A k-mer based transcriptomics approach for antisense drug discovery targeting the Ewing's family of tumors.

Patrick L Iversen,Andrew J Annalora,Craig B Marcus,Shawn O’Neil,Jeremy D Bushman,James E Summerton

doi:10.18632/oncotarget.25736

Patrick L Iversen, Andrew J Annalora + Show 4 more

Open Access

https://doi.org/10.18632/oncotarget.25736

Copy DOI

Abstract

Ewing’s sarcoma treatment failures are associated with high mortality indicating a need for new therapeutic approaches. We used a k-mer counting approach to identify cancer-specific mRNA transcripts in 3 Ewing’s Family Tumor (EFT) cell lines not found in the normal human transcriptome. Phosphorodiamidate morpholino oligomers targeting six EFT-specific transcripts were evaluated for cytotoxicity in TC-32 and CHLA-10 EFT lines and in HEK293 renal epithelial control cells. Average morpholino efficacy (EC50) was 0.66 ± 0.13 in TC-32, 0.25 ± 0.14 in CHLA-10 and 3.07 ± 5.02 µM in HEK293 control cells (ANOVA p < 0.01). Synergy was observed for a cocktail of 12 morpholinos at low dose (0.3 µM) in TC-32 cells, but not in CHLA-10 cells. Paired synergy was also observed in both EFT cell lines when the PHGDH pre-mRNA transcript was targeted in combination with XAGE1B or CYP4F22 transcripts. Antagonism was observed when CCND1 was targeted with XAGE1B or CYP4F22, or when IGFBP-2 was targeted with CCND1 or RBM11. This transcriptome profiling approach is highly effective for cancer drug discovery, as it identified new EWS-specific target genes (e.g. CYP4F22, RBM11 and IGBP-2), and predicted effective antisense agents (EC50 < 1 µM) that demonstrate both synergy and antagonism in combination therapy.

Highlights

Identification of genes or gene segments that are expressed exclusively in tumor cells represents a novel approach to discovery of anti-cancer therapeutics [1]
The initial goal of this project was to assess whether open source transcriptome profiling could rapidly elucidate a potent, antisense therapeutic regimen for cancer with comparable or superior efficacy to conventional chemotherapeutic agents
We set out to characterize the complexity of the tumor-specific transcriptome for the Ewing’s family of Tumors (EFT), which include Ewing’s sarcoma (EWS)

Summary

Introduction

Identification of genes or gene segments that are expressed exclusively in tumor cells represents a novel approach to discovery of anti-cancer therapeutics [1]. A smaller fraction of genes are expressed in normal tissues, but expression is lost in tumors. These normal-specific genes are not likely candidates for therapeutic discovery. Using transcriptomics [2], we can study the transcriptome of a single tumor, or the sum of all RNA transcripts within an individual cell, tissue, organ or complete organism. This approach captures a snapshot of the target’s functional genome, and allows comparisons across different experimental conditions and time points, elucidating information on gene function, gene regulation and underlying changes to an organism’s biology. There are two primary approaches for transcriptome profiling, including microarrays and RNA-sequencing (RNA-Seq), which employs highthroughput sequencing (HTS) [3, 4]

Objectives

Results

Conclusion