The new Bruton's tyrosine kinase inhibitors SPA8007 and SPA8009 reduce stemness and invasiveness of patient-derived glioblastoma tumorspheres

Abstract

Glioblastoma (GBM), the most prevalent primary brain tumor, remains incurable due to the presence of cancer stem cells (CSCs), which can be isolated as tumorspheres (TSs) that exhibit classical characteristics of CSCs, including stemness and invasiveness. The significantly elevated expression of Bruton's tyrosine kinase (BTK) in GBM tissues identifies BTK as a potential therapeutic target in GBM. Consequently, ibrutinib, an FDA-approved BTK inhibitor for hematological malignancies, has been repurposed as a candidate for GBM treatment, demonstrating inhibitory effects on the proliferation, stemness, and invasiveness of GBM cell lines and TSs. However, its broad-spectrum activity targeting other Tec family kinases and members of the epidermal growth factor receptor family, poses potential life-threatening risks, necessitating the development of more selective alternatives. To address this shortcoming, we synthesized BTK-selective analogs (SPA1758, SPA1763, SPA8004, SPA8007, and SPA8009) specifically designed to target two hallmark features of GBM TSs: stemness and invasiveness. WST and ATP assays identified SPA8007 and SPA8009 as the most effective candidates with superior cytotoxic effects in TSs. Additionally, both SPA8007 and SPA8009 significantly inhibited neurosphere formation and reduced invasiveness in GBM TSs. Furthermore, SPA8007 demonstrated improved survival rates in a GBM xenograft mouse model and significantly reduced the expression of invasiveness markers, as observed in immunohistochemistry analysis. These findings highlight SPA8007 as a potential novel chemotherapeutic agent with high specificity and efficacy to enhance GBM therapy.