Contributions
Abstract: S179
Type: Oral Presentation
Session title: Basic and translational myeloma research
Background
Multiple myeloma (MM) is a malignancy of the antibody secreting plasma cells, that is characterized by the accumulation and localisation of tumour cells within the bone marrow microenvironment (BMM). The mitochondrial genome belongs to a group of mitochondrial damage associated molecular patterns (mtDAMPs) and contains islands of unmethylated CpG nucleotide motifs, which have been shown to activate and promote memory B cell antibody secretion and proliferation. Studies have indicated that mitochondrial DNA (mtDNA) is elevated in the circulation of trauma and cancer patients.
Aims
Here we aim to explore the hypothesis that multiple myeloma cells secrete mitochondrial DNA and other mitochondrial DAMPs into the bone marrow microenvironment, leading to the promotion of a state of chronic inflammation that supports multiple myeloma disease progression and expansion.
Methods
Immunocompromised NSG mice were engrafted with human myeloma cell line (MMIS), blood serum samples were taken and analysed using real-time PCR (RT-PCR) to detect the presence of human mtDNA. We established syngeneic mouse models of multiple myeloma using the murine 5TGM1 myeloma cell line, flow cytometry was used to analyse the haematopoietic stem and progenitor cell (HSPC) and macrophage populations within the bone marrow. Bone marrow derived macrophages were sorted from these mice and RT-PCR was used to analyse pro-inflammatory gene expression. To further investigate the role of mtDNA in HSPC expansion, C57BL/6 mice were treated with multiple doses of CpG oligodeoxynucleotides to mimic the mtDNA released by MM. in vitro, BMDM were treated with CpG and mtDNA and pro-inflammatory gene expression was analysed via RT-PCR. To further understand the role of mtDAMPs in regulating proinflammatory signals in the MM microenvironment, we used an inhibitor to block TLR9 (toll-like receptor), the key mtDNA sensing receptor.
Results
We detected elevated levels of MM derived mtDNA in the blood serum of immunocompromised NSG mice engrafted with human myeloma cell line (MM1S). Flow cytometry analysis of the HSPC populations showed that 5TGM1 induced an expansion of the stem cell niche, and real-time PCR analysis of the blood serum showed an elevated level of cell free mtDNA in 5TGM1 engrafted mice. Moreover, the frequency of tumour associated macrophages (TAM) was increased in 5TGM1 engrafted mice and RT-PCR analysis of sorted BMDM showed a pro-inflammatory phenotype. Flow cytometry analysis of CpG treated mice revealed an expansion of HSPC populations similarly found in 5TGM1 engrafted mice. Furthermore, in vitro analysis of BMDM treated with mtDNA and CpG showed increased expression of pro-inflammatory cytokines including IL-6. Lastly, blocking of the TLR9 receptor resulted in reduced MM tumour burden in 5TGM1 engrafted mice treated with TLR9 inhibitor compared to untreated mice.
Conclusion
Here we ascertain that MM releases mtDNA into the microenvironment and show the involvement of mtDAMPs in promoting the disease progression of MM via a pro-inflammatory BMM. This data proposes targeting potential receptor signalling pathways as a novel therapeutic intervention for multiple myeloma.
Keyword(s): Bone Marrow, Microenvironment, Multiple myeloma
Abstract: S179
Type: Oral Presentation
Session title: Basic and translational myeloma research
Background
Multiple myeloma (MM) is a malignancy of the antibody secreting plasma cells, that is characterized by the accumulation and localisation of tumour cells within the bone marrow microenvironment (BMM). The mitochondrial genome belongs to a group of mitochondrial damage associated molecular patterns (mtDAMPs) and contains islands of unmethylated CpG nucleotide motifs, which have been shown to activate and promote memory B cell antibody secretion and proliferation. Studies have indicated that mitochondrial DNA (mtDNA) is elevated in the circulation of trauma and cancer patients.
Aims
Here we aim to explore the hypothesis that multiple myeloma cells secrete mitochondrial DNA and other mitochondrial DAMPs into the bone marrow microenvironment, leading to the promotion of a state of chronic inflammation that supports multiple myeloma disease progression and expansion.
Methods
Immunocompromised NSG mice were engrafted with human myeloma cell line (MMIS), blood serum samples were taken and analysed using real-time PCR (RT-PCR) to detect the presence of human mtDNA. We established syngeneic mouse models of multiple myeloma using the murine 5TGM1 myeloma cell line, flow cytometry was used to analyse the haematopoietic stem and progenitor cell (HSPC) and macrophage populations within the bone marrow. Bone marrow derived macrophages were sorted from these mice and RT-PCR was used to analyse pro-inflammatory gene expression. To further investigate the role of mtDNA in HSPC expansion, C57BL/6 mice were treated with multiple doses of CpG oligodeoxynucleotides to mimic the mtDNA released by MM. in vitro, BMDM were treated with CpG and mtDNA and pro-inflammatory gene expression was analysed via RT-PCR. To further understand the role of mtDAMPs in regulating proinflammatory signals in the MM microenvironment, we used an inhibitor to block TLR9 (toll-like receptor), the key mtDNA sensing receptor.
Results
We detected elevated levels of MM derived mtDNA in the blood serum of immunocompromised NSG mice engrafted with human myeloma cell line (MM1S). Flow cytometry analysis of the HSPC populations showed that 5TGM1 induced an expansion of the stem cell niche, and real-time PCR analysis of the blood serum showed an elevated level of cell free mtDNA in 5TGM1 engrafted mice. Moreover, the frequency of tumour associated macrophages (TAM) was increased in 5TGM1 engrafted mice and RT-PCR analysis of sorted BMDM showed a pro-inflammatory phenotype. Flow cytometry analysis of CpG treated mice revealed an expansion of HSPC populations similarly found in 5TGM1 engrafted mice. Furthermore, in vitro analysis of BMDM treated with mtDNA and CpG showed increased expression of pro-inflammatory cytokines including IL-6. Lastly, blocking of the TLR9 receptor resulted in reduced MM tumour burden in 5TGM1 engrafted mice treated with TLR9 inhibitor compared to untreated mice.
Conclusion
Here we ascertain that MM releases mtDNA into the microenvironment and show the involvement of mtDAMPs in promoting the disease progression of MM via a pro-inflammatory BMM. This data proposes targeting potential receptor signalling pathways as a novel therapeutic intervention for multiple myeloma.
Keyword(s): Bone Marrow, Microenvironment, Multiple myeloma