Targeting breast cancer brain metastasis through novel combination of FDA-approved orally active BBB-permeable RET and tGLI1 inhibitors

Breast cancer is the most diagnosed cancer in American women, and breast cancer brain metastasis (BCBM) exhibits the worst prognoses, with a life-expectancy averaging 8 months. The short survival stems largely from the lack of effective treatment. Major challenges remain, including the limited number of actionable targets for targeted therapy and the scarcity of effective blood-brain barrier (BBB)-permeable drugs. Hence, identifying new therapeutic targets and BBB-permeable agents is urgently needed. Our lab identified the Re arranged during T ransfection receptor tyrosine kinase (RET) as a potential BCBM target. RET inhibitors Pralsetinib and Selpercatinib, FDA-approved for lung and thyroid cancers, exhibit intracranial activity. We previously reported overexpression of RET in BCBM and found that RET monotherapy significantly suppressed BCBM incidences in mouse models but failed to halt the progression of established BCBM. Thus, we sought to identify potential secondary pathways contributing to the observed resistance to RET monotherapy. Through pathway correlation analysis using publicly available breast cancer datasets, T runcated Gli oma-Associated Oncogene Homolog 1 (tGLI1) was identified as positively correlated with RET. tGLI1 is an alternatively spliced, gain-of-function variant of the GLI1 transcription factor that is overexpressed in and promotes BCBM. Consequently, we examined IHC-stained patient samples of matched primary and BCBM tumors and observed concurrent elevation in RET activation and tGLI1 expression in over 80% of BCBM tumor samples. Western blot analysis also revealed increased activated RET and tGLI1 levels in brain-tropic breast cancer cell lines compared to parental counter parts. Datamining analysis with patient-derived datasets indicated significantly higher RET and tGLI1 co-activation in patients with brain metastasis, associated with worse clinical outcomes. Finally, tGLI1-knockdown via antisense oligonucleotides sensitized resistant BCBM subline to Pralsetinib. Thus, we hypothesized that RET and tGLI1 functionally crosstalk to mediate BCBM progression and co-targeting them with FDA-approved orally active BBB-permeable inhibitors overcomes the RET monotherapy resistance and synergistically inhibits BCBM. In vitro cell proliferation assays using tGLI1-overexpressing BCBM sublines demonstrated increased Pralsetinib resistance but enhanced sensitivity to tGLI1 inhibitor, Ketoconazole. Pralsetinib+Ketoconazole combination treatment yielded synergistic inhibition of BCBM cell viability and migration in vitro . The underlying mechanism is under analysis through RNA-sequencing, and in vivo efficacy of the Pralsetinib+Ketoconazole combination therapy on BCBM remains to be validated with mouse models. Together, we report a novel mechanism and combination treatment for BCBM patients.