Multiple myeloma (MM) is a hematologic cancer in which malignant plasma cells amass within bone marrow (BM). Clinical symptoms include hypercalcemia, renal failure, anemia and osteolytic bone lesions. Despite improvements in management of the disease and the efficacy of current treatment options, MM is still incurable for the majority of patients. In fact, most of the patients relapse due to acquired drug resistance and/or persistence of residual myeloma cells. The cancer dormancy model implies that cancer cells in cell cycle arrest, also called quiescent or dormant cancer cells, can be the cause of relapse because of their aptitude to exit the dormancy status, re-proliferate and repopulate the tumor. In this project, we investigated myeloma cell dormancy and developed nanobodies against AXL (receptor tyrosin kinase) and CS1 (plasma cell marker), two cell surface antigens highly expressed by dormant myeloma cells. First, we confirmed the expression of AXL and CS1 on dormant MM cells in vivo using the 5TGM1-GFP DiD labeled MM cells. We observed that AXL was only present on dormant MM cells, while CS1 was expressed by both proliferating and dormant MM cells. Secondly, we tried to generate an in vitro dormancy model by coculturing of MM cells with osteoblasts. We demonstrated that in the presence of osteoblasts, myeloma cells enter in cell cycle arrest (G0/G1 phase) and express higher levels of the cell cycle inhibitors p21 and p27. Coculture experiments of osteoblasts (MC3T3) and myeloma cells (5TGM1-GFP/DiD) revealed higher AXL levels in the dormant MM cell subpopulation compared to proliferating cells. In addition, we found that melphalan treatment increased AXL expression in MM cells in vitro. In a second part of the project, we demonstrated that CS1 was preferentially expressed by MM cells. The increased expression levels of AXL and CS1 in dormant MM cells derived to the production of specific Nbs against these potential targets. In vitro binding studies demonstrate the successful generation of anti-AXL and anti-CS1 nanobodies. In vivo biodistribution studies performed on the 5TMG1 and 5T33MM model, resulted in the selection of Nb U6216 as a lead compound to target specifically CS1 expressing myeloma cells and allow in vivo imaging. We can conclude that osteoblasts are able to induce dormancy and increase AXL expression in MM cells. We were able to generate AXL targeting nanobodies, however further biodistribution studies are necessary to identify a lead compound. In addition, we found a high expression of CS1 on dormant and proliferating MM cells. Moreover, we were able to generate a CS1-specific Nb and in future we will use this as a tool for specific eradication of residual cancer cells by means of targeted radionuclide therapy.
|Date of Award||21 Jun 2018|