Understanding and overcoming resistance of breast cancer brain metastases to RET inhibition - Project Abstract Breast cancer brain metastasis (BCBM) is associated with poor prognoses and dismal survival of 4-6 months following diagnosis. Management of BCBM remains a major clinical challenge due to inadequate understanding of the mechanisms underlying BCBM and the lack of drugs with effective blood-brain barrier (BBB) penetration. Thus, there is an urgent unmet need to identify actionable drug targets for BCBM and develop novel therapies for treating patients with BCBM. Rearranged during transfection (RET) receptor tyrosine kinase is hyperactivated in thyroid and non-small cell lung carcinomas (NSCLC); two RET inhibitors, selpercatinib and pralsetinib received FDA approval in 2020 for treating thyroid cancer and NSCLC. Both RET inhibitors reduced brain metastases in the NSCLC clinical trials; however, their efficacy in BCBM is still unknown. Since it has been reported that RET is overexpressed in BCBM and RET inhibitors (selpercatinib and pralsetinib) cross the BBB, we conducted preliminary studies and found that selpercatinib significantly reduced BCBM incidence when administered one day following intracardiac inoculation of tumor cells. However, selpercatinib did not significantly suppress progression of developed BCBM in our treatment mouse model, which suggests the need to identify pathways/genes that confer the resistance of established BCBM to RET inhibition. To identify pathways co- activated with RET, we analyzed publicly available breast cancer datasets and found correlations between RET and truncated glioma-associated oncogene homolog 1 (tGLI1), a known mediator of BCBM progression. Through chemical screens followed by preclinical validations, a previous study identified FDA-approved antifungal ketoconazole (KCZ) selectively inhibited tGLI1-driven BCBM in vivo with excellent BBB permeability, leading to a window-of-opportunity clinical trial with BCBM. Our preliminary studies further revealed that (i) activated RET and tGLI1 are preferentially co-overexpressed in BCBM tissues compared to matched primary tumors, (ii) tGLI1 overexpression enhances resistance of brain-tropic breast cancer cells to RET inhibitors in vitro, (iii) tGLI1 knockdown sensitizes brain-tropic breast cancer cells to RET inhibition, and (iv) RET inhibitors synergize with KCZ in vitro. Based on these findings, we hypothesize that tGLI1 mediates BCBM resistance to RET inhibition, and dual targeting of RET and tGLI1 synergistically inhibits BCBM. In this R21 project, we will determine the role of tGLI1 in BCBM resistance to RET monotherapy (Aim 1), determine if dual targeting of RET and tGLI1 synergistically inhibits BCBM using intracardiac injection mouse model, and elucidate the pathway crosstalk between RET and tGLI1 (Aim 2).
