Concurrent mutations associated with trastuzumab-resistance revealed by single cell sequencing

Purpose: HER2-positive breast Cancer patients benefit from HER2-targeted therapies, among which the most commonly used is trastuzumab. However, acquired resistance typically happens within one year. The cellular heterogeneity of it is less clear.

Methods: Here we generated trastuzumab-resistant cells in two HER2-positive breast Cancer cell lines, SK-BR-3 and BT-474. Cells at different time points during the resistance induction were examined by exome Sequencing to study changes of genomic alterations over time. Single cell-targeted Sequencing was also used to identify resistance-associated concurrent mutations.

Results: We found a rapid increase of copy number variation (CNV) regions and gradual accumulation of single nucleotide variations (SNVs). On the pathway level, non-synonymous SNVs for SK-BR-3 cells were enriched in the MAPK signaling pathway, while for BT-474 cells they were enriched in mTOR and PI3K-Akt signaling pathways. However, all of the three signaling pathways were in the downstream of the HER2 kinase. Putative trastuzumab-resistance-associated SNVs included AIFM1 P548L and ERBB2 M833R in SK-BR-3 cells, and ADAMTS19 V451L, OR5M9 D230N, COL9A1 R627T, and ITGA7 H911Q in BT-474 cells. Single-cell-targeted Sequencing identified several concurrent mutations. By validation, we found that concurrent mutations (AIFM1 P548L and IL1RAPL2 S546C in SK-BR-3 cells, MFSD11 L242I and ANAPC4 E16K in BT-474 cells) led to a decrease of trastuzumab sensitivity.

Conclusion: Taken together, our study revealed a common pathway level trastuzumab-resistance mechanism for HER2-positive breast Cancer cells. In addition, our identification of concurrent SNVs associated with trastuzumab-resistance may be indicative of potential targets for the treatment of trastuzumab-resistant breast Cancer patients.