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A k-mer based transcriptomics approach for antisense drug discovery targeting the Ewing's family of tumors
Andrew J Annalora1, Shawn O'Neil2, Jeremy D Bushman3
1Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331, USA.
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.
Insights
New antisense agents targeting Ewing
Area of Science:
- Oncology and Molecular Biology
- Cancer Genomics and Therapeutics
Background:
- Ewing's sarcoma (EWS) treatment failures contribute to high mortality rates.
- Novel therapeutic strategies are urgently needed for effective EWS management.
- Identifying cancer-specific molecular targets is crucial for drug discovery.
Purpose of the Study:
- To identify novel mRNA transcripts specific to Ewing's Family Tumors (EFTs).
- To evaluate the therapeutic potential of antisense agents targeting these EFT-specific transcripts.
- To investigate synergistic and antagonistic interactions of morpholino oligomers in combination therapy.
Main Methods:
- Utilized k-mer counting to profile transcriptomes of EFT cell lines and identify cancer-specific mRNA.
- Designed and synthesized phosphorodiamidate morpholino oligomers targeting six identified EFT-specific transcripts.
- Assessed morpholino cytotoxicity and combination effects in EFT cell lines (TC-32, CHLA-10) and control cells (HEK293).
Main Results:
- Identified several EFT-specific transcripts, including CYP4F22, RBM11, and IGFBP-2.
- Morpholino oligomers demonstrated significant cytotoxicity in EFT cells (EC50 < 1 µM), with minimal impact on control cells.
- Observed synergistic effects with a morpholino cocktail and specific paired combinations (e.g., PHGDH with XAGE1B/CYP4F22).
- Identified antagonistic interactions between certain morpholino combinations (e.g., CCND1 with XAGE1B/CYP4F22).
Conclusions:
- Transcriptome profiling is an effective strategy for discovering novel cancer drug targets.
- Identified novel EWS-specific targets and validated antisense agents with potent anti-cancer activity.
- Demonstrated the potential of morpholino oligomers in combination therapy, highlighting the importance of evaluating drug interactions.