Estrogen receptor-positive (ER+) breast tumors adapt to hormone deprivation and acquire resistance to aromatase inhibitors (AIs). The proliferative rate of tumor cells measured by Ki67 after short-term treatment with an AI has been proposed as a surrogate endpoint of long term outcome. The purpose of this study was to identify kinase mutations associated with resistance to endocrine therapy as identified by a high Ki67 score after two weeks of pre-surgical therapy with letrozole. We performed deep-kinome sequencing on 4 ER+/HER2– breast tumors that retained high Ki67 scores (14.8 to 24.5%) following short-term letrozole treatment. Genomic DNA from the surgically excised tumors was deep sequenced using a capture approach with 120-bp biotinylated oligonucleotides hybridizing to 612 genes, including 517 kinases with ≥300x coverage.

All 4 tumors contained a ‘hot spot’ mutation in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K. One tumor also contained a novel D189Y somatic mutation in LYN, a member of the Src family kinases (SFKs; variant frequency of 8%). D189Y (565G>T) is located in the LYN SH2 domain. Reverse-phase protein array (RPPA) analysis available in 10 tumors in this study revealed a significant correlation (p = 0.006) between Y416 P-Src (which detects all SFKs) and a high post-letrozole Ki67 score. A siRNA screen targeting 779 kinases identified LYN as one of the top hits whose knockdown significantly reduced growth of MCF-7 breast cancer cells with acquired resistance to estrogen deprivation. We next analyzed the Cancer Genome Atlas (TCGA) SNP data to detect copy number changes for 444 tumors where corresponding gene expression data were available. Copy number increases (>0.8 log2 ratio over normal matched DNA) in LYN were present in approximately 10% of breast cancers, with the highest copy number gains observed in luminal B tumors. In contrast, other SFKs showed less frequent copy number increases (YES1 <1%, SRC 2%, FYN 1%). Analysis of the 550 breast cancers in the TCGA identified four additional LYN mutations (E159K, K188N, G418R, A503D), with the first two located in the SH2 domain.

Finally, we investigated the role of D189Y LYN in endocrine-resistant breast cancer using ER+ MCF-7 cells transduced with GFP (control), wild-type (WT) LYN, or D189Y LYN vectors. Overexpression of WT or mutant LYN resulted in increased phosphorylation of Src (at Y416), IGF-IR, EGFR, STAT3, AKT, and MAPK. D189Y LYN overexpression also induced phosphorylation of IRS-1. Although stable transduction of either WT or D189Y LYN accelerated MCF-7 cell growth in estrogen-depleted medium, the mutant was more potent than WT LYN at inducing this effect. Further, D189Y LYN overexpression rendered treatment with the ER downregulator fulvestrant or the PI3K inhibitor BKM120 less effective. These results suggest, first, that LYN may play a role in escape from estrogen deprivation in a subset of ER+ breast cancers. Second, ER+ breast cancers harbor multiple molecular alterations capable of mediating hormone-independent growth.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-04.