Gene expression profiling of hematologic malignant cell lines resistant to oncolytic virus treatment

Abstract

Pexa-Vec (pexastimogene devacirpvec; JX-594) has emerged as an attractive tool in oncolytic virotherapy. Pexa-Vec demonstrates oncolytic and immunotherapeutic mechanisms of action. But the determinants of resistance to Pexa-Vec are mostly unknown. We treated hemoatologic malignant cells with Pexa-Vec and examined the gene-expression pattern of sensitive and resistant cells. Human myeloid malignant cell lines (RPMI-8226, IM-9, K562, THP-1) and lymphoid cancer cell lines (MOLT4, CCRF-CEM, Ramos, U937) were treated with Pexa-Vec. Pexa-Vec was cytotoxic on myeloid cell lines in a dose-dependent manner, and fluorescent imaging and qPCR revealed that Pexa-Vec expression was low in RAMOS than IM-9 after 24 hrs and 48 hrs of infection. Gene expression profiles between two groups were analyzed by microarray. Genes with at least 2-fold increase or decrease in their expression were identified. A total of 660 genes were up-regulated and 776 genes were down-regulated in lymphoid cancer cell lines. The up- and down-regulated genes were categorized into 319 functional gene clusters. We identified the top 10 up-regulated genes in lymphoid cells. Among them three human genes (LEF1, STAMBPL1, and SLFN11) strongly correlated with viral replication. Up-regulation of PVRIG, LPP, CECR1, Arhgef6, IRX3, IGFBP2, CD1d were related to resistant to Pexa-Vec. In conclusion, lymphoid malignant cells are resistant to Pexa-Vec and displayed up-regulated genes associated with resistance to oncolytic viral therapy. These data provide potential targets to overcome resistance, and suggest that molecular assays may be useful in selecting patients for further clinical trials with Pexa-Vec.

Keywords: gene expression profiling; hematologic malignancy; oncolytic virus.

Figure 1. Vaccinia virus induces cytolysis in myeloid leukemia cell lines

Percent viability of four myeloid leukemia cell lines at 72 hours of post infection of Pexa-Vec and NYCBH is compared to untreated control. Multiplicity of infection ranges from 8.1 to 0.0001 and represented in log scale. Each assay was tested in triplicate. Error bars = SD.

Figure 2. Lymphoid cell lines are resistance to the vaccinia virus infection

Percent viability of four lymphoid cell lines at 72 hours of post infection of Pexa-Vec and NYCBH shows their resistance to viral infection. Multiplicity of infection ranges from 8.1 to 0.0001 and represented in log scale. Each assay was tested in triplicate. Error bars = SD.

Figure 3. Microscopic images of myeloid leukemia cell line over time following viral infection

The myeloid cell line IM-9 was infected with 1 MOI of Pexa-Vec or NYCBH and incubated for 72 hours. The representative microscopic images were captured every 24 hours post-infection. Mock infected cells were used as control.

Figure 4. Microscopic images of lymphoid leukemia cell line over time following viral infection

The lymphoid cell line Ramos was infected with 1 MOI of Pexa-Vec or NYCBH and incubated for 72 hours and the representative microscopic images were captured at every 24 hours post-infection. Mock infected cells were used as control.

Figure 5. Physical viral particle count by qPCR assay

The number of physical DNA copies targeting E9L gene of vaccinia virus is plotted against time after viral infection on IM-9 and Ramos cell lines. The total DNA was isolated from infected cell harvest using QIAamp DNA blood mini kit. Data is shown on average of duplicate runs. JX-594:Pexa-Vec, WR: western reserve virus.

Figure 6. Fluorescence images after viral infection on leukemia cell lines

The GFP expression was visualized by fluorescence microscopy at 4, 8, 24 and 48 hours post-infection on IM-9 and Ramos cell lines. GFP fluorescence imaging showed that the virus infected on myeloid cell line IM-9 was replicated successfully after 24 hours. The representative images were shown here.

Figure 7. Hierarchical cluster analysis of microarray on virus infected leukemia cell lines

In a microarray raw data set, cell lines are ordered in columns and gene expression value are given in rows. The columns labeled in red (Ramos, CCRF-CEM, MOLT-4 and U937) are data from the lymphoid cell lines infected with Pexa-vec, and in blue (RPMI-8226, IM-9, K562 and THP-1) are from myeloid cell lines. Red cells indicate high expression and green cells indicate low expression