Amplification of the HER2 oncogene results in breast cancers with an aggressive clinical course. Therapeutic agents targeting the HER2 receptor provide objective responses in some patients and have improved overall survival. However, many tumours are refractory to the HER2 blockade or acquire resistance during treatment. Mechanisms of resistance to these agents are poorly understood, particularly for the antibody-drug conjugate trastuzumab emtansine (T-DM1), which is comprised of the potent microtubule inhibitor mertansine (DM1) conjugated to trastuzumab.
This project utilised CRISPR-mediated functional genomics to identify genetic determinants of sensitivity and resistance to T-DM1 in breast cancer. A panel of human breast cancer cell lines showing varying patterns of sensitivity to the HER2 blockade was studied. HER2 amplification and protein expression were assessed by FISH and immunoblotting, respectively. Drug sensitivity was characterised by CellTiter-Glo and clonogenic cell survival assays. Streptococcus pyogenes Cas9 and the whole-genome single guide RNA libraries (GeCKOv2) were stably transduced using lentiviral vectors. The resulting genome-scale knockout libraries were functionally validated using 6-thioguanine, for which resistance-conferring mutations are known. Library complexity and treatment-induced sgRNA enrichment were assessed by next-generation sequencing (Illumina NextSeq500). Saturation-scale knockout libraries were used in screens with T-DM1 and free DM1.
The cell lines displayed patterns of sensitivity to T-DM1, trastuzumab, lapatinib and neratinib reflective of clinical response rates. Genome-scale knockout libraries showed resistance to 6-thioguanine following drug challenge. Deep sequencing of treated MDA-MB-453 libraries revealed enrichment of mutations in the 6-thioguanine sensitivity gene HPRT1. MDA-MB-453 and MDA-MB-361 libraries showed resistance to T-DM1 but not to DM1 after eight and thirteen weeks of escalating drug treatment, respectively. Deep sequencing revealed a number of genes, including SLC46A3, KEAP1, TRIAP1, ERLIN2, and CUL3 that are potential regulators of T-DM1 sensitivity. The genome-wide knockout libraries developed in this study are anticipated to enable identification of genetic mechanisms of treatment resistance, potentially leading to more personalised management of HER2-positive breast cancer.