Project Summary Up to 1 in 8 women in the United States will develop invasive breast cancer in their lifetime, with 70-80% of cases being estrogen receptor positive (ER+). These cancers rely on hormonal signaling, allowing for treatment with endocrine therapy (ET), including inhibitors of the aromatase enzyme, lowering estrogen levels, or targeting of the ER itself using modulators and degraders. While these therapies reduce mortality by up to 40%, the disease often recurs and progresses to metastasis as a result of ET resistance. The main mechanism of resistance is the acquisition of estrogen receptor gene (ESR1) mutations. These mutations confer constitutive activation to ER, exhibiting reduced sensitivity to endocrine therapies, and are found in up to 38% of metastases. There is currently no approved therapy specific to patients harboring these mutations, which should be a clinical priority given their prevalence in metastatic disease. The Fuqua laboratory has identified distinct transcriptional changes between primary and metastatic site tumors, and between therapy-resistant ESR1 mutants and wild type ER cells. In a study of 900 metastatic breast cancer patients, ESR1 mutations were mutually exclusive with other ER+ breast cancer mutations including Myc and MAPK pathways at the DNA level, but current data in the Fuqua laboratory and others indicates ESR1 mutations activate these same growth and proliferation pathways at the transcriptional level. Given the ESR1 mutant’s proclivity for metastasis and therapy-resistance, this proposal’s central hypothesis is ESR1 mutations generate a transcriptionally reprogrammed phenotype promoting both metastasis and ET resistance in breast cancer. The rationale for this proposal is that identifying transcriptional changes in ESR1 metastases and resistant tumors, improves understanding of ESR1 mutant disease evolution. By targeting transcriptional changes occurring during this evolution, patients can remain on ET longer and extend metastasis-free survival. To test this central hypothesis, we propose two Specific Aims. Aim 1: What clonal and transcriptional changes accompany metastasis in ESR1 mutant-containing tumors subjected to ET pressure? This Aim uses computational methods to identify clonal evolution and transcriptional drivers of metastasis using single cell RNA/DNA-seq and ChIP-seq. Transcriptional drivers will be validated using FDA-approved inhibitors of these candidates in combination with ET using ex vivo assays. Aim 2: Does ET enhance the acquisition of ESR1 mutations, promoting subclonal evolution to drive therapy resistance? This Aim is addressed by developing models with ESR1 mutations acquired during long-term ET and utilizing computational biology via a combination of single cell DNA-seq and RNA-seq to determine the clonal evolution and transcriptional driver candidates of ET resistance. Driver candidates will be validated using selective inhibitors in combination with ET in vitro and in vi...