Abstract 397: Myeloma derived mitochondrial DNA activates TLR9 driven pro-tumoral expansion

Abstract

Abstract Multiple myeloma (MM) is reported to cause 85,000 global deaths per year which is estimated to double by 2040. MM is an incurable malignancy of antibody (Ig) secreting differentiated B cells (plasma cells) characterized by the accumulation and localisation of tumor cells in the bone marrow microenvironment (BMM). Mitochondrial genome (mtDNA) contains CpG DNA repeats and CpG DNA has been shown to activate and induce proliferation of memory B cells to secrete Ig. Here we determine if malignant plasma cells maintain their proliferative advantage by releasing mtDNA into the tumour microenvironment which acts as feedback mechanism to induce further proliferation and Ig production by the malignant plasma cell. Human MM cell lines U266 and MM1S were engrafted into NSG immunocompromised mice. Blood sample from control and engrafted mice were collected at various times and plasma was isolated and spun at 15,000g to remove cell debris and large extracellular vesicles. Human and mouse mtDNA was determine by real-time PCR. Results show that human mtDNA can be detected in the plasma from mice engrafted with U266 and MM1S at 2 weeks and 3 weeks post injection of cells. Next, CpG oligonucleotides were used to determine if MM cells can be activated by mtDNA CpG repeats. Primary MM, U226 and MM1S cells were all treated with CpG and CpG control for 4 and 24 hours. Real-time PCR was then used to detect the pro-survival signature showing that IL-1beta, IL-6 and IL-8 were all upregulated by CpG. We then analysed primary MM and MM cell lines for TLR9 expression by flow cytometry and showed that all samples tested had high levels of cytosolic TLR9 expression. Using TLR9 antagonist (ODN TTAGGG (A151)) we show that primary MM cells and MM cell lines have reduced IL-6 and IL-8 expression and decreased cell proliferation. Finally, U266 and MM1S transduced with TLR9 knockdown lentivirus had reduced engraftment, as measured by bioluminescence, in NSG mice. Here we show that MM release mtDNA in vivo and this can induce a tumour directed proliferative response through the engagement of TLR9. This process is necessary for optimum tumor growth and forms part of the malignant phenotype of MM. Citation Format: Aisha Jibril, Jayna J. Mistry, Prakrit Kumar, Jamie A. Moore, Charlotte Hellmich, Cesar Gomez, Kristian Bowles, Stuart A. Rushworth. Myeloma derived mitochondrial DNA activates TLR9 driven pro-tumoral expansion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 397.