ABSTRACT Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. “Drug-tolerant persisters” (DTPs), a sub-population of cancer cells that survive via reversible, non-genetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKIs) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single cell RNA-sequencing reveal that HER2+ breast cancer cells cycle stochastically through a “pre-DTP” state, characterized by a G 0 -like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting show that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation. STATEMENT OF SIGNIFICANCE DTPs are implicated in resistance to TKIs, other targeted therapies, and chemotherapy, but their ontogeny and vulnerabilities remain unclear. We find that HER2 TKI-DTPs emerge from stochastically arising primed cells (“pre-DTPs”) that preferentially engage either of two distinct transcriptional programs upon TKI exposure. Our results provide new insights into DTP ontogeny and identify potential therapeutic vulnerabilities.

Ontogeny and Vulnerabilities of Drug-Tolerant Persisters in HER2+ Breast Cancer