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Codon Specific Readthrough as a Mechanism of BRCA2 Restoration in Acquired PARP Inhibitor and Chemotherapy Resistance [WES]

ABSTRACT: Deleterious alterations of the gene encoding BRCA2, a protein involved in homologous recombination (HR), contribute to the pathogenesis of a subset of ovarian, breast, prostate and pancreatic cancers as well as their increased sensitivity to platinating agents and poly(ADP-ribose) polymerase inhibitors (PARPis). While secondary mutations that restore a functional BRCA2 protein are detected in a fraction of cases that become drug resistant, the causes of resistance in other cases are poorly understood. Here we characterized clones derived from the BRCA2-mutant ovarian cancer line PEO1 by PARPi selection. Colony forming assays demonstrated cross-resistance of these clones to multiple PARPis, cisplatin and doxorubicin. Despite persistence of the signature 4965C>G (p.Y1655X) BRCA2 nonsense mutation, HR was restored in the resistant cells. Immunoprecipitation followed by immunoblotting or mass spectrometry demonstrated expression of low levels of BRCA2 protein, including peptides distal to the premature termination codon. Reporter assays demonstrated readthrough selective for the UAG stop codon in the resistant clones but not parental PEO1 cells. Mass spectrometry and immunoblotting provided evidence for readthrough of stop codons, particularly UAGs, in additional proteins in the resistant clones. BRCA2 gene interruption either 5’ or 3’ to the stop codon restored sensitivity of the resistant clones to PARPis and cisplatin, implicating the low level BRCA2 expression in resistance. Accordingly, the present results identify low level readthrough of nonsense codons as a potential mechanism of drug resistance that might need to be considered when assessing the impact of BRCA2 nonsense mutations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE243142 | GEO | 2024/09/01

REPOSITORIES: GEO

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Project description:Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 and mRNA secondary structure in the 3’-UTR had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features important for translation termination and readthrough.

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Codon Specific Readthrough as a Mechanism of BRCA2 Restoration in Acquired PARP Inhibitor and Chemotherapy Resistance [WES]

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