TAF12 Enhances the Biological Reponses of Myeloma Cells to Low Concentrations of 1,25-(OH)₂dvitamin D₃

Multiple myeloma (MM) is currently incurable, and its associated bone destruction is a major source of morbidity that significantly contributes to patient mortality. Development of novel treatments for MM is imperative, since myeloma patients continue to relapse and suffer the consequences of the bone disease, even when they are in remission. Thus new targets for treating and preventing MM induced bone destruction are needed.

We found that TAF12 is highly expressed in CD138⁺ primary MM cells and bone marrow stromal cells (BMSC) from MM patients compared to their normal counterparts. TAF12 acts as a co-activator of VDR, that permits very low concentrations of 1,25-(OH)₂D₃ or analogs that bind VDR to enhance MM cell growth and bone destruction. The molecular responses of myeloma cells to vitamin D are unknown, as are its effects on the marrow microenvironment and myeloma bone disease.

To assess the role of TAF12 in MM, we co-cultured JJN3 myeloma cell line with the human BMSC cell line (SAKA-T). The experiments showed that MM cells directly upregulates TAF12 in SAKA-T cells; this increase in TAF12 was mediated by IL-6 secreted by MM cells. Co-culture of MM cells with BMSC from TAF12 heterozygous knockout mice (TAF12^+/-) blocked VCAM1 induction and the enhanced MM cell growth and osteoclast (OCL) formation in response to physiologic levels 1,25-(OH)₂D₃ (10^-10 M) induced by BMSC. Thus, TAF12 plays an important role in increased cytokine production and adhesive interactions of MM cells/ BMSC induced by 1,25-(OH)₂D₃.

We determined the role of TAF12 in the sensitivity of several MM cell lines to physiological concentrations of 1,25-(OH)₂D₃. TAF12 expression was tested in MM cell lines (MM1.S, JJN3, ANBL6, RPMI8226, U266 and OPM2). Five of 6 cell lines (except RPMI8226) expressed high levels of TAF12. In 3 of these five, 1,25-(OH)₂D₃ increasedRANKL. Interestingly, both MM1.S and JJN3 cells produced RANKL at very low concentrations of 1,25-(OH)₂D₃ (10^-12-10^-10 M), which also induced VEGF, DKK-1 and α4 integrin.

To further investigate the mechanisms responsible the effects of low concentrations of 1,25-(OH)₂D₃ mediated by TAF12 in MM, we stably knocked-down (k/d) TAF12 in JJN3 myeloma cells using a TAF12 shRNA lentivirus (TAF12k/d-JJN3). The TAF12k/d-JJN3 cells demonstrated about 80% reduction in TAF12 protein. JJN3-Wt cells treated with 10^-8M 1,25-(OH)₂D₃ increased CYP24A1 mRNA levels 60 fold compared to untreated cells. In contrast, CYP24A1 mRNA in TAF12k/d-JJN3 cells was not significantly induced even when treated with 10^-8 M of 1,25-(OH)₂D₃. ChIP analysis using an anti-VDR or TAF12 antibody and primers flanking the two VDREs in the CYP24A1 promoter showed that 1,25-(OH)₂D₃ induced VDR and TAF12 binding to the CYP24A1 promoter in JJN3-Wt cells. In contrast, basal and induced levels of VDR binding were much lower in TAF12k/d-JJN3 cells.

Further, VDR content in TAF12k/d-JJN3 cells treated with 1,25-(OH)₂D₃ (10^-10-10^-8M) was markedly decreased compared to JJN3-Wt cells. VDR content was quantified by Western blot. In addition, RANKL, VEGF, DKK1 and α4 integrin production by TAF12k/d-JJN3 cells treated with10^-10 M of 1,25-(OH)₂D₃ were decreased 60% compared to JJN3-Wt cells.

These results demonstrate that increased TAF12 in MM cells enhances transcriptional responses of a subset of VDR target genes, leading to hyper-responsivity to 1,25-(OH)₂D₃ in MM cells - suggesting that TAF12 is a potential therapeutic target for MM and MM bone disease.