Preclinical study of novel targeted agents on nasopharyngeal carcinoma (NPC)

Abstract

Nasopharyngeal carcinoma (NPC) has a solid geographic distribution and is endemic in Southern China and Southeast Asia, including in Hong Kong. Around one-third of NPC patients will eventually experience progression five years after receiving radiotherapy ± adjuvant chemotherapy standard treatment. No targeted therapy is available for advanced-stage NPC, and preclinical research on new therapeutic agents is much needed. The proteasome inhibitor has successfully controlled multiple myeloma in the last two decades. It induces apoptosis, cell cycle arrest, and endoplasmic reticulum stress in cancer cells by blocking the ubiquitin- proteasome system. Ixazomib (MLN2238) is a newly FDA-approved second- generation oral proteasome inhibitor for multiple myeloma treatment with fewer side effects and improved selectivity than the first-generation proteasome inhibitor. Through blocking the protein degradation activity of the proteasome, ixazomib inhibits the nuclear factor-kB (NF-kB) signaling pathway, which is found constitutively activated in almost all Epstein-Barr virus (EBV)-associated NPC. In this study, we aim to examine the antitumor effect and the mechanism of action of ixazomib - alone and in combination with other therapeutic agents - in NPC in-vitro and in-vivo. Three authentic human EBV-positive NPC cell lines (C666-1, NPC43, and C17) and patient-derived xenografts (xeno113, xeno76, and xeno76- CR) were used as NPC preclinical models. In this study, ixazomib effectively inhibited cell proliferation in a time- and dose-dependent manner in these three NPC cell lines with relatively low concentrations compared to other solid tumors. Primary and advanced-stage NPC patient-derived xenografts were tested in mice with oral ixazomib weekly for three weeks. Significant tumor growth suppression was observed in all NPC models, with no noticeable toxicities in all treated mice. Combination treatment with cisplatin, radiotherapy, a CDK4/6 inhibitor, or lenalidomide was tested with ixazomib on NPC in-vitro and in-vivo, in which additive effect was found between ixazomib and cisplatin. We then further examined the molecular mechanism of actions of ixazomib in NPC cells. We demonstrated that the cell death induced by ixazomib is mediated by apoptosis, autophagy, and cell cycle arrest. In addition, the expression of EBV lytic genes and proteins was examined, and it was indicated that ixazomib induced EBV lytic cycle reactivation in NPC. Consistent with the results from previous studies, the inhibition of the canonical NF-kB signaling pathway was seen, with an accumulation of phosphorylated-IkB after ixazomib treatment in NPC cells. The expression of p50/p105 was also diminished after treatment, suggesting that ixazomib may also inhibit the atypical NF-kB signaling pathway, although further investigation is needed. This study provides preclinical evidence revealing that ixazomib may be a potent therapeutic agent for NPC patients, and the findings support the need for further testing in clinical trials.