Oncolytic Virotherapy in Multiple Myeloma: A Possible Alternative Role of Bovine Viruses.
Authors: Marchica Valentina , Donofrio Gaetano , Vescovini Rosanna , Tebaldi Giulia , Rosamilia Alfonso , Guasco Daniela , Storti Paola , Bolzoni Marina , Costa Federica , Schifano Chiara , Bonomini Sabrina , Accardi Fabrizio , Piazza Francesco , Aversa Franco , Giuliani Nicola 
Published: October 2019

Abstract
Multiple Myeloma (MM) is a plasma cell malignancy characterized by the tight dependence to the bone marrow (BM) microenvironment that supports MM cells survival. Despite significant therapeutic progress in the recent years with the introduction of several new drugs, MM remains an incurable disease. Oncolytic virotherapy is an alternative therapeutic technology in the cancer treatment exploiting naturally or genetically engineered viruses able to infect, transduce and consequently kill cancer cells directly or indirectly through the delivery of the microenvironment cells. Several oncolytic viruses have shown promising pre-clinical results for the treatment of MM, in particular Measles virus and Reovirus. However, the use of human viruses such as Measles could be limited by the antiviral immune response of the patients due to vaccination or natural infection. In order to avoid these potential limits of the human viruses, the aim of this study was to investigate the development of bovine viruses as an alternative oncolytic strategy in MM by checking these viruses that showed an anti-tumoral activity in different solid tumors. Thus, we investigated the potential lytic effect on human MM cells of the Bovine Viral Diarrhea Virus (BVDV), known to bind CD46 as reported for human measles virus, and the Oncolytic Bovine Herpesvirus type 4 (BoHV-4).
Firstly, we treated several human myeloma cell lines (HMCLs) with BVDV or the heat-inactivated virus for 24, 48 and 72 hours. The infection efficiency was verified by nested multiplex PCR. We showed a significant increase of cell mortality, checked by trypan blue count and flow cytometry analysis, already after 48 hours of infection in JJN3 (mean±SD % of dead cells: BVDV 45±11 % vs inactive virus 16±2.5 %, p=0.013), and in OPM2 (BVDV 43±1.4 % vs inactive virus 28±2.1 %, p= 0.015) but not in U266 (BVDV 25±23 % vs inactive virus 18±12 %, p=NS. However, BVDV pre-treatment for 12 hours and followed by 48 hours bortezomib (bor) treatment (concentration ranging: 5-10nM) significantly restored bor sensitivity in U266 resistant cells (mean±SD % of dead cells: BVDV plus bor 10 nM 69±8 % vs inactive virus + bor 10 nM 36±1 %, p= 0.031). Interestingly, the cytotoxic effect of BVDV treatment in HMCLs was associated by a significantly increase of apoptotic markers evaluated by flow cytometry. Subsequently, we infected BM primary purified CD138+, showing a significant increase of the mortality rate after treatment with BVDV as compared to the inactivated virus. On the contrary, BVDV was not able to infect human BM mesenchymal stromal cells (MSCs) not showing any lytic effect.
Thereafter the capacity to induce MM cell lysis by a recombinant BoHV-4 virus, delivering a Red Fluorescent Protein (RFP) expression cassette as reporter gene, was also evaluated. As observed by the percentage of RFP-positive cells, BoHV-4 was unable to infect and consequently to kill several HMCLs tested. Then we used BM MSCs as in vitro model for oncolytic virus delivery in co-culture systems with MM cells. BoHV-4 infected hTERT-MSCs, expressing RFP at 24, 48 and 72 hours. Consistently, hTERT-MSCs viability was progressively reduced at 24 and 48 hours after infection, as compared to controls, (mean±SD % reduction of cell viability: -22±8 %, p=0.0254 and -49±2 %, p=0.0001, respectively), reaching the highest effect at 72 hours (-70±1.5 %, p=0.0003). Thus we evaluated the effect of BoHV-4 in a co-culture system between human MSCs and two stroma-dependent HMCLs as INA-6 and saMMi. In both cases the percentage of dead HMCLs increased in co-culture with BoHV-4 infected hTERT-MSCs, as compared to hTERT-MSCs untreated controls (INA-6: BoHV-4 61±2.1 % vs control 12±2.1 %, p= 0.0018; saMMi: BoHV-4 48±1.9 % vs control 14±1.4 %, p= 0.0027).
Overall our data indicate both direct and MSC-mediated oncolytic effects of bovine viruses on MM cell, suggesting their possible use as novel alternative anti-MM virotherapy strategy.

Disclosures
Giuliani: Celgene: Research Funding; Janssen: Research Funding.