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

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 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.

Fig. 1. TAS4464 induces apoptosis in AML cell lines.

A AML cell lines were seeded in 96-well plates and treated the next day with various concentrations of TAS4464. After 3 days, cell viability was determined by measurement of the cellular ATP contents. B Apoptotic cell death was evaluated by flow cytometric analysis. HL-60 cells were treated with 0.1 μmol L⁻¹ TAS4464 for 0, 8, and 24 h. C Total percentage of cells with each status shown in (B). D Heatmap showing differentially expressed molecules identified by proteomics analysis in HL-60 cells. Cells were treated with TAS4464 (0.1 μmol L⁻¹) for 0, 1, 4, 8, 16, and 24 h (left). Pathways enriched with time-dependently upregulated or downregulated molecules, as identified by Gene Ontology analysis, are listed (right).

Fig. 2. TAS4464 activates both intrinsic and extrinsic apoptotic pathways.

A HL-60 and THP-1 cells were treated with 0.1 μmol L⁻¹ TAS4464 for 1, 4, 8, 16, and 24 h, and total protein was extracted. The active form of each caspase was detected with the indicated antibodies. B Cells were treated with 1 μmol L⁻¹ z-IETD-FMK alone, 1 μmol L⁻¹ z-LEHD-FMK alone, and a combination of these inhibitors for 1 h prior to exposure to TAS4464. Then, cells were treated with 0.1 μmol L⁻¹ TAS4464 for 16 h. Caspase-3/7 activity levels are expressed as relative luminescence units (RLU), which were normalized to the number of viable cells under each condition (relative fluorescence units, RFU). Data are presented as the mean ± SEM values of data from three independent experiments.

Fig. 3. TAS4464 increases NOXA expression and decreases c-FLIP expression at the mRNA transcriptional level.

A HL-60 and THP-1 cells were treated with 0.1 μmol L⁻¹ TAS4464 for 1, 4, 8, 16. and 24 h, and total protein was extracted. Immunoblotting for neddylated Ubc12 and cullin1 was performed with the anti-NEDD8 antibody. Parallelly, immunoblotting for Bcl-2 family proteins in the intrinsic apoptotic pathway and death receptor signal-related proteins in the extrinsic apoptotic pathway was performed with the indicated antibodies. B qRT-PCR was performed to compare the mRNA levels of PMAIP1 and CFLAR in cells treated with 0.1 μmol L⁻¹ TAS4464 and control cells (0 h) at the indicated time points (hours). PMAIP1 and CFLAR mRNA expression levels were normalized to 18S rRNA expression levels. Data are presented as the mean ± SEM values from three independent experiments.

Fig. 4. RNA-seq results for TAS4464-treated HL-60 cells.

A Principal component analysis showing the clusters of HL-60 cells depending on the duration of TAS4464 treatment. DMSO (control) was used for the untreated condition. B RNA-seq data for each population. Differentially expressed mRNAs are listed using hierarchical clustering. Representative c-Myc target genes are shown in the list. C Upstream regulators were predicted using Ingenuity Pathway Analysis based on the difference in gene expression between untreated cells and cells treated with TAS4464 for 24 h. qRT-PCR was performed to compare the mRNA levels of CDKN1A, TNF, E2F1, E2F2 (D) and IDH2 (E) in cells treated with 0.1 μmol L⁻¹ TAS4464 and control cells. 18S rRNA expression was used for normalization and data are presented as the mean ± SD values of data from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5. TAS4464 increases the protein stability of c-Myc.

A The top 100 differentially expressed proteins between untreated cells and treated with 0.1 μmol L⁻¹ TAS4464 for 4 h identified by non-target proteomic analysis are listed in the heatmap. B Changes in c-Myc protein expression levels identified in proteomic analysis during treatment with 0.1 μmol L⁻¹ TAS4464 for up to 24 h. Data are presented as the mean ± SD values of data from three independent experiments. C Immunoblotting for c-Myc in HL-60 and THP-1 cells treated with 0.1 μmol L⁻¹ TAS4464. Samples were harvested at 1, 4, 8, 16, and 24 h after treatment. D Cycloheximide (CHX)-chase analysis for HL-60 cells. The HL-60 cells were treated with DMSO (Untreated) or TAS4464 (0.1 μmol L⁻¹) in the presence of CHX (100 μg mL⁻¹) for the indicated time points (hours). Immunoblotting for c-Myc was performed to evaluate the protein stability. E Immunoblotting for HA-tagged ubiquitin and FLAG-tagged c-Myc after immunoprecipitation of c-Myc. 3×FLAG-c-Myc and HA-ubiquitin were transfected into HEK293T cells. After 8 h of transfection, cells were treated with MG132 (1 mM) and TAS4464 (0.1 μmol L⁻¹) for the last 16 h, and the extracted proteins were subjected to ubiquitination assay for c-Myc.

Fig. 6. TAS4464-induced c-Myc activation transcriptionally mediates apoptotic gene expression.

A Schematic representation of the PMAIP1 promoter region and c-Myc enrichment at each site. The regions targeted by the primer pairs are indicated as “BS1” to “BS5” and “3′ UTR”. B Enrichment of c-Myc protein in the BS4 region in (A). The fold enrichment value compared to the untreated condition was used to evaluate the effect of TAS4464 induction. HL-60 cells were treated with TAS4464 (0.1 μmol L⁻¹) for 4 h. C MYC siRNAs were transfected into HL-60 cells. After 48 h, cells were treated with 0.1 μmol L⁻¹ TAS4464 for 16 h except for c-Myc detection (1 h). Total protein was extracted and then immunoblotting for NEDD8-cullin1, c-Myc, c-FLIP, NOXA and cleaved caspase-3 was performed. D Apoptotic cell death was evaluated by flow cytometric analysis. HL-60 cells were treated with NAE1 siRNAs for 16 h. E Immunoblotting for c-Myc, Cleaved caspase-8 and Cleaved caspase-9 after NAE1 siRNAs transfection. Cells were harvested 8 h after transfection. F qRT-PCR to measure the level of PMAIP1 mRNA was performed in HL-60 cells after NAE1 siRNA transfection at 16 h time point. Data are presented as the mean ± SD values of data from three independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 7. TAS4464 exerts strong antitumor activity via activation of both the intrinsic and extrinsic apoptotic pathways accompanied by an increase in the NOXA level and a decrease in the c-FLIP level in a human AML xenograft model.

A TAS4464 (100 mg kg⁻¹) was administered intravenously. Tumors were harvested at the indicated times after administration of TAS4464 and prepared for immunoblotting. B TAS4464 was administered intravenously twice weekly at 100 mg kg⁻¹ per day. Cytarabine was administered intravenously twice a week for 3 weeks at 100 mg kg⁻¹ per day. Data are presented as the mean ± SD values. *P < 0.05 in the treated group compared with the control group (Dunnett’s test).