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T. Hitchman
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MINI 27 - Biology and Other Issues in SCLC (ID 152)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:P.A. Bunn, Jr, J. Sage
- Coordinates: 9/09/2015, 16:45 - 18:15, 605+607
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MINI27.02 - RPS25 Is Essential for the Translation of the Seneca Valley Virus Genome and Proliferative Capacity of Small Cell Lung Cancer Cell Lines (ID 3278)
16:45 - 18:15 | Author(s): T. Hitchman
- Abstract
- Presentation
Background:
Small cell lung cancer (SCLC) is an extremely aggressive and lethal disease for which there is a desperate need for novel and more effective treatments. A recently discovered oncolytic picornavirus, Seneca Valley Virus (SVV), infects tumors with neuroendocrine features, including SCLC with high selectivity. SVV is highly effective in the eradication of solid tumors in multiple in vivo models; however the mechanism of selective tropism for SVV is unknown. Because of the strong selectivity of the virus for SCLC, we hypothesize the host determinants of SVV permissivity could constitute future druggable targets for the treatment of SCLC.
Methods:
A retroviral gene trap mutagenesis screen was utilized in HAP1, a haploid human cell line permissive to SVV, HAP1. Once mutagenized, resistant cells, or cells with retroviral insertion in a gene essential to the viral life cycle, were selected for by incubation of the pool with SVV at a high multiplicity of infection (MOI). Hits from this screen were deconvoluted using an insertion mapping approach. Illumina sequencing provided quantitative counts of each insertion site in each gene. Hits from the screen were validated using various mechanistic approaches.
Results:
Our screen identified multiple unique insertion sites in the gene RPS25 on Chromosome 11. The RPS25 protein is a ribosomal protein that is a component of the 40S subunit of the ribosome. RPS25 has been previously shown to be important for IRES-dependent translation of multiple viral genomes as well as cellular mRNAs containing IRES elements. Using the CRISPR-Cas9 approach, we knocked out the RPS25 gene in the SVV-permissive SCLC cell line, NCI-H446. Upon total knock-down of RPS25, H446 cells become completely resistant to cell killing by SVV at high MOI. Surprisingly, these cells also show a severely marked decrease in doubling time and robustness in culture. In contrast, RPS25 CRISPR knock-down in HEK293T cells has been previously shown to have no distinguishable phenotype other than defects in IRES-dependent translation. Further studies to fully characterize the interaction of RPS25 with the SVV genome as well as the importance of RPS25 in other SCLC cell lines are ongoing.
Conclusion:
We have identified a host protein that is essential for SVV replication and infection using a genome wide mutagenesis screen. SCLC cells completely defective in RPS25 are resistant to SVV-dependent cell killing. RPS25 appears to not only be important for the life cycle of SVV but may be important in proliferative capacity in SCLC. As SVV is highly selective for SCLC, we hypothesize that the host determinants of SVV tropism may be very specific to SCLC cells. Proteins important in the SVV life cycle may be novel “druggable” targets for the treatment of SCLC.
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