PERSISTENCE OF EBV IN THE CANCER STEM CELL FRACTION OF MULTIPLE MYELOMA
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Date
2013-10-31
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Johns Hopkins University
Abstract
Epstein Barr Virus (EBV) is a human herpesvirus that infects most of the world’s adult population. It is associated with several malignancies including Burkitt lymphoma and nasopharyngeal carcinoma. EBV infects B cells and establishes a latent life cycle as an extra-chromosomal plasmid or produces mature virions by undergoing lytic replication. We show that EBV is present in some multiple myeloma cell lines and patients and when present, it is detected in a subpopulation of cells. Previous investigators, noting the phenotypic heterogeneity of clonal myeloma cell lines, have presented evidence that cells with a plasma cell-like phenotype develop from cells with a B cell-like phenotype in culture and that a similar process may occur in patients.
In this report, we present evidence that EBV persists in a phenotypically distinct cellular fraction. We have investigated cells with a cancer stem cell-like mature B cell phenotype in the peripheral blood of patients with multiple myeloma (MM) and in MM cell lines. In peripheral blood lymphocytes from MM patients, EBV DNA is enriched 10 fold in aldehyde dehydrogenase rich-mature memory B cells (CD20+CD27+) expressing the same light chain type as malignant plasma cells, and enriched approximately 100 fold over the CD19+ peripheral B cell population. The presence of EBV is confirmed by fluorescence in situ hybridization and immunofluorescence. In contrast EBV is absent from these patients plasma cells. The phenotypic characteristics of this fraction overlap with the putative cancer stem cell fraction.
We investigated further in tissue culture and found that in commonly studied MM cell lines (MM1S, RPMI8226, NCIH929, KMS11), EBV is also present, but almost exclusively in the subpopulation of cells with a mature B cell phenotype. Limiting dilution analysis shows that the frequency of cells that harbor EBV in these lines is less than 1 in 100. However, the presence of rare EBV infected cells is confirmed by fluorescence in situ hybridization and immunofluorescence for viral nuclear antigens.
Gene expression of EBV nuclear antigen (EBNA) – 1 and 2 as well as latency membrane protein (LMP)-2 was detected by RT-PCR and confirmed by immunofluorescence. The subpopulation of cells that harbor virus were phenotypically distinct CD19+CD138- cells. Blocking plasma cell differentiation led to a higher frequency of CD19+ B cells and subsequently an increased frequency of EBV infected cells , thereby confirming that in MM, EBV was harbored in the B cell-like population. Introduction of a dominant negative inhibitor of the EBV latency protein EBNA-1 affected cell kinetics in EBV(+) MM cells but not in EBV(-) cell lines. These results suggest that in some MM cell lines, EBV is harbored in a B cell-like progenitor fraction that may overlap with the putative cancer stem cell fraction. EBV may be lost during differentiation to plasma cells.
Treatment with antiviral drugs like acyclovir stabilized EBV viral copy number over months showing that the virus was present in a latent plasmid form. Targeting the EBNA-2 protein function slowed down the growth kinetics of the myeloma cell lines that were EBV positive.
Knock down of a pro-differentiation transcription factor (XBP-1) led to an increase in the frequency of EBV-infected cells. Expression of a transdominant inhibitor of latency viral replication and segregation was accompanied by a slowing of cell growth as measured by doubling time in myeloma cell lines that harbor EBV but not in cell lines lacking the virus consistent with the interpretation that the viral genome plays an important role in the growth kinetics of the MM cell lines in which it is found. Eviction of the EBV nuclear plasmid or inhibition of EBV-growth signaling slows the growth of the culture as a whole consistent with the possibility that EBV plays a role in maintaining the growth of MM cancer stem cells.
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Keywords
multiple myeloma cancer stem cells, Epstein-Barr virus