Bcl‐2/Bcl‐xL inhibition predominantly synergistically enhances the anti‐neoplastic activity of a low‐dose CUSP9 repurposed drug regime against glioblastoma. (30th July 2019) - Halatsch, Marc‐Eric
Abstract : Background and Purpose: Drug repurposing represents a promising approach to safely accelerate the clinical application of therapeutics with anti‐cancer activity. In this study, we examined whether inhibition of the anti‐apoptotic Bcl‐2 family proteins Bcl‐2 and Bcl‐xL enhances the biological effects of the repurposed CUSP9 regimen in an in vitro setting of glioblastoma. Experimental Approach: We applied 3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyltetrazolium bromide assays to assess cellular proliferation. Annexin V/propidium iodide and tetramethylrhodamine, ethyl ester staining were used to examine apoptosis. Western blotting, RT‐PCR, and specific knockdown experiments using siRNA were employed to examine molecular mechanisms of action. Key Results: Bcl‐2/Bcl‐xL inhibition exerted synergistic anti‐proliferative effects across established, primary cultured, and stem‐like glioblastoma cells when combined with CUSP9 which had been reduced to only one tenth of its proposed original concentration (CUSP9‐LD). The combination treatment also led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, CUSP9‐LD counteracted ABT263‐mediated up‐regulation of Mcl‐1. Silencing of Mcl‐1 enhanced ABT263‐mediated apoptosis which indicates that down‐regulation of Mcl‐1 is crucial for the induction of cell death by the combination treatment. Conclusion and Implications: These data suggest that Bcl‐2/Bcl‐xL inhibition enhances the susceptibility of glioblastoma cells towards CUSP9, allowing dramatic dose reduction and potentially decreased toxicity when applied clinically. A clinical trial involving the original CUSP doses (CUSP9v3) is currently ongoing in our institution (NCT02770378). The Bcl‐2/Bcl‐xL inhibitor ABT263 is in clinical trials and might represent a valuable adjunct to the original CUSP.