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

Diana Corallo,Elena Mariotto,Sanja Aveic,Giampietro Viola,Gian Paolo Tonini,Mirco Ponzoni,Fabio Pastorino,Marcella Pantile,Federico Caicci

doi:10.1186/s13046-020-01692-x

Abstract

BackgroundDespite reported advances, acquired resistance to tyrosine kinase inhibitors still represents a serious problem in successful cancer treatment. Among this class of drugs, ponatinib (PON) has been shown to have notable long-term efficacy, although its cytotoxicity might be hampered by autophagy. In this study, we examined the likelihood of PON resistance evolution in neuroblastoma and assessed the extent to which autophagy might provide survival advantages to tumor cells.MethodsThe effects of PON in inducing autophagy were determined both in vitro, using SK-N-BE(2), SH-SY5Y, and IMR-32 human neuroblastoma cell lines, and in vivo, using zebrafish and mouse models. Single and combined treatments with chloroquine (CQ)—a blocking agent of lysosomal metabolism and autophagic flux—and PON were conducted, and the effects on cell viability were determined using metabolic and immunohistochemical assays. The activation of the autophagic flux was analyzed through immunoblot and protein arrays, immunofluorescence, and transmission electron microscopy. Combination therapy with PON and CQ was tested in a clinically relevant neuroblastoma mouse model.ResultsOur results confirm that, in neuroblastoma cells and wild-type zebrafish embryos, PON induces the accumulation of autophagy vesicles—a sign of autophagy activation. Inhibition of autophagic flux by CQ restores the cytotoxic potential of PON, thus attributing to autophagy a cytoprotective nature. In mice, the use of CQ as adjuvant therapy significantly improves the anti-tumor effects obtained by PON, leading to ulterior reduction of tumor masses.ConclusionsTogether, these findings support the importance of autophagy monitoring in the treatment protocols that foresee PON administration, as this may predict drug resistance acquisition. The findings also establish the potential for combined use of CQ and PON, paving the way for their consideration in upcoming treatment protocols against neuroblastoma.

Highlights

Despite reported advances, acquired resistance to tyrosine kinase inhibitors still represents a serious problem in successful cancer treatment
Ponatinib triggers autophagy in human neuroblastoma cells In a previously established highthroughput screening (HTS) assay among 349 screened small molecule inhibitors, PON was identified as the most promising candidate for abrogating neuroblastoma growth [13]
The cleavage of PARP protein and CASPASE 3 were evident for the IC50 doses of PON; more importantly, the activation of autophagy was already observed at the sub-lethal doses of drugs, while the pro-apoptotic signals were visible at doses approaching the IC50 of PON (Fig. 1a)

Summary

Introduction

Despite reported advances, acquired resistance to tyrosine kinase inhibitors still represents a serious problem in successful cancer treatment. Among this class of drugs, ponatinib (PON) has been shown to have notable long-term efficacy, its cytotoxicity might be hampered by autophagy. We examined the likelihood of PON resistance evolution in neuroblastoma and assessed the extent to which autophagy might provide survival advantages to tumor cells. With respect to the first and second generations of BCR-ABL tyrosine kinase protein inhibitors, PON was more successful in eliminating both BCR-ABL wild-type and mutant (BCR-ABLT315I) CML cells, reducing the possible evolution of resistance due to drug exclusion [2]. In a previous highthroughput screening (HTS) study, among 349 compounds tested, PON gave the best results in impeding the growth of neuroblastoma cells [13]

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