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J. Poirier
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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)
16:50 - 16:55 | Author(s): J. Poirier
- 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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ORAL 25 - Biology and Other Issues in SCLC (ID 125)
- Event: WCLC 2015
- Type: Oral Session
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:J. Sage, L. Montuenga
- Coordinates: 9/08/2015, 10:45 - 12:15, 605+607
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ORAL25.07 - DNA Methylation in Small Cell Lung Cancer Defines Distinct Disease Subtypes and Correlates with High Expression of EZH2 (ID 3031)
11:50 - 12:01 | Author(s): J. Poirier
- Abstract
- Presentation
Background:
Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor characterized by extreme plasticity, high metastatic potential, and capacity for acquired resistance to chemotherapy. Despite significant advances in our understanding of SCLC genetics and etiology, the epigenetics of this deadly disease remain under studied. This study profiles DNA methylation in primary SCLC, patient-derived xenografts (PDX) and cell lines at single-nucleotide resolution.
Methods:
This study profiled DNA methylation at single-nucleotide resolution in 47 extensively characterized SCLC samples, including 34 fresh frozen primary SCLC tumors as well as 6 distinct primary patient-derived xenografts and 7 cell lines using the Illumina Human Methylation 450k Bead Chip array. Importantly, 24 primary SCLC in this study have previously been analyzed by whole exome sequencing and RNAseq, allowing integrated analysis of these data types with measurements of DNA methylation. We applied unsupervised clustering, discrete and locally clustered differential methylation analysis, correlation with gene expression, spacial correlation with genomic features, and interrogated the role of the EZH2 methyltransferase in SCLC using bioinformatic and pharmacologic approaches.
Results:
Unsupervised clustering of all samples revealed that PDX clustered with primary SCLC, while cell lines were easily discriminated. We explored this phenomenon further and found that while the top differentially methylated CpGs in both PDX and cell lines were >80% concordant with primary SCLC, only PDX maintained high concordance across larger probe lists. Unsupervised clustering of primary SCLC revealed three distinct subgroups at both the DNA methylation and gene expression levels that correlated with expression of the neurogenic transcription factors ASCL1 and NEUROD1. The chromatin modifier EZH2 was expressed >12-fold higher in SCLC than in normal lung. In addition to the high expression observed in SCLC compared to normal lung, we observed a significant correlation between median EZH2 gene expression and promoter methylation using data from The Cancer Genome Atlas (TCGA). Overall, EZH2 expression in SCLC is greater than or comparable to that of any other tumor type represented in TCGA. EZH2 protein expression was detected by Western blot in 15/17 SCLC PDXs (88%). We assessed the efficacy of the potent EZH2 inhibitor EPZ-5687 in the LX92 SCLC PDX in vivo. EPZ-5687 was well-tolerated and demonstrated remarkable efficacy at 100 mg/kg either QD or BID.
Conclusion:
DNA methylation patterns in primary SCLC are more closely mirrored by those found in PDX, compared to cell lines, including PDX lines of very high passage. Distinct epigenetic subtypes could be observed in SCLC, even among histologically indistinguishable samples with similar mutation profiles. SCLC is notable for consistent high level DNA methylation clustered in promoters containing CpG islands. Promoter methylation in SCLC is distinct from other lung cancers and correlates strongly with high-level expression of the histone methyltransferase gene EZH2. Pharmacologic inhibition of EZH2 in a SCLC PDX markedly inhibited tumor growth. These findings point to a critical role of EZH2 in SCLC tumor biology and support further preclinical efficacy studies in models of SCLC.
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