103 Generation and Characterisation of Neratinib-resistant Breast Cancer Cell Line Variants

Burkitt’s lymphoma cell line BJAB-LexR that has been made naturally resistant to TRAIL through long-term culture in the presence of increasing concentrations of the drug Lexatumumab, an anti-DR5 antibody, as well as BJAB cells with Six1 overexpression. Performing experiments in both these contexts would allow us to distinguish between resistance genes that arise spontaneously versus resistance genes that are a direct effect of Six1 expression. We performed a genome-wide loss of function screen using the GeneNet lentiviral shRNA library containing 200 000 shRNAs with sequencing tags, allowing for high throughput deep sequencing. After selection for a particular phenotype, in this case TRAIL resistance, the shRNAs that promote or prevent this phenotype can be deduced from the frequency in which the shRNA is present in the selected population versus the unselected population. A library was constructed using sequence tag specific primers and deep sequencing was performed using Illumina sequencing technology. Bioinformatic analysis yielded a list of putative resistance genes that are currently being validated in the BJAB system and also in MDAMB231 breast cancer cells that have been made resistant to TRAIL in a similar fashion. With this unbiased approach, we aim to identify not only known components of the TRAIL pathway that may be deregulated but also completely novel pathways of TRAIL resistance that could be targeted for improved cancer therapy.