Malignant Plasma Cell Derived Mitochondrial Damps Activate Macrophages Via Sting Signalling to Promote Myeloma Development

Mitochondrial damage-associated patterns (mtDAMPs) include proteins, lipids, metabolites, and DNA and have various context specific immunoregulatory functions. Cell free mitochondrial DNA (mtDNA) contains islands of unmethylated cytosine-guanine dinucleotide (CpG) motifs which are recognised via pattern recognition receptors and are potent activators of the innate immune system. Cell free mtDNA is elevated in the circulation of trauma and cancer patients, however the functional consequences of elevated mtDNA are largely undefined.

Multiple myeloma (MM) relies upon cellular interactions within the bone marrow (BM) microenvironment for survival and progression. A key feature of myeloma is the homing and retention of MM cells to the BM, and it can be assumed that this homing process is mediated by chemotactic signals provided by the BM microenvironment. Here, using in-vivo models, we describe the role of MM cell derived mtDAMPs in the pro-tumoral BM microenvironment, and the mechanism and functional consequence of mtDAMPs in myeloma disease progression.

Initially, we identified elevated levels of mtDNA in the peripheral blood serum of myeloma patients compared to healthy controls. This was also consistently observed in the serum of mice, using two distinct myeloma mouse models: KaLwRij-5TGM1 and NSG-MM1S. Using the MM xenograft model, we established that mtDNA was derived from myeloma cells. Blood serum samples were taken and analysed by RT-qPCR to detect the presence of human mtDNA in the serum of engrafted mice and correlated to disease progression. Since macrophages sense and respond to mtDAMPs we isolated BM macrophages from myeloma engrafted mice and showed they had an increased gene expression signature indicative of STING pathway activation. The same signature was observed when bone marrow-derived macrophages (BMDMs) were treated with mtDAMPs in culture, suggesting that this activation was mediated via myeloma-derived mtDAMPs.

To understand the role of macrophage STING signalling in MM, we engrafted KaLwRij mice with murine 5TGM1^{(GFP+ Luci+)} cells. After tumour was established, we treated the mice with STING inhibitor H-151 (750nM). Using live imaging, we observed that tumour progression was reduced in the STING inhibited group compared to the non-treated group. Flow cytometry of the bone marrow confirmed a significant reduction in 5TGM1^{(GFP+ Luci+)} cell burden in the STING inhibited group. Furthermore, cytokine array analysis of BMDMs treated with mtDAMPs revealed an upregulation of chemokines involved in chemotaxis, suggesting that mtDAMPs induce a migratory signature in BM macrophages. To investigate this, we performed a transwell assay to assess the chemotaxis of 5TGM1 cells towards conditioned media from BMDMs treated with mtDAMPs. We found that 5TGM1 migration was enhanced with mtDAMPs treatment and reduced with H-151 treatment. Furthermore, RT-qPCR analysis of FACs isolated macrophages from KaLwRij-5TGM1 mice showed increase expression of genes associated with chemotaxis. Moreover, the expression of the chemotaxis gene signature was attenuated with H-151 inhibitor treatment.

Finally, using our syngeneic 5TGM1-KaLwRij mouse model we found that depletion of macrophages via clodronate-liposome treatment resulted in egress of myeloma cells from the BM and into the peripheral blood.

Here, we demonstrate that malignant plasma cells release mtDNA, a form of mtDAMPs, into the myeloma BM microenvironment, which in turn activates macrophages via STING signalling. We establish the functional role of these mtDAMPs activated macrophages in reducing disease progression and retaining MM cells in the pro-tumoral BM microenvironment.

No relevant conflicts of interest to declare.