Abstract
Rational: Multiple myeloma (MM) is the second most common hematologic malignancy and is incurable for most patients. We reported that MM cells induce expression of the transcriptional repressor, Growth independent factor 1 (Gfi1), in bone marrow stromal cells (BMSC) repressing Runx2 gene transcription, and results in prolonged suppression of osteoblast differentiation. 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 can attenuate the pro-apoptotic effects of bortezomib.
Methods: CD138+ cells isolated from MM patients, healthy donors and human MM cell lines (HMCLs) (H929, JJN3, 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. HMCLs were treated with IL-6, S1P or TNFa or co-cultured with a human BMSC line (SAKA-T) to assess their effects on Gfi1 expression. The anti-apoptotic effects of Gfi1 overexpression (o/e) in MM1.S and H929 were tested by transduction with a pUC2 lentivirus encoding Gfi1 or with the empty vector followed by bortezomib (2 - 5nM) or vehicle (DMSO) treatment for 24 and 48 hours. MTT assays and cleaved caspase 3 protein levels were used to assess cell viability and cell death. Since acetylation of Gfi1 and p53 affects their activity and ability to bind each other, we also characterized HDAC inhibitors (HDACi)-induced changes on p53 enrichment at the Noxa, PUMA and p21 gene promoters by ChIP assays and the effects of acetylation of Gfi1 on its p53 binding capacity in MM cells.
Results:We found that Gfi1 is highly expressed, at the mRNA and protein level, in CD138+ cells from MM patients and cell lines than 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 (o/e) in MM cells conferred a survival advantage over their respective empty vector transduced controls as assessed by cell counts and MTT assays. Further, Gfi1 o/e protected MM cells from apoptosis induced by treatment with bortezomib as measured by MTT and cleaved caspase 3 protein levels. Since SphK1 activitycan also prevent apoptosis of MM cells, we next determined if Gfi1 regulated SphK1 in MM cells.CD138+ cells from MM patients had increased SphK1 mRNA levels compared with normal donors, and SphK1 mRNA levels and protein activity were further increased in MM cells by exogenous IL-6 and S1P. Co-culture of MM cells with BMSC also 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. Gfi1's inhibition of apoptosis resulted in part from binding of Gfi1 to p53, which blocked p53's access to its pro-apoptotic target gene promoters. HDACi treatment resulted in acetylation of Gfi1 and inhibited Gfi1's suppression of apoptosis by preventing Gfi1-p53 binding and subsequent enrichment of p53 at the Noxa, PUMA and p21 promoters in MM cells.
Conclusion: Taken 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.
Disclosures
Giuliani: Celgene: Research Funding; Janssen: Research Funding. Roodman:Amgen: Consultancy.
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