Introduction: We have reported that fenretinide (4-HPR) is active against high-risk neuroblastoma (NB) in vitro and in vivo; our Phase I trials of novel 4-HPR formulations evidenced clinical activity. Mechanisms of activity include increase of cytotoxic dihydroceramides. Fenretinide activity in vitro is enhanced by safingol (S), the L-threo diastereomer of sphinganine. Here, we delineate stress pathways activated in response to 4-HPR±S and identify ixazomib, a proteasomal inhibitor, and antimalarial, mefloquine, a disruptor of autophagy, as new potential synergizing agents for 4-HPR±S. Methods: Sphingolipids were assessed by LC/MS/MS; cytotoxicity by fluorescence-based plate assay in 2% and 5% oxygen; apoptosis by TUNEL assay. ER stress markers, unfolded protein response (UPR), and autophagy, were assessed using immunoblotting, immunoprecipitation, and electron/confocal microscopy. Target validation was by gene silencing. Results: 4-HPR rapidly increased D-erythro-dihydroceramides; safingol was catabolized to L-threo-dihydroceramides. Safingol (2-3 µM) caused multi-log cytotoxic synergy of 4-HPR in eight of ten GBM and five NB cell lines (CI<0.7). Treatments resulted in golgi fragmentation (+6-12h) without decrease of stack proteins, and preceded increase of ER stress transducer, GRP78, and pro-apoptotic CHOP protein; UPR was evidenced by increase of poly-ubiquitinated proteins and increased autophagic flux (+12-48h). Cell death was apoptotic (cleaved caspase-3/PARP; TUNEL-positive) and non-apoptotic (+12-48h). Disruption of autophagy by mefloquine, or siRNA-silencing of BECN1 or ATG7, temporally-accelerated cytotoxicity and increased total apoptosis (p<0.05) in GBM cells. Consistent with cytotoxicity being dependent on misfolded protein stress, ixazomib further increased ER stress markers and accelerated/increased cytotoxicity (p<0.05). Treatment produced early inhibition (+4h) of tyrosine phosphorylation of p97/VCP, an AAA+ ATPase critical for the fusion of tER membrane vesicles into golgi stacks. Knockdown of p97/VCP recapitulated features of 4-HPR±S treatment, including golgi fragmentation, increase of ubiquitinated proteins and autophagic vacuoles. Treatment with D-erythro-sphinganine plus dihydroceramide desaturase inhibitor, GT-11, plus safingol increased both D-erythro and L-threo-dihydroceramides and recapitulated morphological, biochemical, and cytotoxic effects. Conclusion: Delineation of response pathways allowed the identification of mefloquine and ixazomib as new potential co-drugs to synergize 4-HPR±S. Confirmatory studies in NB xenografts are in progress. Intravenous fenretinide is currently in a Phase 2 trial in adult refractory/relapsed Peripheral T-Cell Lymphoma. A Phase I trial of fenretinide + safingol is in progress in the South Plains Oncology Consortium.
Citation Format: Nikhil Vad, Dong Wang, Hwangeui Cho, Dattesh Verlekar, Charlie Linch, C Patrick Reynolds, Min Kang, Barry J. Maurer. Dihydroceramide increase precedes golgi dispersal, pro-survival autophagy, ER stress, and UPR in fenretinide + safingol treated neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2885.