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

Abstract

Background 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, 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. Methods The 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. Results Our 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. Conclusions Together, 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.

Dual inhibition of autophagy and PI3K/mTOR pathway as a potential therapeutic strategy against laryngeal squamous cell carcinoma

“…In our study, an increase of LC3II/I ratio was found in both LSCC cell lines treated with PI3K or mTOR inhibitors, among which the dual inhibitor NVP-BEZ235 is the most significant, supporting its induction of cell autophagy. Based on accumulating evidence that inhibition of autophagy can enhance the efficiency of antitumor drugs ( 22 ), we combined NVP-BEZ235 with autophagy inhibitors for further verification.…”

Efficacy of Dual PI3K/mTOR Inhibitor Combined With Autophagy Suppression Against Laryngeal Squamous Cell Carcinoma

“…In this study, an increase of LC3II/I ratio was found in both LSCC cell lines treated with PI3K or mTOR inhibitors, among which the dual inhibitor NVP-BEZ235 is the most signi cant, supporting its induction of cell autophagy. Based on accumulating evidence that inhibition of autophagy can enhance the e ciency of antitumor drugs [33][34][35], we combined NVP-BEZ235 with autophagy inhibitors for further veri cation. Surprisingly, the combination of NVP-BEZ235 with two autophagy inhibitors (3-MA, CQ) showed a synergetic antitumor effect in both cell lines.…”

Roles of AMPK and Its Downstream Signals in Pain Regulation

“…During this process, microtubule-associated protein 1A/1B-light chain 3 (LC3) is transformed from the LC3-I (cytosolic form) to the LC3-II (autophagosome membranes-associated form) [ 79 , 80 ]. Increased LC3-II and decreased p62, a substrate of autophagy, indicate the normal autophagic flux that induces protein degradation and damaged organelle removal [ 81 ] ( Figure 3 ). In the SNL model, beclin-1, which is a key factor in the VPS34 complex, and LC3, in the ipsilateral spinal dorsal horn, are upregulated on day 7 and maintained for 14 days, suggesting that nerve injury triggers autophagy [ 82 , 83 ].…”