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 patient outcome. To identify kinase mutations associated with resistance to endocrine therapy, we performed deep-kinome sequencing on 4 ER+/HER2– breast tumors that retained high Ki67 scores (14.8 to 24.5%) after two weeks of pre-surgical therapy with letrozole. Genomic DNA from the surgically excised tumors was deep sequenced using a capture approach with oligonucleotides hybridizing to 612 genes, including 517 kinases with ≥300x coverage.

All 4 tumors contained an activating mutation in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K. One tumor also contained a novel D189Y (565G>T) somatic mutation in the SH2 domain of LYN, a member of the Src family kinases (SFKs; variant frequency of 8%). 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 ER+ MCF-7 cells with acquired resistance to estrogen deprivation. LYN mRNA expression was also upregulated in these cells. 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%).

Finally, we investigated the role of D189Y LYN in antiestrogen-resistant breast cancer using ER+/PIK3CA mutant 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), FAK, IGF-IR, EGFR, STAT3, AKT and MAPK. LYND189Y increased phosphorylation of FAK, EGFR, and IRS-1 as compared to LYNWT. Although stable transduction of either LYNWT or LYND189Y accelerated MCF-7 cell growth in estrogen-depleted medium, the mutant was more potent than LYNWT at inducing this effect. Further, LYND189Y overexpression rendered treatment with the ER downregulator fulvestrant or the PI3K inhibitor BKM120 less effective, as well as rescued apoptosis induced by BKM120. These results suggest, first, that LYN plays 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 Format: Emily M. Fox, Justin M. Balko, Carlos L. Arteaga. Deep kinome sequencing identifies a novel D189Y mutation in the Src family kinase LYN as a possible mediator of antiestrogen resistance in ER+ breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2013-3563