Abstract P6-03-06: <i>ESR1</i> mutant alleles induce differential sensitivity to combination endocrine and targeted therapies
Authors: Sunil Kumar, Gaorav P. Gupta
Journal: Cancer Research (2020)

Abstract Background: Estrogen receptor alpha (ESR1) mutations in estrogen receptor positive (ER+) metastatic breast cancer drive the development of acquired resistance to endocrine therapy. However, ESR1 mutation status does not currently guide treatment decisions due to a lack of comparative data on sensitivity to clinically approved or investigational combination therapies. The goal of our study was to quantify how the two most common ESR1 mutations, p.D538G and p.Y537S, impact therapeutic sensitivity to different types of endocrine therapy without or with targeted inhibition of cyclin-dependent kinase 4 and 6 (CDK4/6), mammalian target of rapamycin (mTOR) or phosphoinositide 3-kinase (PI3K). A preclinical competition-based drug treatment platform was developed to monitor expansion of ESR1 mutant subclones as a quantitative measure of therapeutic resistance. Methods: CRISPR/Cas9-based targeting was used to generate two ESR1 mutant (p.D538G or p.Y537S) subclones of the PIK3CA mutation positive ER+ breast cancer cell line, T-47D. Mutant cellular mixtures were generated by spiking-in one or both ESR1 mutant subclones into the parental T-47D cell population at approximately 10% mutant allele frequency (MAF). The mixtures of wild-type and mutant cells were grown for a duration of five weeks in the presence of clinically approved endocrine therapy (50nM Fulvestrant or 1µM Tamoxifen) alone or in combination with inhibitors of CDK4/6 (Palbociclib), mTOR (Everolimus), or PI3K (Alpelisib). Cells were sampled every week to quantify changes in overall cell numbers and in ESR1 wild-type and mutant allele frequencies by allele-specific digital PCR assays (Bio-Rad). Cumulative population doublings of wild-type and mutant cells were calculated. Two-tailed t-tests were performed to assess statistical significance. Results: Tamoxifen and fulvestrant treatment of mixed cell populations resulted in clonal selection for both p.D538G and p.Y537S ESR1 mutant alleles, relative to the wild-type allele. However, mixing of both mutant alleles revealed a greater resistance to fulvestrant induced by the p.Y537S allele, which was not observed for tamoxifen. Combination treatment with fulvestrant and inhibitors of CDK4/6, mTOR, or PI3K resulted in greater than 3-fold reduction in the rate of clonal selection for ESR1 p.D538G, relative to fulvestrant alone. In contrast, clonal expansion of ESR1 p.Y537S expressing cells exposed to fulvestrant was only attenuated by co-treatment with a CDK4/6 inhibitor, and not impacted by inhibitors of either mTOR or PI3K. Thus, cellular mixtures containing both p.D538G and p.Y537S subclones became overtaken (i.e., &amp;gt;90% MAF) by p.Y537S expressing subclones within five weeks of treatment with either fulvestrant plus everolimus or fulvestrant plus alpelisib. Conclusions: A preclinical competition-based model of metastatic breast cancer reveals ESR1 mutant allele-specific differences in therapeutic susceptibility. The ESR1 p.Y537S allele induces greater resistance to fulvestrant, fulvestrant plus everolimus, and fulvestrant plus alpelisib, relative to the ESR1 p.D538G allele. In contrast, ESR1 p.Y537S and ESR1 p.D538G alleles exhibit comparable sensitivity to tamoxifen and fulvestrant plus palbociclib. Clinical validation of these findings may warrant investigation of novel treatment strategies to overcome therapeutic resistance induced by the ESR1 p.Y537S allele. Citation Format: Sunil Kumar, Gaorav P. Gupta. ESR1 mutant alleles induce differential sensitivity to combination endocrine and targeted therapies [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-06.