Background: We recently reported two ESR1 fusions (ESR1-YAP1 and ESR1-PCDH11X) that drive endocrine therapy (ET) resistance and metastasis in estrogen receptor positive (ER+) metastatic breast cancer (MBC) (PMC6171747). Here we report additional ESR1 fusions with diverse C-terminal partner genes – ESR1-DAB2, ESR1-GYG1, ESR1-SOX9, ESR1-ARNT2, ESR1-PCMT1 and ESR1-ARID1B. Their functional characteristics and effects on kinase biology will be described.

Methods: ESR1 fusions were identified by RNA-seq in MBC using ChimeraScan to detect fusion junction reads. ESR1 fusion cDNA constructs were expressed in ER+ breast cancer cell lines. An alamarBlue assay assessed cell proliferation. RNA-seq followed by mRNA-qPCR assessed the transcriptional properties. A scratch wound assay assessed cell motility. A Kinase Inhibitor Pulldown (KIP) mass spectrometry-based assay was conducted to examine ESR1 fusion-driven druggable kinases.

Results: All fusions retained the first 6 exons (e6) of ESR1, fused in-frame to C-terminal sequences of diverse partner genes. In addition to ESR1-YAP1 and ESR1-PCDH11X fusions, ESR1-SOX9 and ESR1-ARNT2 drove fulvestrant-resistant growth. RNA-seq revealed fusion-specific transcriptional signatures indicating enrichment of estrogen responsive and epithelial-to-mesenchymal transition (EMT) pathways that were confirmed by mRNA-qPCR. Transcriptionally active ESR1 fusions also promoted hormone-independent cell motility. KIP profiling demonstrated an increase in protein abundance of multiple receptor tyrosine kinases including RET and insulin like growth factor 1 receptor (IGF1R) in T47D cells expressing active ESR1 fusion proteins, both of which have been previously implicated in driving ET and MBC. Both proteins were also elevated in a patient-derived xenograft naturally harboring the ESR1-YAP1 fusion. Combinatorial inhibition of RET and IGF1R significantly suppressed ESR1 fusion-driven cell growth in vitro. Neither transcriptional activation nor kinase upregulation was observed in other ESR1-e6 fusions. Lastly, we report recurrent examples of specific known active ESR1 fusions: ESR1-PCDH11X (3 examples) (TEMPUS unpublished data) and ESR1-ARNT2 (2 examples) (PMC6872491).

Conclusion: A subset of ESR1-e6 fusions are active, drive ET resistance and metastasis/EMT in experimental models. When ESR1 is fused in-frame with another transcriptional regulator, activity is predictable. However, recurrent partners that have non-transcriptional roles (PCDH11X) suggests cDNA expression-based functional screens should be continued. A common pattern of kinase activation indicates that ESR1 fusion specific therapeutic strategies could be devised.

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