Estrogen receptor fusions genes as drivers of endocrine resistance in breast cancer

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Anti-estrogen therapy is key to the prevention and treatment of estrogen receptor (ER, ESR1) positive breast cancer, but therapy resistance occurs in up to a half of cases resulting in incurable advanced disease. We recently reported recurrent ESR1 fusions in advanced endocrine-resistant breast cancers, with loss of ligand binding representing a canonical model of drug resistance. Intriguingly, cases with ESR1 fusions co-occurred with ESR1 base-pair mutations, likely pointing towards convergent evolution under prolonged anti-estrogen therapy. Evidence from our group and others suggest that ESR1 fusions are found in ~10-20% of advanced breast cancers, however, many ESR1 fusions harbor different 3’ partner genes, making detection challenging. To determine prevalence, we will comprehensively re-analyze the ESR1 genomic locus in data from metastatic breast cancers. Further, we have developed and optimized a target capture sequencing assay capable of comprehensively detecting ESR1 fusions in both tumor biopsies and ctDNA. We have identified endogenous ESR1 fusions that are expressed in two patient derived xenografts (PDX) (ESR1-LPP and ESR1-NFkB) and in an ER-positive breast cancer cell line MDA-MB-435 (ESR1-SYNE1). We will express ESR1 fusions in recently generated patient-derived organoids (PDO) which show ER expression and response to estrogen that is equal or greater than traditional cell lines. Intriguingly, different ESR1 fusions show stark differences in activity, implicating a role for the 3’ gene partner in modulating activity of the ESR1 N terminal domain (NTD). The NTD is an intrinsically disordered protein within the transactivation domain, and using a novel integrative-biophysics approach, we recently reported that while it is mostly disordered, it can adopt an unexpectedly compact conformation which in turn directs interaction with coregulators. In this proposal we seek to test the hypothesis that ESR1 fusions are key mediators of endocrine-resistance and breast cancer mortality. We will employ EnRich to determine the prevalence of ESR1 fusions (and point mutations) in national and international clinical cohorts of advanced endocrine resistant breast cancer and monitor when they arise during therapy using longitudinal collection of plasma and ctDNA analyses. We will use patient-derived organoid models of breast cancer to compare and contrast the activity and function of ESR1 fusions and point mutations relative to wild- type ER and translate this into in vivo patient derived organoid xenograft (PDOX) and (PDX) models to assess endocrine resistance. We will study mechanism of action, including the effects on DNA binding and transcriptional activity, and the role of the 3’ partner gene in any gain of function activities. Finally, we will examine the structure-function of ESR1 fusions focusing on the NTD and determining the altered structure in the context of the fusion partner before assessing the function of specific NTD amino acids and how they change structure and subsequent target gene expression. We believe our multi-disciplinary studies will provide mechanistic insight to aid in the development of new therapies against breast cancers with ESR1 fusions.