The Polycomb Group Transcriptional Repressor Bmi-1 Is Constitutively Expressed in Multiple Myeloma (MM) Cells and Modulates Proliferation through a Mechanism Independent of the Ink4a/ARF Locus.

The polycomb group protein Bmi-1 is required for self-renewal of adult hematopoietic stem cells by inhibiting cell senescence and apoptosis through repression of p16^INK4a and p14/p19^ARF expression. Based on gene expression profile studies showing absence of Bmi-1 expression in normal plasma cells versus upregulation in all MM cell lines and primary patient MM cells, we evaluated its role in MM pathobiology. Bmi-1 expression in MM cell lines is constitutive, and is not modulated by IL-6 and IGF-1. Immunoprecipitation and immunoblotting studies confirmed both Bmi-1 overexpression and nuclear localization. Bmi-1 was found to be tyrosine- and serine-phosphorylated. We next inhibited Bmi-1 expression in MM cell lines (ARP and MM.1S) by stably transfecting a PU6 vector expressing siRNA targeting Bmi-1. Following G418 selection, cell growth, as measured by H³ thymidine incorporation and MTT assay, was stimulated 1.4 fold in MM.1S and 2 fold in ARP cells compared to control cells transfected with a PU6 vector. Gene expression profiling showed: up-regulation of IL-6 and IGF-1 receptors, as well as Gab-1, which plays a critical role in MM cell proliferation and survival induced by IL-6; Phospholipase D1 and proteinase 3 which potentiate cell proliferation by repression and cleavage of p21^cip1/waf1; stem cell growth factor, HOXA9, which modulate bone marrow stem cell survival and proliferation. In contrast, p21^cip1/waf1 and GADD45a, GADD45b which moderate cell growth arrest were down regulated. However, no change in expression of either p16^INK4a or p14/p19^ARF was detected by microarray and immunoblotting. We therefore conclude that Bmi-1 is abnormally expressed in MM cells and contrary to its classic action, modulates MM cell growth and survival by modulating cytokine signaling rather than INK4a/ARF. Our studies further suggest that Bmi-1 represents a promising target for novel MM therapeutics.