Abstract
Obesity-induced increases in bone marrow adipocyte (BMAd) numbers and volume are associated with an increased risk of multiple myeloma (MM). We analyzed gene expression from previously published public datasets and found that 11 out of 47 genes associated with fatty acid (FA) metabolism showed increasing trend from MGUS (monoclonal gammopathy of undetermined significance), MM, to plasma cell leukemia. These genes included ACC1, the first and rate-limiting step of de novo fatty acid biosynthesis and ECHS1, the second step of the mitochondrial fatty acid beta-oxidation pathway. We have previously shown that adipocytes support the growth of MM cells. However, the molecular mechanisms of interaction between MM cells and BMAd remain largely unknown. Here, we hypothesize that BMAd support MM cells through metabolic reprogramming.
Here we sought to identify the molecular pathways involved in MM/ BMAd interaction. BM aspirates of MGUS, smoldering MM (SMM), and newly diagnosed MM (NDMM) patients were used to isolate fat-enriched BM fraction and BM stromal cells (BMSCs). Murine BMSC cell-line OP9, murine MM cell-line 5TGM1, and human MM cell lines MM.1S and OPM2 were obtained from ATCC or provided by collaborators and cultured as their respective standard procedures. In-vitro adipogenesis was induced in OP9 cells or BMSCs by supplementing media with dexamethasone, indomethacin, insulin, and IBMX. MM cells were co-cultured directly with pre- or mature adipocytes. Cell proliferation was assessed using CyQUANT NF Cell Proliferation Assay. Lipolysis was assessed by High Sensitivity Lipolysis Assay Kit (Sigma). Lipid uptake in MM cells was assessed by flow-cytometry analysis of the incorporation of fluorescent 12- or 16-carbon long-chain fatty acids BODIPY-FL-C12 and BODIPY-FL-C16, respectively, or LipidTox labelled FA from co-cultured adipocytes. In-vivo effects of excess FA on MM cell growth were assessed using a plasmacytoma model in CB17 SCID mice.
In-vitro co-culture revealed that BMSC-derived adipocytes (Ad) from MGUS, SMM and NDMM donors increased the proliferation of MM.1S MM cells significantly. Similarly, mature murine OP9 Ad cells also increased the proliferation of 5TGM1 murine MM cells. Interestingly, co-cultures showed dramatic decrease in LipidTox-stained lipid-droplet size distribution, suggesting increased lipolysis in Ad. In the process of lipolysis, various lipase enzymes hydrolyze stored triglycerides into free fatty acids (FFA) and glycerol. Co-culture of 5TGM1 or OPM2 cells with OP9 mature Ad increased glycerol secretion in the conditioned media by more than 3-fold indicating that MM induce lipolysis in Ad. Consequently, BMAd from MGUS, SMM, and NDMM patients showed increased expression of genes responsible for lipolysis (NR1H3) and increased FA desaturation (SCD1, FASD2). Although MM cells lacked intracellular lipid storage, OPM2 and 5TGM1 MM cells rapidly took up BODIPY-C12 and -C16 FAs. The FA secreted from Ad were directly taken up by MM cells as shown by transfer of LipidTox-labeled lipids from OP9 Ad to unstained 5TGM1 or OPM2 MM cells as assessed by flow cytometry. The addition of Acipomox, a small-molecule inhibitor of lipolysis, decreased LipidTox signal in MM cells compared to untreated OP9 cells. FA are primarily transported into cells through FATP (1-6) or CD36 receptors. Bioinformatic analysis of public database showed that FATP1 and FATP4 were highly expressed in 21 human MM cell lines. Indeed, MM cells from NDMM patients expressed high levels of FATP1 and FATP4. The uptake of BODIPY-C12 and -C16 by 5TGM1 or OPM2 MM cells was significantly reduced in the presence of Lipofermata, a pharmacological small-molecule inhibitor of FATP. Lipidomic analysis of BM aspirates from MM patients showed altered expression of various FA, including arachidonic acid (AA). Low doses of AA (0.125 - 2 µM) increased the proliferation and viability of MM cells whereas high doses (25- 100µM) dramatically decreased it, indicating a bimodal cellular effect of AA. Peritumoral AA treatment in a plasmacytoma model in CB17 SCID mice using MM.1S cells dramatically decreased tumor volume along with the markers of proliferation.
In summary, we show that MM cells induce lipolysis in BMAd and that the released FFA are then taken up by MM cells through FATPs. Inhibition of either BMAd lipolysis or FFA transporter into MM cells could be a potential novel strategy to prevent MM progression.
Disclosures
Fulzele: Constellation Pharma: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other.
Fatty Acid Allosteric Regulation of C-H Activation in Plant and Animal Lipoxygenases
