gfi1's inhibition of apoptosis resulted in part from binding of Gfi1 to p53 which blocked p53's access to its pro‐apoptotic targets promoters. HDACi treatment inhibited Gfi1's suppression of apoptosis by acetylation of Gfi1 at the K292 residue, which prevented Gfi1‐p53 binding and subsequent enrichment of p53 at Noxa, PUMA and p21 promoters in MM cells. Since SphK1 activity can also prevent apoptosis of MM cells, we next determine if Gfi1 regulated SphK1 in MM cells. CD138+ cells from MM patients showed increased levels of SphK1 mRNA compared with normal donors and SphK1 mRNA levels and protein activity were further increased in MM cells by exogenous IL‐6 and S1P. Further, direct co‐culture of MM cells with BMSC enhanced Gfi1, IL6 (3 fold) and SphK1 (2.5 fold) mRNA levels in MM cells. Importantly, Gfi1 KD in MM cells profoundly downregulated SphK1 mRNA levels and reduced expression of phospho‐SphK1, suggesting that Gfi1 enhances MM growth in part via increasing expression and activity of SphK1.ConclusionTaken together, our results suggest that Gfi1 may act as a key regulator of MM growth and survival through its regulation of p53 and SphK1 activity, and that targeting Gfi1 may be a novel therapeutic strategy for MM patients.Support or Funding InformationVA Merit Review funds to GDR