Standard therapy boosts glioblastoma stem-like cell resistance to apoptosis and confers sensitivity to BH3 mimetics

Glioblastoma (GBM) is a lethal brain tumor that almost invariably recurs within 15 months after a standard treatment, which includes surgery, chemotherapy with temozolomide (TMZ), and radiotherapy (IR). GBM is thought to arise from stem-like cells (GSCs) that inherently resist therapies and can evolve under therapeutic pressure, ultimately driving tumor recurrence. Notably, in individual treatment-naïve GBMs, multiple GSC subpopulations can coexist, displaying different genetic and phenotypic profiles, potentially conferring varying levels of intrinsic treatment resistance. These traits may be selected or modulated by treatments, contributing to the therapeutic refractoriness observed in relapsing GBM. Investigating the mechanisms behind GSC resistance is essential to uncovering vulnerabilities that could be targeted in treating recurrences. Due to the rarity of surgically removed recurrent GBMs and their limited availability for study, we developed an original ex vivo protocol to select GSCs that are resistant to conventional therapies, ideally representing the GSC subclones driving recurrence. Leveraging treatment-naïve GBMs removed with conventional or ultrasonic aspiration, we collected bona fide whole tumor cell populations. These were then subjected to chemo- or radio- therapies simulating patient treatment, while maintained in culture conditions that preserve GSC subclonal heterogeneity. From each GBM, we established multiple long-term GSC-enriched cultures (neurospheres, NS), referred to as “res-GSC families”. Each family included at least one member selected by TMZ or IR (TMZ-NS and IR-NS respectively) alongside an untreated control (CTRL-NS). Genetic and functional analysis revealed that resistant GSC family members exhibited properties mechanistically associated with the emergence of secondary therapeutic resistance. Secondary resistance to TMZ was linked to the onset of genetic alterations in mismatch repair genes such as MSH6 and MSH2, while secondary resistance to IR involved adaptive mechanisms sustaining DNA double-strand break repair by homologous recombination, including increased RAD51 expression. Overall, therapy-selected 4 GSCs displayed cytogenetic changes, increased stem cell frequency and tumor-initiating ability, and altered growth factor receptor expression, including increased EGFR and MET activity, two receptors known to support GSC survival and therapeutic resistance. Notably, secondary resistant GSCs exhibited an imbalance in BH3 family protein expression, favoring prosurvival proteins, partly driven by EGFR activity. Pharmacological inhibition of the prosurvival protein BCL-xL using BH3 mimetics restored this balance, effectively inducing apoptosis in resistant GSCs. This evidence paves the way for further investigations with a view toward clinical translation.