Soluble Signals Exchanged between Bone Marrow Adipocytes and Myeloma Cells Alter the Expression of Metabolic Genes in Adipocytes and Increase Pro-Survival Molecules in Myeloma Cells

Multiple myeloma (MM) is a hematologic cancer of the plasma cell with expansion in, and dependence upon, the bone marrow niche. Recent studies from our lab found that MM-associated bone marrow adipocytes (BMAds) have reduced lipid content, a skewed adipokine production profile, a senescence-associated secretory phenotype (SASP), and tumor-supportive properties. However, a better understanding of how BMAds support myeloma cells is necessary to identify novel targets in MM.

To examine BMAd-derived factors and their effects on MM cells, we first treated MM cells with BMAd-derived conditioned media (BMAd-CM). MM.1S cells treated with BMAd-CM for 24 hours exhibited significant increases in cell numbers and proliferation (Ki67) and increases in proportions of cells in the G2/M phase of cell cycle concurrent with a reduction in G0/G1. BMAd-CM also altered pathways related to metabolism in MM.1S cells, based on SWATH mass spectrometry analyses. No differences in either reactive oxygen species (ROS) or intracellular lipid were detected. Surprisingly, Seahorse XF metabolic testing showed significant increases in most glycolytic parameters in MM cells treated with BMAd-CM compared to basal media, with some differences in cellular respiration, suggesting BMAds may induce a glycolytic metabolic shift in tumor cells.

To create a more realistic, dynamic model, MSC-derived BMAds (from 3 different cancer-free, orthopedic surgery patients) and human myeloma cell lines (MM.1S, RPMI-8226, or OPM-2), were cultured either separately or together, divided by 0.4 µm transwell membranes for 4-72 hours. Cell number was quantified using luciferase activity. Flow cytometry was used to measure ROS and cellular lipid content using CellRox Green and LipidSpot 610 respectively. RNA-Sequencing (RNASeq) was used to detect differentially expressed genes (DEGs) in MM and BMAd cells after 72 hours of transwell co-culture. Interestingly, MM.1S^gfp+/luc+ cells in transwell co-culture with BMAds exhibited a significant decrease in luciferase activity after both 4 and 24 hours, although by 72 hours, no differences were detected in cell number or Ki67 detected by flow cytometry. BMAd transwell co-culture also increased the percent of MM cells in G0/G1 phase and induced dexamethasone resistance, including a rescue of cell numbers, proliferation, and progression through cell cycle. No differences in ROS or lipid droplets were detected in MM cells with BMAd co-culture. Similar results were observed in 5TGM1-TK mouse MM cells grown with 3T3-L1 adipocytes, in that no effect was detected on cell number after 72 hours of transwell co-culture.

Interestingly, RNASeq analysis of MM cells in transwell co-culture with BMAds versus MM cells alone, identified 20 transcripts that met the most stringent parameters for determining DEGs (log2FC |1| & padj of <0.05) and were enriched for the Reactome pathways “PTK6 Activates STAT3”, “Signaling by Leptin”, and “Interleukin-6 Signaling”. Many survival-related genes, including BCL6 and BCL3, were also upregulated in all MM cells cultured with BMAds.

Interestingly, RNAseq analysis of BMAds after 72 hours of transwell co-culture with MM cells showed upregulation of fatty acid elongase 6 (ELOVL6) (an enzyme involved in lipogenesis and linked to obesity related malignancies) and other metabolic genes (ACLY, ACACA, DGAT2, FABP4, PLIN2), as well as a cluster of mitochondrial genes. No differences were observed in genes encoding lipolytic enzymes in BMAds cultured with MM cells, despite prior publications reporting this.

Combined, these findings implicate secreted factors from BMAds in upregulating pro-survival pathways in myeloma with consequences for drug resistance. Future studies are required to further delineate the mechanism by which BMAds upregulate these survival molecules. Understanding the metabolic landscape within the bone marrow niche may provide new avenues for MM therapy.

No relevant conflicts of interest to declare.