Arachidonic Acid Induces Ferroptosis-Mediated Cell-Death in Multiple Myeloma

Mounting evidence suggests positive correlation of obesity and bone marrow (BM) adipogenesis with multiple myeloma (MM) initiation and progression. MM is more prevalent in patients with metabolic disorders including obesity and Gaucher's disease. Adipocytes constitute 70% of BM cellular volume in human adults making them one of the prominent players in MM progression. However, the underlying mechanisms involved remain unknown. Fat depots are increasingly recognized as the key endocrine organs for their ability to secrete various hormones (e.g. leptin, adiponectin, resistin), lipid substrates (polyunsaturated fatty acids), and cytokines (e.g. IL-6, IL-1, and TNFα). For example, leptin levels are increased in newly diagnosed MM patients that allow MM initiation by inhibiting NK T-cells (Favreaue 2017, Leukemia). Lipid substrates are also deregulated in the serum of cancer patients, including MM patients. These fatty acids are important initiators of many key downstream drug-targetable signaling pathways such as cyclooxygenase, cytochrome P450, and lipoxygenase pathways. In this study, we hypothesized that the changes in lipid profiles in MM patients contribute to disease progression.

PPARγ is the key transcription factor that potentiates adipocyte differentiation and its key functions including lipid synthesis and transportation. We found that the BM adipose tissue (BMAT) fraction from MGUS, and SMM patients showed significantly increased PPARγ expression compared to the healthy donors. In vitro, MGUS and SMM BM stromal cells (BMSCs) showed increased adipogenic differentiation potential compared to the healthy donors. In-vitro, the proliferation of MM.1S human MM cells was significantly increased when co-cultured with BMSCs from MGUS/SMM patients compared to healthy donors. Taken together these data demonstrate a vicious cycle in which early stage MM cells and BM adipocytes support each other's growth.

Multicomponent gene expression analysis of the BMAT from MGUS and MM patients showed altered genes involved mostly in fatty acid synthesis and metabolism. Lipidomics analysis revealed significantly decreased Arachidonic Acid (AA) levels in the BMSCs of SMM, the BM supernatant (BMS) of NDMM patients, and the blood serum of MGUS and SMM patients. This led us to hypothesize that increasing AA levels around MM cells may decrease MM progression.

Physiological-range AA treatment of human MM cell lines, MM1S, H929, and U266, dose-dependently decreased proliferation and viability after 72-hours in all three cell lines. For in vivo studies, we generated humanized MM tumor growth in SCID mice by growing MM.1S cells in the intrascapular subcutaneous region for 3-weeks. Mice were then treated with either 100µg/g or 500µg/g of AA daily along with vehicle control via subcutaneous injection for 3-weeks near the tumor mass. Tumor volume continued to increase in vehicle control and 100 µg/g AA treatment groups but was significantly decreased in 500µg/g AA treatment group beginning 10-days after starting treatment. Gross examination of the tumor mass showed dramatically increased tumor necrosis. Flow cytometry analysis of CD138+ myeloma cells from non-necrotic part of the tumors showed significantly increased number of dead cells and the cells undergoing apoptosis in 500µg/g AA treatment group.

AA is metabolized to primarily activate cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX) signaling pathways. Recent studies also show that AA induces ferroptosis, a programmed cell death in response to accumulation of lipid peroxides and mediated by iron (Kagan et al., 2017). To identify the primary apoptosis-inducing AA signaling pathway in MM cells, we used various inhibitors of each of these signaling pathways including ibuprofen, baicalein, BW B70C, 1-aminobenzotriazole, and ferrostatin. Among these compounds, ferrostatin completely rescued AA induced apoptosis in the human MM cells.

Taken together, here we show that the adipogenesis is involved in myeloma pathogenesisis. Furthermore, AA signaling induces ferroptosis-mediated cell death in MM cells. Therefore, therapeutically targeting members of this signaling pathway is a potential novel treatment strategy for MM, especially in the MGUS and SMM stages.