A bruton's tyrosine kinase inhibitor reduces stemness and invasiveness of patient-derived glioblastoma tumorspheres
Glioblastoma (GBM), the most prevalent primary brain tumor, remains incurable due to the presence of cancer stem cells (CSC), which can be isolated as tumorspheres (TS) that exhibits 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 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 (EGFR) family, poses potential life-threatening risks, necessitating the development of more selective alternatives. To address this, BTK selective analogs (SPA1758, SPA1763, SPA8004, SPA8007, SPA8009) were synthesized with the aim of targeting two key characteristics 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 reduced the expression of invasiveness markers significantly, as observed in immunohistochemistry (IHC) analysis. These findings highlight SPA8007 as potential novel chemotherapeutic agents with high specificity and efficacy to enhance GBM therapy. Overall design: RNA-seq of patient-derived GBM TS, treated with BTK inhibitors for 72 hours.