Deep sequencing RNA-Analysis of a cell and mouse model of KSHV sarcomagenesis

DARIA SALYAKINA ; JULIÁN NAIPAUER; VYTAS DARGHIS-ROBINSON ; SANTAS ROSARIO ; ENRIQUE A. MESRI

Los Angeles

Workshop; 19th International Workshop on KSHV and Related Agents; 2016

Human viral oncogenesis is the consequence of the transforming activity of virally encoded oncogenes and non-coding RNAs in combination with host oncogenic alterations. In the case of KS and KSHV, there is a lack of experimental systems to dissect viral and host contributions to the malignant phenotype. Deep RNA sequencing of KSHV Bac36 transfected mouse endothelial cells (mECK36) and tumors are a unique model to dissect genetic mechanisms of KSHV dependent and independent sarcomagenesis in an unbiased hi-throughput fashion since the system allows for unique experimental comparisons in the same cell and KS-like tumor types: 1) KSHV+ve vs KSHV-ve mECK36 were used to study KSHV mediated effects ?in vitro? 2) KSHV+ve mECK36 grown in vitro and in tumors were used to study ?in vitro? vs ?in vivo? variations of host and viral gene expression induced by micro-environmental cues as well as the occurrence of host mutations in the tumors 3) KSHV+ve mECK36 vs KSHV-ve mECK36 tumors were used to dissect the role of KSHV genes and non coding RNAs in tumorigenesis by comparing mECK36 tumors driven by KSHV vs mECK36 tumors driven by host mutations. We performed Illumina, stranded, RNA seq analysis of all KSHV stages of this cell and animal model including: KSHV+ve mECK36 cells (N=2), KSHV-ve mECK36 (N=2), KSHV+ve mECK36 tumors (N=6) and KSHV-ve mECK36 tumors (N=3). Analysis of the host and viral transcriptome was used to characterize mechanisms of KSHV dependent and independent sarcomagenesis as well as the contribution of host mutations. Most significant results of analysis indicate that: Transcriptome: As expected by the minimal phenotypic in vitro differences, KSHV+ve vs ?ve cells showed a difference of 150 Differentially expressed (DE). In contrast KSHV+ve mECK36 cells and tumors genes showed close to 4000 DE while KSHV+ve vs KSHV-ve tumors showed more than 6000 DE. Gene enrichment analysis of DEs between KSHV+ve and KSHV-ve tumors using GeneGO or GSEA plattforms show that KSHV-driven in vivo growth display tumorigenesis pathways occurring predominantly by activation of Notch, EMT, TGFbeta and hypoxia while KSHV-ve tumors, known to be driven by PDGFRA D842V activating mutations, occur with a predominance of proliferative pathways including Ras, c-Myc and E2F. Mutations: Mutational analysis of mECK36 cells and tumors revealed a surprising set of mutations, including mutations in three inflammasome related IFN response genes, absent in mECK36 cells but present in all mECk36 tumors in the same location. This indicates that these mutations should be the consequence of ?in vivo? clonal selection of few mutated mECK36 cells of the population. This result suggests that in the context of in vivo tumorigenesis both these mutations and the virus may determine tumor growth. Clustering analysis of mutations driving KSHV-ve tumors reveal a network comprising PDGFRA D842V, Pak1 and Nucleolin mutations implicated in cell proliferation. Non-coding RNAs: Differential analysis of miRNA showed highest DE scores between KSHV+ve and KSHV-ve tumors and for ?in vitro? vs tumor growth of mECK36 cells. Close to 20 miRNA, including 10 well-characterized oncomirs were differentially regulated by KSHV expression in tumors and inversely correlated with regulation of predicted target mRNAs. Our results have uncovered novel specific aspects of the interplay between host oncogenic alterations and virus-induced transcriptional effects in the context of ?in vivo? KSHV sarcomagenesis