Studying synergistic effects of temozolomide and vistusertib in glioblastoma cells
TL;DR
Targeting the mTORC2/AKT/BABAM1 axis with vistusertib worked synergistically with TMZ in GBM by impairing the BRCA1-A complex and HR repair and sustained tumour suppression in both 2D and 3D models.
Abstract
Glioblastoma (GBM) is a highly aggressive malignant brain tumour with an exceptionally poor prognosis, largely driven by intrinsic resistance to standard-of-care chemotherapy with temozolomide (TMZ). Aberrant activation of the mechanistic target of rapamycin complex 2 (mTORC2) signalling plays a crucial role in GBM cell survival and intersects with the DNA damage response (DDR) system. Upstream phosphorylation of BRISC and BRCA1-A complex member 1 (BABAM1) by AKT drives its nuclear translocation, which initiates homologous recombination (HR) repair of double-strand breaks (DSBs) induced by TMZ, leading to promoting cell survival and chemoresistance. Vistusertib (AZD2014) is a dual mTORC1/2 inhibitor, but its potential to act synergistically with TMZ by disabling this specific DSB repair machinery remains unclear. This study aimed to investigate the synergistic effects of TMZ and vistusertib in human glioblastoma (U87MG) cells by targeting the mTORC2/AKT/BABAM1-mediated DSB repair axis. U87MG cells were evaluated in 2D monolayers and 3D multicellular spheroids. Metabolic viability, synergistic effects and cell death dynamics were assessed using MTS assays, Zero Interaction Potency (ZIP) synergy mapping, flow cytometry, and Calcein AM/Propidium Iodide live-dead tracking. Signalling cascades were mapped via Western blot and immunofluorescence. In 2D cultures, combining 1 µM vistusertib with 200 µM TMZ resulted anti-proliferative synergy (ZIP score > 10) and significantly increased apoptotic populations compared to monotherapies. Mechanistically, vistusertib inhibited the signalling cascade, suppressing the phosphorylation of AKT (Ser473) and its downstream substrate, BABAM1 (Ser29). Immunofluorescence confirmed that this blockade disrupted the early nuclear scaffolding and recruitment of the BRCA1-A repair complex. Unlike the late-stage RAD51 inhibitor B02, which allowed upstream repair proteins to accumulate at focal sites, vistusertib suppressed an upstream DDR machinery. In the 3D multicellular model, the combination treatment significantly reduced overall spheroid volumes and suppress necrotic death zone expansion. In conclusion, targeting the mTORC2/AKT/BABAM1 axis with vistusertib worked synergistically with TMZ in GBM by impairing the BRCA1-A complex and HR repair. This combination sustained tumour suppression in both 2D and 3D models, highlighting the critical importance of 3D platforms in preclinical oncology.