Macrophage Migration Inhibitory Factor Drives Multiple Myeloma IL-6/8 Pro-Survival Signals in the Tumor Microenvironment

Background

Multiple myeloma (MM) accounts for 10-15% of all newly diagnosed hematological malignancies in the western world and despite recent significant progress in the treatment of MM, it presently remains an incurable disease. Average survival following diagnosis is currently approximately 5 years. Current treatments are effective at reducing malignant cell number (and therefore alleviating symptoms), however a small sub-population of MM cells will survive within the bone marrow milieu and relapse is inevitable. Interactions between the MM and bone marrow mesenchymal stromal cells (BM-MSCs) have been shown to be highly beneficial to the MM cells, protecting them from chemotherapeutic agents and upregulating the expression of genes associated with increased survival and proliferation. We hypothesize that the myeloma cells initiate the re-programming of the BM-MSCs for this very purpose, providing themselves with a more favorable environment.

The cytokine MIF (Macrophage Migratory Inhibitory Factor) has been shown to be present in abnormally high levels in many cancer types. Here we show that MIF is aberrantly produced by primary myeloma cells and induces transcriptional changes and cytokine release from the BM-MSC. We also show these signals contribute to MM cell survival and drug resistance.

Methods

Primary MM and BM-MSC were obtained from patient's bone marrow as previously described. Primary MM cells at 1x10⁶ were co-cultured on confluent autologous BM-MSC. Conditioned media from MM alone, MM/BM-MSC and BM-MSC cultures were collected and analyzed using Proteome Profiler Human XL Cytokine Array and cytokine specific ELISAs. Real-time PCR was used to analyze the changes in BM-MSC transcriptional levels in response to specific cytokine stimulation or inhibition. Recombinant MIF and the MIF inhibitor ISO-1 were used to verify the MM-BMSC findings.

Results

Here we report that bone marrow, aspirated from MM patients, had a higher level of MIF expression when compared to samples obtained from healthy donors. We examined the cytokine profile in human primary MM monoculture compared to MM cultured with autologous BM-MSC or BM-MSC alone and found that MIF was highly expressed by primary MM and that IL-6 and IL-8 were also increased in MM/BM-MSC co-cultures. RT-PCR confirmed that the majority of MIF transcription within the bone marrow microenvironment occurs within the myeloma cells themselves, with relatively minimal levels found within the bone marrow stromal cells.

Next BM-MSCs from MM patients (n=5) were stimulated with recombinant MIF and cytokine release was measured using the XL cytokine array. The results showed that that recombinant human MIF stimulated the release of IL-6 and IL-8 to the media from BM-MSC monocultures. These observed changes were confirmed with the use of cytokine specific ELISA assays. RT-PCR also showed that IL-6 and IL-8 RNA expression were induced in BM-MSCs in response to recombinant MIF. These results confirm that MM derived MIF is instrumental in the transcriptional regulation of IL-6 and IL-8 in BM-MSCs.

Next we found that the MIF inhibitor ISO-1 inhibited MM and MIF induced BM-MSC IL-6 and IL-8 RNA expression and cytokine release. Finally we found that MIF inhibition in MM/BM-MSC co-cultures reduced MM survival as well as enhancing the therapeutic potential of proteasome inhibition.

Summary

Together, these data suggest that MM signals via MIF to initiate the re-programming of the BM-MSCs, which creates a pro-tumoral environment in which MM can thrive and be protected from anti-MM therapeutics.