P18.01.A ACTIVATION OF HSP27 REPRESENTS A POTENTIAL MECHANISM OF RESISTANCE AGAINST THE DRUG REPURPOSING APPROACH CUSP9V3 IN MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma represents one of the most common brain tumors in children. We previously observed that the drug repurposing approach CUSP9v3 has a strong antineoplastic activity against medulloblastoma cell models in vitro. In this study, phosphoprotein profiling served at identifying phosphorylation of HSP27 as a potential mechanism of resistance against CUSP9v3. We performed a preclinical testing of a combined treatment with suboptimal doses of CUSP9v3 and repurposed inhibitors of HSP27 in vitro. MATERIAL AND METHODS The combinatorial approach was tested on established and primary cultured medulloblastoma cells using MTT assays. BLISS analysis was performed for thorough evaluation of the nature of the drug-drug interaction. Spheroids were used to examine the effects of the combination treatment in a 3-dimensional setting. Annexin V/PI staining followed by flowcytometric analysis was used to detect pro-apoptotic effects. Western blot analyses and knockdown experiments with siRNA were performed for molecular analysis. Extracellular flux analyses were performed to assess metabolic changes. RESULTS Treatment with CUSP9v3 at reduced dosage in combination with HSP27 inhibition led to a predominantly synergistic antiproliferative effect on medulloblastoma cells showing enhanced phosphorylation of HSP27 in response to CUSP9v3. In the 3-dimensional setting, the combination treatment resulted in a significantly enhanced inhibitory effect on the growth of medulloblastoma spheroids. On the mechanistic level, the combination treatment led to a significantly increased fraction of Annexin-V-positive (apoptotic) cells. In addition, specific knockdown of HSP27 by siRNA empowered the antineoplastic effect of CUSP9v3 which reaffirms this potential mechanism of resistance. On the metabolic level inhibition of HSP27 combined with CUSP9v3 leads to suppression of OXPHOS. CONCLUSION Treatment with CUSP9v3 leads to enhanced phosphorylation of HSP27 across a variety of medulloblastoma cell models. Inhibition of HSP27 in combination with CUSP9v3 leads to a predominantly synergistic inhibitory effect on the cell viability of a broad panel of medulloblastoma cells in 2D- and 3D-settings. This effect is associated with a metabolic reprogramming.