Combination Therapy with Reovirus and Immunomodulatory Drugs Induces Direct Oncolytic and Immune-Mediated Killing of Multiple Myeloma Cells and Overcomes Stromal-Mediated Microenvironmental Protection
Authors: Parrish Christopher , Scott Gina B , Coffey Matt , Melcher Alan , Errington-Mais Fiona , Cook Gordon 
Published: October 2019

Abstract
Introduction
Despite considerable advances in therapy over recent years, multiple myeloma (MM) remains largely incurable with a clinical course characterised by ever-shortening periods of treatment and relapse; new therapies with novel mechanisms of action are needed. The immunomodulatory agents (IMiDs) in current clinical practice can augment immune effector function and combination with other immunologically active agents is a rational avenue of investigation – indeed trials of IMiDs with agents such as monoclonal antibody therapies are already underway. The naturally occurring oncolytic virus reovirus serotype 3 Dearing (Reolysin, Oncolytics Biotech Inc) has been shown to be safe in the context of advanced epithelial-derived cancers, and more recently in patients with MM. In this study we demonstrate the potential of reovirus in combination with IMiDs and steroids to induce both direct tumour cell killing and an anti-tumour immune response, particularly in the context of the cytoprotective effect of stromal cell interactions.
Methods
Human myeloma cell lines (HMCLs) were tested for JAM1 expression (the high affinity receptor for reovirus) by flow cytometry (FACS). HMCLs, alone or in co-culture with human stromal cell lines, were treated with reovirus at a range of multiplicities of infection (MOIs), alone or in combination with lenalidomide and/or dexamethasone and examined by flow cytometry to determine time- and concentration- dependent cell death. Peripheral blood mononuclear cells (PBMC) from healthy donors and patients were treated with reovirus/drug combinations and immune effector cell activation determined by surface expression of CD69. Treated PBMC and untreated HMCL were co-cultured and NK cell degranulation and NK cell-mediated HMCL cell death was determined. Bone marrow aspirate samples (BM) were collected from patients with MM, treated with reovirus, lenalidomide and dexamethasone combinations, and examined by FACS for killing of CD138+ cells and immune effector cell activation.
Results
JAM1 was expressed on the HMCLs H929, JIM3, KMS11, KMS18, JJN3 and U266B, but not on OPM2. All lines except OPM2 (which was resistant) were susceptible to reovirus-induced oncolysis in a time- and MOI-dependent manner. Addition of dexamethasone and lenalidomide to these treatments further increased the killing of tumour cells. Reovirus treatment induced upregulation of CD69 on NK cells, CD4+ T cells, CD8+ T cells and NKT cells isolated from the peripheral blood of both healthy donors and patients with MM. Healthy donor NK cells activated ex vivo with reovirus showed augmented degranulation (by surface CD107a expression) and killing of HMCL tumour targets. Importantly, in the case of OPM2, which was resistant to direct viral-induced cell death, reovirus-activated NK cells killed the HMCL. NK cells from MM patients showed enhanced degranulation against autologous tumour cells. Cell contact-independent killing of HMCL by reovirus-treated PBMC was also noted. Coculture of HMCLs with human bone marrow stromal cell lines (HS-5 and HS-27) protected against lenalidomide- and dexamethasone-induced HMCL cell death. However, treatment with reovirus overcame this cytoprotection. In keeping with this, when BM from MM patients was treated with lenalidomide and/or dexamethasone, the addition of reovirus augmented killing of CD138+MM cells.
Conclusions
Our data indicate reovirus has dual modes of action against MM – not only can the virus mediate direct oncolytic cytotoxicity against the malignant clone, but in addition it can activate immune effector cells and thus induce immunologically-mediated tumour killing. Importantly, this immune-mediated action was noted even against HMCLs resistant to the direct viral effects. Furthermore, the cytoprotection and immune evasion afforded by stromal cells was mitigated by reovirus therapy and thus addition of reovirus to established immunomodulatory myeloma therapies augments their efficacy. Reovirus-based immunomodulatory combination therapies are a rational therapeutic approach for patients with relapsed MM and warrant further investigation in the pre-clinical and early phase clinical trial setting.

Disclosures
Coffey: Oncolytics Biotech Inc.: Employment.