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RNA-seq data from TAS4464, a NEDD8-activating enzyme inhibitor, activates both intrinsic and extrinsic apoptotic pathways via c-Myc-mediated regulation in acute myeloid leukemia

Citation

Tanaka, Tomoaki (2021), RNA-seq data from TAS4464, a NEDD8-activating enzyme inhibitor, activates both intrinsic and extrinsic apoptotic pathways via c-Myc-mediated regulation in acute myeloid leukemia, Dryad, Dataset, https://doi.org/10.5061/dryad.hqbzkh1f6

Abstract

TAS4464, a potent, selective small molecule NEDD8-activating enzyme (NAE) inhibitor, leads to inactivation of cullin-RING E3 ubiquitin ligases (CRLs) and consequent accumulations of its substrate proteins. Here, we investigated the antitumor properties and action mechanism of TAS4464 in acute myeloid leukemia (AML). TAS4464 induced apoptotic cell death in various AML cell lines. TAS4464 treatments resulted in the activation of both the caspase-9-mediated in intrinsic apoptotic pathway and caspase-8-mediated extrinsic apoptotic pathway in AML cells; combined treatment with inhibitors of these caspases markedly diminished TAS4464 induced apoptosis. In each apoptotic pathway, TAS4464 induced the mRNA transcription of the intrinsic proapoptotic factor NOXA and decreased that of the extrinsic antiapoptotic factor c-FLIP. RNA-sequencing analysis showed that the signaling pathway of the CRL substrate c-Myc was enriched after TAS4464 treatment. Chromatin immunoprecipitation (ChIP) assay revealed that TAS4464 induced c-Myc bound to the PMAIP1 (encoding NOXA) and CFLAR (encoding c-FLIP) promoter regions, and siRNA-mediated c-Myc knockdown neutralized both TAS4464-mediated NOXA induction and c-FLIP downregulation. TAS4464 activated both caspase-8 and caspase-9 along with an increase in NOXA and a decrease in c-FLIP, resulting in complete tumor remission in a human AML xenograft model. These findings suggest that NAE inhibition leads to anti-AML activity via a novel c-Myc-dependent apoptosis induction mechanism.

Methods

HL-60 cells were either not treated (control) or treated with TAS4464 (0.1 μmol L-1) for 24 or 48 hours. Then, RNA was isolated from the cell samples using a phenol-guanidinium isothiocyanate (P/GTC) reagent according to the manufacturer’s protocol [45]. RNA was precipitated from the aqueous phase with isopropanol, and proteins were precipitated from the phenol/ethanol phase by the addition of acetone. A total of single-end-read RNA-seq tags were generated using a HiSeq 2000 sequencer according to the standard protocol. The generated sequence tags were mapped onto the human genome sequence (hg19 from the University of California Santa Cruz Genome Browser) using the Eland program (Illumina).