A High-Content Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy

Abstract

Novel druggable targets have been discovered in neuroblastoma (NB), paving the way for more effective treatments. However, children with high-risk NB still show high mortality rates prompting for a search of novel therapeutic options. Here, we aimed at repurposing FDA-approved drugs for NB treatment by performing a high-content screening of a 349 anticancer compounds library. In the primary screening, we employed three NB cell lines, grown as three-dimensional (3D) multicellular spheroids, which were treated with 10 μmol/L of the library compounds for 72 hours. The viability of 3D spheroids was evaluated using a high-content imaging approach, resulting in a primary hit list of 193 compounds. We selected 60 FDA-approved molecules and prioritized drugs with multi-target activity, discarding those already in use for NB treatment or enrolled in NB clinical trials. Hence, 20 drugs were further tested for their efficacy in inhibiting NB cell viability, both in two-dimensional and 3D models. Dose-response curves were then supplemented with the data on side effects, therapeutic index, and molecular targets, suggesting two multiple tyrosine kinase inhibitors, ponatinib and axitinib, as promising candidates for repositioning in NB. Indeed, both drugs showed induction of cell-cycle block and apoptosis, as well as inhibition of colony formation. However, only ponatinib consistently affected migration and inhibited invasion of NB cells. Finally, ponatinib also proved effective inhibition of tumor growth in orthotopic NB mice, providing the rationale for its repurposing in NB therapy. .

Autophagic flux inhibition enhances cytotoxicity of the receptor tyrosine kinase inhibitor ponatinib

“…In a previously established HTS assay among 349 screened small molecule inhibitors, PON was identified as the most promising candidate for abrogating neuroblastoma growth [ 13 ]. Here, we assessed a possible functional association between PON and the modulation of autophagy in human neuroblastoma.…”

“…Treatments started 12 days after tumor cell implantation. HCQ (60 mg/kg [ 36 ];) and PON (30 mg/kg [ 13 ];)—as single agents or in combination (COMBO)—were administered i.p. and by gavage, respectively, every day for 16 days total.…”

“…However, some studies have found an increased rate of resistance to PON in either pre-clinical or clinical settings [ 8 – 10 ], and a similar finding may therefore be possible in neuroblastoma, in which the efficacy of PON emerged from both in vitro and in vivo pre-clinical assessments [ 11 , 12 ]. In a previous high-throughput screening (HTS) study, among 349 compounds tested, PON gave the best results in impeding the growth of neuroblastoma cells [ 13 ].…”

Developing preclinical models of neuroblastoma: driving therapeutic testing

“…Compared to cells grown in monolayer culture, spheroid cultures exhibited increased expression of metabolic markers, cell stress response proteins, cell structure proteins, and transport polypeptides [148]. Sidarovich et al used spheroids for high throughput drug screening of over 300 FDAapproved anti-cancer compounds or compounds undergoing clinical trials [149]. From this screen, the authors identified, and later evaluate in vivo, two compounds, ponatinib and axitinib, as potential new therapies for NB based on toxicity, molecular target, and side effects [149].…”

“…Sidarovich et al used spheroids for high throughput drug screening of over 300 FDAapproved anti-cancer compounds or compounds undergoing clinical trials [149]. From this screen, the authors identified, and later evaluate in vivo, two compounds, ponatinib and axitinib, as potential new therapies for NB based on toxicity, molecular target, and side effects [149].…”