Abstract 2864: RET as a novel therapeutic target in breast cancer brain metastases

Brain metastases (BrM) are reported in approximately 10-30% of breast cancer patients with a poor median overall survival of 8.5 months. Current therapeutic strategies are mostly limited to surgery, chemotherapy and radiotherapy, making the study of novel druggable targets and brain-permeable small molecule inhibitors an urgent clinical need. Our lab recently reported a hybrid-capture exome RNA-sequencing study on 21 patient-matched pairs of primary breast tumors and brain metastases and identified upregulation of several receptor tyrosine kinases including RET in BrM. The ligand for RET is Glial Cell Derived Neurotrophic Factor (GDNF), which is released by astrocytes and microglia in response to neuroinflammation and brain tissue damage. Clinical BrM cell growth in ex vivo cultures was reduced by the multikinase inhibitor Cabozantinib, indicating a potential role for RET in the growth of BrM lesions. We hypothesize that the high level of brain-derived GDNF provides a permissive microenvironment for RET overexpressing breast cancer cells to form brain metastases, and will evaluate the therapeutic potential of RET targeted therapies for breast cancer BrM patients. Our preliminary data show that in RET-overexpressing (RET-OE) breast cancer cell models, 30-minute GDNF treatment induces RET phosphorylation at two distinct tyrosine residues, along with downstream signaling illustrated by ERK1/2, AKT, and STAT3 phosphorylation. This induction was effectively abrogated after shRNA-mediated RET knockdown. We employed a phospho-kinase array to identify novel downstream targets regulated by the GDNF-RET signaling axis, and found that GDNF induces phosphorylation of multiple targets (including p27 and RSK) in RET-OE cells. Interestingly, phosphorylation of multiple Src family kinases (Lck, Lyn, Fgr) was downregulated with GDNF treatment. We will further examine these downstream targets via reverse phase protein array (RPPA). GDNF triggers migration of RET-OE cell lines in both 2D wound scratch and 3D transwell migration assays. Additionally, we present results obtained through an ex vivo organotypic coculture system, in which RET-OE cells are cultured on top of 300-micron thick murine brain, kidney, or liver slices. RET overexpression increases colony formation compared with empty vector control, suggesting a role for RET signaling in the initial establishment of metastatic brain lesions. Differences in colonization between empty vector transfected and RET-OE cells remain minimal in kidney and liver sections, suggesting a role for RET specific to the brain microenvironment. We will examine RET-driven brain metastasis in vivo by employing RET-OE and RET-deficient cell lines in intracranial, intracardiac, and mammary fat pad injection. These models of BrM will be randomized to RET inhibitor or vehicle treatment to evaluate the efficacy of RET-targeted therapy in the setting of breast cancer brain metastasis. Citation Format: Simeng Liu, Geoffrey Pecar, Jagmohan Hooda, Jennifer M. Atkinson, Fangyuan Chen, Adrian V. Lee, Steffi Oesterreich. RET as a novel therapeutic target in breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2864.