Preclinical evaluation of the novel, potent diarylisoxazole resorcinol HSP90 inhibitor NVP-AUY922 in adult and pediatric glioblastomas

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Introduction. The dismal prognosis of glioblastoma (GB) in adult and pediatric population indicates the urgent need for new therapies. Heat shock protein 90 (HSP90) inhibitors induce proteasome-mediated degradation of several client proteins (eg. ERBB2, CDK4, C-RAF) involved in all of the cancer hallmark characteristics. Our new, potent diarylisoxazole amide resorcinol HSP90 inhibitor, NVP-AUY922 has just entered Phase I clinical trials in adults. NVP-AUY922 is devoid of the limitations (eg. cumbersome formulation, extensive metabolism) of 17-AAG (17-allylamino-17-demethoxygeldanamycin). Objective. To perform in vitro and in vivo evaluation of NVP-AUY922 in both adult and pediatric GB. Material and methods. In vitro growth inhibition of adult (U87MG, SF268) and pediatric (SF188, KNS42) GB cell lines was determined by sulforhodamine B assay. Viable and dead (trypan blue-positive) cells were analysed by counting (hemacytometer), cell cycle analysis (FACS), and apoptosis analysis (PARP cleavage in western blot, WB). The molecular signature of HSP90 inhibition, comprising depletion of client proteins and induction of HSP72 was analysed by WB. Results. Despite different origins and different genetic backgrounds (eg. EGFR, mutant P53, PTEN), all GB cell lines were sensitive to both 17-AAG and NVP-AUY922, with a higher potency (2.5-fold) for NVP-AUY922. IC₅₀ values for 17-AAG were 13.9±2.0, 12.4±0.6, 11.2±0.6, 29.7±2.3nM and for NVP-AUY922 were 7.8±1.2, 6.1±1.5, 6.6±2.5, and 6.6±1.3nM in U87MG, SF268, SF188 and KNS42, respectively. After 48hr of NVP-AUY922 or 17-AAG treatment, a cytostatic effect was observed with a decrease in S phase. After 72hr, cell growth recommenced in adult cells. However, increased cell death occurred in both the pediatric cell lines when treated with NVP-AUY922, compared to untreated controls. This was only observed in KNS42 cells with 17-AAG. Cell death was associated with a sub-G1 phase and cleaved PARP, both indicative of apoptosis. The HSP90 client proteins (eg. ERBB2, IGF1R, C-RAF, CDK4, AKT and mutant p53) were depleted from 8-16hr, and recovered at 48-72hr. Inhibition of AKT and ERK was maintained only in the pediatric cell lines undergoing cell death, suggesting a possible role of concomitant inhibition of these 2 pathways in the pro-apoptotic effect of HSP90 inhibition. HSP72 and HSP90 levels were induced. Daily dosing with NVP-AUY922 (50mg/kg i.p) to mice bearing established U87MG glioma xenografts gave significant regression (T/C=7%). Therapeutic effects were concordant with changes in pharmacodynamic changes including depletion of p-AKT, p-S6, HIF-1α and induction of HSP72. Microvessel density was significantly reduced in NVP-AUY922 treated tumors, consistent with an antiangiogenic effect. Conclusion. These results indicate the therapeutic potential of NVP-AUY922 in the treatment of adult and pediatric GB.