Seeking genetic modifiers to the drug trastuzumab emtansine (T-DM1) to advance HER2-targeted therapy

Abstract

Background: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the HER2-targeted antibody trastuzumab linked to DM1, a potent tubulin inhibitor. T-DM1 shows effective clinical activity in HER2-positive breast cancer, however, intrinsic and acquired resistance remains a major challenge. Greater understanding of the resistance mechanisms could identify predictive biomarkers for T-DM1 therapy, leading to improved response rates in the stratified patients or provide a therapeutic target for combination therapy. Genome-wide CRISPR screens offer a powerful tool to identify key modifiers of drug resistance. This thesis aimed to validate candidate genes identified in CRISPR screens for genetic determinants of T-DM1 sensitivity in HER2-positive breast cancer cell lines. Methods: This study utilised MDA-MB-361 NUMA1, SLC46A1 and IRF2BP2 knockout clonal cell lines generated outside of this project. Meanwhile, MIEN1 knockout and MIEN1 silencing were achieved in MDA-MB-361 and HCC1954 cell lines. The efficiency of gene knockdown was assessed by western immunoblot analysis and Sanger sequencing. Sensitivity of the isogenic cell lines to T-DM1, DM1 and neratinib was measured by growth inhibition sulforhodamine B assay. Furthermore, an in vivo T-DM1 CRISPR knockout screen using a focused library in MDA-MB-361 was carried out in NOD scid gamma mice. Bioinformatic analysis using model-based analysis of genome-wide CRISPR knockout was carried out to determine the gene knockouts enriched or depleted in response to T-DM1 treatment. These results identified potential genes implicated in resistance to T-DM1. Results: Growth inhibition assays revealed NUMA1 knockout may promote resistance to TDM1 in MDA-MB-361 cells, while SLC46A1 knockout did not appear to influence T-DM1 response. The validation of CRISPR-mediated MIEN1 knockout clones revealed off-target unintended excision of nearby genes. Similarly, investigation into IRF2BP2 was halted due to reduced HER2 expression in the clones. However, gene silencing suggests MIEN1 knockdown may promote sensitivity to T-DM1. The in vivo T-DM1 CRISPR screen revealed high library representation and identified potential candidate genes involved in T-DM1 sensitivity or resistance. Conclusion: Taken together, these candidate genes provide a foundation that further biomarker discovery can build upon for T-DM1 therapy in HER2-positive breast cancer.