Polyunsaturated Fatty Acid (PUFA) Signaling Induces Ferroptosis-Mediated Cell-Death in Multiple Myeloma
Authors: Panaroni Cristina , Fulzele Keertik , Soucy Rosemary , Huang Cherrie , Mukaihara Kenta , Chattopadhyay Shrikanta , Raje Noopur S. 
Published: November 2019

Altered cellular metabolic pathways are the hallmark of tumor cells. Multiple myeloma (MM) is positively correlated with metabolic disorders such as obesity and Gaucher's disease. The local bone marrow (BM) microenvironment (TME) majorly influences the initiation and progression of MM. In a typical MM patient, BM adipocytes make up 70% of the cellular volume. The abundance of adipocyte-secreted free fatty acids (FFA) may shift myeloma cellular metabolism from aerobic glycolysis to more energy-producing fatty acid oxidation. The FFAs are important catalysts of key downstream drug-targetable signaling pathways such as cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX) pathways. In this study, we hypothesized that altered lipid profile in the local BM TME contributes to MM progression.
BM-Fat enriched tissue isolated from BM aspirates of Monoclonal Gammopathy of Undetermined Significance (MGUS) and smoldering MM (SMM) patients showed a significant increase in adipogenic PPARγ gene expression compared to aged-matched healthy donors (N≥3). The BM mesenchymal stem/progenitor cells (BMSCs) from MGUS/SMM patients expressed normal levels of BMSC markers CD271, CD105, CD44, CD106, CD29, CD90, CD49e, and Notch4 but showed significantly increased expression of adipogenic markers including Preadipocyte factor 1, Leptin Receptor, and Perilipin A (N=6). This also translated into significantly increased adipogenic differentiation of patient BMSCs when cultured alone or with the human MM cell-line MM.1S (N≥3). Furthermore, MM.1S showed significantly increased proliferation when co-cultured with BMSCs from MGUS/SMM patients (N=5). These data demonstrate a vicious cycle where adipogenesis is increased in early precursor MM stages that further support the growth of myeloma cells.
We performed gas chromatometry based lipidomics analysis on the supernatant of BM aspirates from MGUS, SMM, and newly diagnosed MM (NDMM) patients. The analysis identified significant decreases in key polyunsaturated fatty acids (PUFA) including Arachidonic Acid (AA) and Docosatetraenoic acid (N≥5). Lipid metabolism specific gene array on RNA from adipose tissue fraction of BM aspirates from MGUS, SMM and NDMM patients showed altered changes in genes responsible for fatty acid synthesis and metabolism. PUFA are involved in anti-inflammatory mechanisms in cancer. We hypothesized that increased levels of certain PUFA, such as AA, in the BM TME may decrease MM progression.
To test this hypothesis, we treated MM cells with physiological doses of AA. AA dose-dependently decreased proliferation and viability of human MM cell lines, MM1S, H929, and U266, and CD138+ patient myeloma cells. For in vivo studies, humanized MM tumor model was generated in SCID mice by growing MM.1S cells in the intrascapular subcutaneous region for 3-weeks. Mice were then treated with daily localized injections of vehicle, 100µg/g AA, 500µg/g of AA, or IV with 2mg/kg/biweekly Carfilzomib (CFZ), or CFZ with 500µg/g of AA (COMBO). Tumor volume significantly decreased in 500µg/g AA treatment group beginning 10-days and was comparable to the CFZ treatment. Gross examination and flow cytometry analysis of CD138+ myeloma cells showed dramatically increased tumor-cell apoptosis in 500µg/g AA and COMBO treatment groups.
To identify the primary apoptosis-inducing AA signaling pathway in MM cells, we used specific inhibitors of each of these signaling pathways including ibuprofen (Cox inhibitor), baicalein (12-LOX inhibitor), BW B70C (5,15-LOX inhibitor), 1-aminobenzotriazole (CYP450 inhibitor), and ferrostatin (Ferroptosis/lipid peroxidation inhibitor). Among these compounds, ferrostatin treatment completely rescued AA induced apoptosis in the human MM.1S cells. Ferroptotic cell death is the result of an accumulation of lipid peroxides which is generally prevented by the enzyme Glutathione peroxidase 4 (GPX4). We, therefore investigated the role of AA on GPX4 and found that all MM cell lines partially or completely lost the expression of GPX4 when exposed to AA and that this effect was completely prevented when cotreated with Ferrostatin.
Taken together, we show that BM adipocytes promote myeloma cell proliferation at least in part through secreted FFAs. Therapeutically targeting members of this signaling pathway, such as ferroptosis, is a potential novel treatment strategy for MM especially in the MGUS and SMM stages.

Disclosures
Raje: Celgene Corporation: Consultancy; Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy.