Understanding The Differential Response Of Multiple Myeloma To Reovirus Treatment
Authors: Stiff Andrew , Badway Joseph , Nuovo Gerard , Sborov Douglas W. , Smith Emily M , Talabere Tiffany , Hofmeister Craig C , Pichiorri Flavia 
Published: October 2019

Abstract

Introduction
Multiple myeloma (MM) is a currently incurable malignancy arising from post germinal, terminally differentiated plasma cells. Despite the success of proteasome inhibitors and immune-modulating drugs such as bortezomib and thalidomide in the treatment of relapsed disease patients eventually become refractory to these drugs making new therapeutic agents a critical need.
One potential new agent is Reovirus Serotype 3- Dearing Strain. While infection of immunocompetent hosts results in only minor respiratory illness it has been demonstrated that reovirus selectively replicates in and is cytopathic to Ras activated cells. This specificity is due at least in part to the inhibition of auto-phosphorylation of PKR. The minimal side effects and dependence of reovirus on Ras activation make it an interesting candidate for relapsed/refractory MM as it has been reported that at least 50% of relapsed MM patients have activating Ras mutations and most patients show activation at other Ras checkpoints including p38. While there has been limited investigation of reovirus treatment of MM cell lines there has been less analysis of resistance to reovirus treatment in MM.

Methods
MM cell lines were screened via MTS assay to identify cell lines sensitive, intermediate, and resistant to reovirus treatment. Cell lines were then treated at a MOI of 10 or 25 for 9 and 24 hours and analyzed by immunohistochemistry (IHC) for p38, reoviral protein, and caspase-3 as well as in situ hybridization (ISH) and qRT-PCR for reoviral RNA. Total RNA isolates of sensitive, intermediate, and resistant cell lines were used to perform a screening analysis of mRNA, microRNA (miRNA), and long non-coding RNA to identify differential expression. In addition, expression of the reovirus high affinity receptor JAM-1 was assessed in these cell lines by western blot and IHC. Following this analysis mRNA, miRNA, and long non-coding RNA expression was analyzed in bone marrow samples from relapsed/refractory MM patients enrolled in a reovirus monotherapy clinical trial before and after treatment. These results were correlated with patient response and compared to the results from the cell lines.

Results
MTS results clearly identified several cell lines as being sensitive (RPMI), intermediate (H929), and resistant (OPM2) to reovirus treatment according to percent viability. IHC revealed over 98% positivity for p38 in all cell lines indicating Ras activation and permissibility to reovirus infection regardless of sensitivity. IHC also showed a higher percentage of cells positive for reoviral protein in sensitive and intermediate cell lines compared to resistant cell lines. The increased reoviral protein expression in these cell lines correlated with these lines having a higher percentage of caspase-3 positive cells compared to both the controls and the resistant cell lines. ISH demonstrated that the relative signal intensity of reoviral RNA was proportional to the sensitivity of each cell line, and these results were consistent with qRT-PCR results.

Conclusions
Despite susceptibility to reovirus infection, as determined by p38 positivity, substantial variation was seen in response to reovirus treatment amongst cell lines. Given the correlation of cell line sensitivity with reoviral RNA levels and reoviral protein production it will be important to identify factors that drive these differences. Ongoing analysis is focused on determining the role of viral entry including expression of the reoviral receptor JAM-1 and subsequent viral trafficking and degradation. In addition, evaluation of differential expression of RNA molecules in cell lines and patient samples is ongoing as miRNA have been identified in the establishment of productive reoviral infections and cancer cell death (Nuovo et al. 2012).

Disclosures:
No relevant conflicts of interest to declare.