Inducible Knockdown of t6A Pathway Genes in PEO1 and PEO4 Ovarian Cancer Cells
Session Number
2
Advisor(s)
Daniel Arango, Khulood Abdulaziz Alzahrani, Northwestern University
Location
A151
Discipline
Biology
Start Date
15-4-2026 11:10 AM
End Date
15-4-2026 11:55 AM
Abstract
Ovarian cancer is often classified as a cold tumor due to its low tumor mutation burden (TMB) and limited neoantigen expression, which results in poor responses to immune checkpoint inhibitor therapy. One possible strategy to increase neoantigen production is to disrupt the fidelity of protein translation. The tRNA modification N6-threonylcarbamoyladenosine (t6A) plays an essential role in maintaining translation accuracy by stabilizing codon-anticodon interactions and preventing frameshifting during protein synthesis. Elevated levels of t6A modifications have been observed in ovarian cancer, suggesting that cancer cells may rely on enhanced translational fidelity to limit the production of aberrant peptides. This study aims to establish an inducible CRISPR-Cas9 knockdown system to investigate how depletion of t6A-related enzymes may affect translation and neoantigen production. Two ovarian cancer cell lines, PEO1 (cisplatin-sensitive) and PEO4 (cisplatin-resistant), were used to compare gene expression and validate the experimental system in both wild-type and Cas9-expressing cells. Protein concentrations were quantified using the Bradford assay to ensure equal loading for downstream analyses. RNA samples were treated with DNase to remove genomic DNA contamination before amplification. PCR was performed to confirm the presence and gene expression, and Western blot analysis was used to evaluate protein expression levels. These experiments provide a foundation for generating inducible knockdown models to study how disruption of t6A-mediated translational fidelity may increase non-canonical ORF translation and promote neoantigen expression in ovarian cancer cells.
Inducible Knockdown of t6A Pathway Genes in PEO1 and PEO4 Ovarian Cancer Cells
A151
Ovarian cancer is often classified as a cold tumor due to its low tumor mutation burden (TMB) and limited neoantigen expression, which results in poor responses to immune checkpoint inhibitor therapy. One possible strategy to increase neoantigen production is to disrupt the fidelity of protein translation. The tRNA modification N6-threonylcarbamoyladenosine (t6A) plays an essential role in maintaining translation accuracy by stabilizing codon-anticodon interactions and preventing frameshifting during protein synthesis. Elevated levels of t6A modifications have been observed in ovarian cancer, suggesting that cancer cells may rely on enhanced translational fidelity to limit the production of aberrant peptides. This study aims to establish an inducible CRISPR-Cas9 knockdown system to investigate how depletion of t6A-related enzymes may affect translation and neoantigen production. Two ovarian cancer cell lines, PEO1 (cisplatin-sensitive) and PEO4 (cisplatin-resistant), were used to compare gene expression and validate the experimental system in both wild-type and Cas9-expressing cells. Protein concentrations were quantified using the Bradford assay to ensure equal loading for downstream analyses. RNA samples were treated with DNase to remove genomic DNA contamination before amplification. PCR was performed to confirm the presence and gene expression, and Western blot analysis was used to evaluate protein expression levels. These experiments provide a foundation for generating inducible knockdown models to study how disruption of t6A-mediated translational fidelity may increase non-canonical ORF translation and promote neoantigen expression in ovarian cancer cells.