The microtubule-destabilizing agent AUS₀01 as a candidate for glioblastoma therapy.

3114 Background: The development of novel drugs targeting glioblastoma (GBM) is crucial due to the aggressive nature of this brain cancer, which often resists current treatments. Microtubules are a well-established target for cancer treatment, however there is a need to identify microtubule targeting agents (MTAs) with decreased toxicity and the ability to also retain efficacy in drug-resistant tumors. The novel MTA, AUS₀01 has been shown to impede the growth of 15 established glioma cell lines with a half-maximal inhibitory concentration in the range of 0. 04-0. 246uM. The goal of the current study was to explore its potential in GBM treatment. Methods: Co-cultures of primary Human brain endothelial cells, pericytes and astrocytes layered in an insert were applied for the assessment of Blood Brain Barrier (BBB) permeability. AUS₀01 efficacy against GBM was studied in vitro and in an ex vivo platform of advanced 3D cell culture systems. Differences in drug response were investigated between a Temozolomide (TMZ) -resistant human GBM cell line and parental cells. The ability of AUS₀01 to overcome taxane-associated drug resistance was explored in P-glycoprotein (P-gp) and βIII-tubulin over-expression models of drug resistance. A microelectrode array system was used to assess the electrical activity and functionality of human Pluripotent Stem Cell (hPSC) -derived midbrain and cortical neurons, upon dose escalation drug treatments followed by a full media change to allow the recovery period. Results: By employing a 3D Human BBB Model, we demonstrated that AUS₀01 is a good BBB permeability substrate. AUS₀01 prevents the growth of Parental Primary Patient-Derived GBM and Patient-Derived Glioma Lines enriched in Cancer Stem cells under 2D culturing conditions with high potency. To this end, we evaluated the effect of AUS₀01 in 3D spheroids originating from primary GBM patient-derived tumors that include all cells present in each patient’s primary tumor. AUS₀01 efficiently prevented the growth of these tumors, including cases where the standard of care, TMZ was not effective. Notably, 2D GBM cultures with acquired TMZ resistance retained sensitivity to the cytotoxic effects of AUS₀01. We also found that AUS₀01 is less prone to mechanisms of resistance to currently approved MTAs, including increased expression of the βIII-isotype of tubulin or the P-gp drug efflux pump. An in vitro neurotoxicity model revealed that functional damage caused by AUS₀01 was reversible, while paclitaxel-treated neurons suffered sustained neurotoxicity. Conclusions: Collectively, we show that AUS₀01 has the potential to circumvent significant limitations of clinically approved MTAs, including brain penetration, drug resistance and peripheral neuropathy, making it a promising approach for the treatment of GBM.