The Gfi1-SphK1 Axis Regulates the Growth and Survival of Myeloma Cells

Abstract In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable hematologic malignancy due to emergence of drug-resistant clones. We previously reported that MM cells upregulate expression of the transcriptional repressor, Growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSC), which induces prolonged inhibition of osteoblast differentiation. We recently showed that Gfi1 levels are also increased in the majority of CD138+ cells from MM patients and cell lines ,and Gfi1 levels significantly correlated with Mcl-1 protein expression. Further, Gfi1 repressed MM cell death by inhibiting expression of apoptosis-inducing genes. Importantly, Gfi1 overexpression in MM cells enhanced MM cell growth and conferred resistance to proteasome-inhibitor- induced apoptosis. However, the mechanisms responsible for these effects of Gfi1are unknown. Sphingosine kinase 1 (SphK1) is overexpressed in many cancers including MM, and catalyzes the phosphorylation of sphingosine (SPH) to sphingosine-1-phosphate (S1P). S1P is a pleiotropic lipid mediator that regulates cell survival, migration, the recruitment of immune cells and angiogenesis, all of which contribute to cancer progression. Therefore, we hypothesized that adhesive interactions between MM cells and BMSC stimulate survival and growth of MM cells in part through the Gfi1-SphK1 axis by modulating their sphingolipid profile (Ceramide/SPH/S1P ratio). We found that SphK1 mRNA is highly expressed in CD138+ cells from MM patients and cell lines compared with normal donors. Further, Gfi1 protein expression correlated significantly with SphK1 protein level in CD138+ cells from MM patients and MM cell lines. Soluble factors (IL-6 and S1P) that are increased in the MM microenvironment, hypoxia (1% O2) and adhesive interactions of MM cells with BMSC further increased Gfi1 and SphK1 mRNA and protein levels in MM cells. Gfi1 Knockdown (KD) in MM cells induced a profound decrease of SphK1 mRNA and protein activity and inhibited MM cell growth and viability. In contrast, over-expression of Gfi1 in MM cells increased SphK1 levels that conferred a survival advantage to MM cells over empty vector-transduced control cells. Increased Gfi1 expression also resulted in increased intracellular S1P and decreased sphingosine levels, as measured by LC-MS/MS. Further, treatment of MM cells with a SphK1 specific inhibitor (SKI2), dose-dependently reduced MM cell viability at 24h, regardless of their p53 status. Interestingly, p53 null MM cells were more resistant to SK12, as compared with p53 replete cells. In p53 replete MM cells, SphK1 inhibition significantly reduced c-Myc protein expression, induced autophagy (as shown by increased LC3 II protein levels) and increased total ceramides levels. Moreover, BMSC protected p53 replete MM cells from the anti-survival effects of SKI2 in 3D cultures. These data suggest that Gfi1 regulates MM growth in part by enhancing the expression and activity of SphK1. Taken together, our results support that Gfi1 acts as a key regulator of MM growth and survival, at least partially through modulation of SphK1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. Disclosures Roodman: Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.