MLN4924-resistant cells are sensitive to the pan-E1 inhibitor Compound 1, but are resistant to NAE-selective Compound 1 analogues.

<p><b>A)</b> Cells were seeded in 96-well plates (3×10³ cells/well) and treated with increasing concentrations of various selective NAE inhibitors for 72 hours. After treatment, cell viability was assessed by the CellTiter Glo assay. Values shown are the mean percentage ± SD of viable cells relative to vehicle controls. EC₅₀ values calculated from the dose-response curves of the parental K562 cells presented here have been presented in a previous publication by Lukkarila <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093530#pone.0093530-Lukkarila1" target="_blank">[35]</a>. <b>B)</b> Parental K562 and MLN4924-resistant R-K562_MLN cells were plated in 96-well plates (5×10³ cells/well) and treated with increasing concentrations of compound 1 for 72 hours. After incubation, cell viability was measured by the CellTiter Glo assay. Values shown are the mean percentage ± SD of viable cells relative to controls. <b>C)</b> K562 and R-K562_MLN cells were treated with increasing concentrations of compound 1 for 24 hours. After treatment, total cellular proteins were analyzed by SDS-PAGE and immunoblotting with anti-NEDD8, anti-ubiquitin (Ub) and anti-α-tubulin antibodies.</p

MLN4924-resistant K562 (R-K562_MLN) leukemia cells show decreased sensitivity to MLN4924, but not all chemotherapeutics.

<p><b>A</b>) Cells (5×10³ cells/well) were seeded in 96-well plates and treated with increasing concentrations of MLN4924 for 72 hours. After treatment, cell growth and viability were determined by the CellTiter-Glo luminescence assay. Values shown are the mean percentage ± SD of viable cells relative to controls. <b>B</b>) Cells (1×10⁵/ml) were plated in 6-well plates. After incubation, cells were stained with trypan blue and counted at the time points indicated. R-K562_MLN cells were incubated in the presence or absence of 250 nM MLN4924 as indicated. Values shown represent the means ± SD of viable cells. <b>C</b>) Cells (5×10³/well) were seeded in 96-well plates and treated with increasing concentrations of MLN4924 for 72 hours. After incubation, cell viability was assessed by the CellTiter Glo assay. Values shown represent the mean percentage ± SD of viable cells relative to vehicle controls. R-K562_MLN-5W, R-K562_MLN cells that were maintained in MLN4924-free medium for 5 weeks. <b>D</b>) Cells (5×10³/well) were plated in 96-well plates and treated with increasing concentrations of chemotherapeutic drugs as indicated for 72 hours. Cell growth and viability was assessed by the MTS assay. Values shown represent the mean percentage ± SD of viable cells relative to vehicle controls.</p

cDNA sequencing reveals missense point mutations in the UBA3 coding region of MLN4924-resistant K562 cells which decrease the sensitivity of NAE to MLN4924 in vitro.

<p><b>A)</b> Total cellular RNA was isolated from parental and MLN4924-resistant R-K562_MLN cells. UBA3 cDNA spanning its entire coding region was amplified by RT-PCR and then sequenced. The nucleotide sequence from codon 307 to 313 for K562 (<i>left panel</i>) and R-K562_MLN (<i>right panel</i>), three letter amino acid codes, and codon numbers are each shown above the sequence electropherogram tracing. Arrows indicate the position of the second nucleotide of the codon 310 in which the single nucleotide shift (T → A) in R-K562_MLN occurred. <b>B)</b> Stereoscopic views of NAE and SAE active sites. Enzymes are shown in cyan ribbon format with either NEDD8 or SUMO UBL in green stick format. Secondary structure elements are labelled. <b>1)</b> The NAE active site from the PDB = 2NVU structure is shown with NEDD8 and ATP. Also in stick format is I310, which mutated to asparagine renders the enzyme more active than wild type and resistant to MLN4924. For comparison, four other mutations that render resistance are shown in red line format. <b>2)</b> The PDB = 3GZN structure is shown with the NEDD8-MLN4924 adduct. Also shown in line format are side chains of residues nearby. <b>3)</b> The SUMO-AMP adduct of SAE from PDB = 3KYC shows that a similar isoleucine (I384) is positioned near the diglycine motif of the UBL. <b>4)</b> The covalent cysteine intermediate, captured by PDB = 3KYD, shows that I384 must be displaced for reaction to proceed. <b>C)</b> Sequence alignment of human UBA3, UBA1, UBA2, and UBA6 was performed, and residues corresponding to UBA3’s I310 are shaded. <b>D)</b> NEDD8 (2-fold dilutions from 50 μM) was added to ATP:PPi exchange reactions containing 20 nM NAE or NAE (UBA3 I310N), 1 mM ATP, and 100 μM PPi (supplemented with 50 cpm/pmol [³²P] PPi). After 30 min at 37°C, radiolabeled ATP generated was measured. Error bars represent SEM (n = 3). Bar graphs indicate the NEDD8 concentration and maximum observed rate for the enzymes. The effects of ATP (<b>E</b>) and PPi (<b>F</b>) on ATP synthesis were evaluated using either 1.56 μM NEDD8 (NAE) or 6.25 μM NEDD8, determined from (<b>D</b>). ATP concentrations tested were 2-fold dilutions from 2 mM with 100 μM PPi. PPi concentrations were 2-fold dilutions from 0.5 mM with 1 mM ATP. <b>G)</b> MLN4924 was tested (3-fold dilutions from 200 μM, 2% DMSO final concentration) in reactions containing 20 nM NAE or NAE (UBA3 I310N), 1 mM ATP, 100 μM PPi, and NEDD8. The measured IC₅₀ from non-linear regression analyses of triplicate experiments is shown. Error bars represent SEM.</p

MLN4924-resistant K562 cells show decreased inhibition of cullin neddylation by MLN4924 but remain sensitive to knockdown of NEDD8.

<p><b>A)</b> Cells were treated with increasing concentrations of MLN4924 as indicated for 24 hours. After treatment, total cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting using anti-NEDD8 and anti-α-tubulin antibodies to detect NEDD8-cullin complexes and equal protein loading, respectively. <b>B)</b> Cells were infected with lentiviral vectors expressing three independent shRNA sequences targeting NEDD8 (shNEDD8) or a control sequence (shControl), and successfully transduced puromycin-resistant populations were selected. Total cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting with antibodies against NEDD8 and α-tubulin. <b>C)</b> Cells infected with vectors containing shNEDD8 or control sequences and selected for puromycin resistance were seeded in 6-well plates (1×10⁵ cells/ml). After incubation for 2, 3, and 6 days, cell growth and viability were assessed by trypan blue exclusion. Values represent the mean percentage ± SD of viable cells relative to cells infected with control sequences.</p

MLN4924-resistant U937 leukemia cells show reduced sensitivity to MLN4924 and are heterozygous for a UBA3 mutation Y352H.

<p><b>A)</b> Cells (1×10⁴ cells/well) were seeded in 96-well plates and incubated with increasing concentrations of MLN4924 for 72 hours. After incubation, cell viability was measured by the CellTiter-Glo assay. Values shown are the mean percentage ± SD of viable cells relative to controls. <b>B)</b> Cells were treated with increasing concentrations of MLN4924 as indicated for 24 hours followed by isolation of total cellular proteins. Equal amounts of proteins were fractionated by SDS-PAGE and analyzed by immunoblotting with anti-NEDD8 and anti-α-tubulin antibodies. <b>C)</b> UBA3 cDNA in parental and MLN4924-resistant R-U937_MLN cells was sequenced as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093530#pone-0093530-g004" target="_blank">Figure 4</a>. Shown are the nucleotide sequence from codons 347 to 354 for U937 (left panel) and R-U937_MLN cells (right panel), three letter amino acid codes, and codon numbers. Arrows depict the position of the first nucleotide of the codon 352 in which the single nucleotide shift (T → C) in R-U937_MLN occurred. <b>D)</b> Sequence alignment of UBA3s from different organisms was performed, and residues corresponding to the Y352 are shaded. <b>E)</b> The Y352H mutation. To the left the NAE heterodimer surface is shown in grey and its subdomains labelled. Phe352 is shown in magenta. Bound NEDD8 is shown in green ribbon format. On the right, a close-up of the interface between NAE (cyan) and NEDD8 (green) is shown in ribbon format. Shown in line format are residues within proximity.</p