Targeting multiple myeloma with oncolytic viral therapy

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Introduction: Multiple Myeloma (MM) is an incurable cancer that accounts for 10% of hematological malignancies. Stem cell rescue following high-dose chemotherapy with autologous or allogenic transplants have become standard therapy for a subset of good performance patients with MM. Graft versus host disease and the need for a matching donor are limitations of allogenic transplants whereas minimal residual disease or contaminating tumor cells within the autograft leading to relapse is a concern with autotransplants. Previously we have shown that reovirus, an ubiquitous double stranded RNA virus could successfully purge RPMI 8226 MM cell line as well as a patient tumor specimen with minimal effects on stem cells (Thirukkumaran et al. 2004 : Blood. 102:377-387). In the present study we investigate 1) The oncolytic ability of reovirus against a larger number of MM cell lines and ex vivo patient specimens in order to assess MM sensitivity to reovirus in a community population, 2) Reovirus purging efficacy in a murine model of transplantation.
 Methods: MM cell lines RPMI 8226, RPMI 8226-GFP, U266, H929 and HUNS1 were incubated with live or UV-inactivated reovirus at a multiplicity of infection of 40 for 24, 48 and 72 hours. Percent cytotoxicity was assessed using the WST assay and apoptosis was monitored via flow cytometry using the Annexin V and 7-AAD. To investigate human stem cell (CD34+) re-population and MM tumor take in SCID/NOD mice, they were sublethally irradiated (2Gy) and intraperitoneally injected with 50µg anti-asialo antibody to eradicate NK cells. Mice in 3 groups were injected (tail vein, 200 µl) of either 5 X 10⁶ RPMI 8226-GFP cells, 5 X 10⁶ human CD34+ stem cells or PBS. On day 21 mice were sacrificed and subjected to whole body fluorescence imaging using a GFP Imaging System. Bone marrow cells were collected from mouse femurs after flushing with PBS and analyzed for CD38+/138+/GFP+ MM cells as well as CD34+ human stem cells.
 Results: To date, 70% of MM cell lines tested showed reovirus sensitivity and reovirus induced cell death was mediated through apoptosis. Human stem cells showed engraftment in SCID/NOD animals and RPMI 8226-GFP cells showed excellent tumor take in mice. In addition, GFP+ MM cells homed to mouse bone marrow, reminiscent of human disease making it an ideal murine purging model system for our studies.
 Conclusions: The sensitivity of reovirus towards MM and its lack of effect human stem cells highlight the potential of reovirus as a purging agent during autologous hematopoietic stem cell transplants for MM. The SCID/NOD model system used in the present study appears suitable for evaluating reovirus purging efficacy in vivo. Currently, we are in the process of studying reovirus purging efficacy in vivo and its purging capacity of ex vivo MM patient tumor in order to generate data for a future phase I clinical trial.