Omomyc Reveals New Mechanisms To Inhibit the MYC Oncogene

Abstract

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.

Keywords: DNA binding; Max; Myc; Omomyc; cotranslational; rRNA; transcription.

FIG 1

Omomyc affects cell proliferation and MYC-mediated transcription. (A) Purification and characterization of recombinant Omomyc. Shown is an SDS-PAGE gel of bacterially expressed Omomyc under nonreduced (NR) and reduced (Red) conditions. (B) Myc levels of cells used for cell proliferation and other experiments. (C) Effect of both recombinant Omomyc and synthetic Omomyc on proliferation of Ramos and HCT116 cells over 3 days. (D) Gene set enrichment analysis (GSEA) comparing gene expression between untreated and 10 μM Omomyc-treated HCT116 cells. Normalized enrichment scores (NES), false discovery rate (FDR) q values, and numbers of genes for MYC signatures are shown. (E and F) Q-PCR showing the effect of 10 μM Omomyc on the expression of several Myc target genes identified by RNA-Seq in HCT116 cells. Genes tested were the ASNS, SAT1, ID3, EGR2, and CD274 (PD-L1) genes.

FIG 1

Omomyc affects cell proliferation and MYC-mediated transcription. (A) Purification and characterization of recombinant Omomyc. Shown is an SDS-PAGE gel of bacterially expressed Omomyc under nonreduced (NR) and reduced (Red) conditions. (B) Myc levels of cells used for cell proliferation and other experiments. (C) Effect of both recombinant Omomyc and synthetic Omomyc on proliferation of Ramos and HCT116 cells over 3 days. (D) Gene set enrichment analysis (GSEA) comparing gene expression between untreated and 10 μM Omomyc-treated HCT116 cells. Normalized enrichment scores (NES), false discovery rate (FDR) q values, and numbers of genes for MYC signatures are shown. (E and F) Q-PCR showing the effect of 10 μM Omomyc on the expression of several Myc target genes identified by RNA-Seq in HCT116 cells. Genes tested were the ASNS, SAT1, ID3, EGR2, and CD274 (PD-L1) genes.

FIG 2

Omomyc interacts with Myc and Max in cells and also affects Myc stability and protein levels. (A) Immunoprecipitation (IP) of Myc, Max, and biotinylated Omomyc in HCT116 cells. HCT116 cells were treated with 10 μM Omomyc, 2.5 μM Omomyc with ProteoJuice, or no Omomyc at all and then lysed after 24 h. Lysates were then incubated with antibodies against Myc and Max, control IgG, and streptavidin; pulled down with magnetic beads; run on an SDS-PAGE gel; and then blotted with antibodies to Myc, Max, or biotin. (B) Effect of treatment with 10 μg/ml cycloheximide in the presence and absence of 10 μM Omomyc over time. HCT116 cells were treated with 10 μg/ml cycloheximide in the presence or absence of 10 μM Omomyc for up to 4 h. Cells were lysed, subjected to SDS-PAGE, and then Western blotted with antibodies to Myc, Max, and Gapdh. (C) Effect of cycloheximide or the proteasome inhibitor MG132 on Myc, Max, and Omomyc interactions. HCT116 cells were treated with 0 or 10 μM Omomyc in the absence or presence of 20 μg/ml cycloheximide or 10 μM MG132 for 4 h; lysed; incubated with antibodies (ab) to Myc and Max, control IgG, and streptavidin; and then pulled down with magnetic beads. The material was run on an SDS-PAGE gel and Western blotted with antibodies to Myc, Max, and biotin. (D) Treatment of Ramos cell lysates with Omomyc induces thermal destabilization of Myc. Shown are aggregation temperature (T_agg) curves and corresponding Western blot images for Myc in Ramos cell lysates after incubation at 4°C with Omomyc (20 μM) or PBS for 2 h. All data were normalized to the Myc band intensity for the lowest temperature, 39°C. The T_agg shifts were analyzed using the Boltzmann sigmoidal equation and are 50.3°C for the buffer-treated lysate and 43.9°C for the Omomyc-treated lysate. (E) Omomyc decreases the level of Myc in the cell. Ramos cells were treated with increasing concentrations of Omomyc for 24 h, lysed, run on an SDS-PAGE gel, and Western blotted with antibodies to Myc and Gapdh. (F) Time course of Myc reduction by 10 μM Omomyc. Ramos cells were treated with Omomyc, and samples were collected at 0, 2, 4, and 24 h. The samples were lysed, subjected to SDS-PAGE, and Western blotted for Myc and Gapdh. (G) Myc reduction is due to proteosomal degradation of Myc. Ramos cells were treated with or without 10 μM Omomyc in the presence or absence of 100 nM MG132 for 24 h, lysed, subjected to SDS-PAGE, and Western blotted for Myc and Gapdh.

FIG 3

Omomyc binds DNA in vitro. (A) Fluorescence polarization assay using 5′-FAM-labeled 20-mer E box DNA and synthetic Omomyc, demonstrating DNA binding activity in millipolarizations (mP) of Omomyc (22 nM). (B) Competitive binding fluorescence polarization assay combining 5′-FAM-labeled Myc E box DNA with either competitive (unlabeled E box) DNA or noncompetitive (scrambled E box) DNA. (C) Dose-resolved proteomic pulldown experiments using immobilized Myc E box DNA. As expected, both MYC and MAX are competed off the DNA with increasing concentrations of free Omomyc spiked into the Ramos cell lysate (Myc EC₅₀ = 321 nM; MAX EC₅₀ = 297 nM). Owing to the sequence overlap between Omomyc and Myc, we show quantitative values for a representative MYC peptide. The 10 nM Max data point was excluded because it was an obvious outlier. (D) Omomyc can compete for binding to the E box with Myc, Max, and other dimerization partners (Mxi1, Mxd3, Mnt, Mga, and Maz). The experiment was performed using a method similar to the one described above for panel C. All dimerization partners have similar EC₅₀s. (E) WDR5 and KMT2A, two core components of the MYC-associated MLL complex, are potently competed by Omomyc (WDR5 EC₅₀ = 213 nM; KMT2A EC₅₀ = 196 nM).

FIG 4

Omomyc can bind to DNA in cells (A) Chromatin immunoprecipitation assay demonstrating that Omomyc can displace Myc/Max heterodimers from binding to DNA and can bind to DNA directly. Immunoprecipitations were done with anti-Myc antibody, a control IgG, and streptavidin beads, which pull down biotinylated Omomyc. Genes assayed were the BOP1, RRS1, NCL1, FBXW8, LYAR, PUS1, ATDA3, VEGFA, FBX32, and HSBPA1 genes, along with a control region that does not contain any E box (data not shown). The statistical significance of data (P value) was calculated using two-tailed unpaired Student’s t test, which was done with GraphPad Prism. ****, P < 0.0001; ***, P < 0.005; **, P < 0.01; *, P < 0.05. (B) Chromatin immunoprecipitation assay to determine at which promoters WDR5 colocalizes with Myc and Omomyc. Antibodies to WDR5 and Myc and a control IgG were used along with streptavidin beads to immunoprecipitate DNA/protein complexes. Genes assayed were the NCL1, NPM1, RRS1, PUS1, BOP1, VEGFA-1, FBX32, and HSBAP1 genes, along with a control region that does not contain any E box. (C) Sequences of the consensus E box and E boxes found in the VEGFA promoter region. VEGFA E boxes 1 and 2 were used in the FP assay in Table 3 to determine the affinities of various dimers for each sequence.

FIG 5

Omomyc is cell penetrant and localizes to the nucleolus. (A) Omomyc is cell penetrant. Fluorescein-labeled Omomyc was used to treat HCT116 cells for 24 h. Cells were fixed, stained with Hoechst 3342, and then visualized with a Molecular Dynamics ImageXpress high-content imager using 20× (left) and 60× (right) air objectives. Bars, 100 μm (left) and 10 μm (right). (B) Localization of Omomyc to the nucleolus. HCT116 cells were treated with 10 μM Alexa Fluor 647-Omomyc for 24 h, fixed, permeabilized, and stained with anti-UBF for the nucleolus, anti-histone H3 for DNA, and Hoechst 33342 for the nucleus. Cells were visualized with a Nikon SIM-E microscope at a ×100 magnification. Bar, 10 μm. (C) Omomyc binds to rDNA promoters and replaces Myc at these promoters. Cells were treated with 2.5 μM Omomyc plus ProteoJuice for 24 h and then fixed, nuclei were isolated, and chromatin was then sheared by sonication. Chromatin was then immunoprecipitated with antibodies to Myc, control IgG, and streptavidin. The chromatin was then eluted, treated with proteinase K, purified, and subjected to Q-PCR using Sybr green, using probes for transcribed rRNA regions for 45S rRNA, 18S rRNA, and 5.8S rRNA and an untranscribed rRNA region. All regions were previously shown to be bound by Myc and Mad1 (19, 28).

FIG 6

Interaction of differently labeled Omomyc, Myc, and Max both in the cell and at E boxes in promoters. (A) HCT116 cells were plated and then treated with a 10 μM mixture of biotinylated (5 μM) and 6×His-tagged (5 μM) Omomyc for 24 h. Cells were fixed in 3.9% formaldehyde, permeabilized, and then stained with antibodies to Myc, Max, biotin, or penta-His. The cells were then processed according to the instructions of the Sigma Duo-link kit. Cells were imaged on a Perkin-Elmer Opera Phenix high-content imager using a 40× water objective. Bar, 10 mm. (B) Omomyc colocalizes with Max in the nucleus and nucleolus. HCT116 cells were treated with 10 mM Alexa Fluor 647-Omomyc for 24 h, fixed, permeabilized, and stained with anti-UBF and Max antibodies as well as Hoechst 3342. Cells were imaged with a Nikon SIM-E microscope using a 100× objective. Bar, 10 mm. (C) ReCHIP assay for binding of Max and biotinylated Omomyc. HCT116 cells were treated with 2.5 μM Omomyc plus ProteoJuice for 24 h and then processed for chromatin immunoprecipitation by fixing the cells, isolating the nuclei, and shearing the chromatin. The initial chromatin immunoprecipitation was performed with antibodies to Myc and Max, control IgG, and streptavidin. The chromatin was eluted and then reimmunoprecipitated (ReCHIP) with the indicated antibodies. After the second chromatin immunoprecipitation, the eluted chromatin was treated with proteinase K, purified, and then subjected to Q-PCR with NPM1, RRS1, VEGFA-1, FBX32, and a control region that does not contain an E box. (D) ReCHIP assay demonstrating the interaction of Omomyc monomers on DNA. ReCHIP assays were performed as described above for panel B, using anti-6×His tag antibody and streptavidin for immunoprecipitation.

FIG 7

Myc/Max and Omomyc/Max heterodimers form cotranslationally. (A) Interaction of Omomyc with Myc and Max in HCT116 cell lysates. A total of 10 μM biotinylated Omomyc was added to an HCT116 cell lysate; incubated for 4 h; immunoprecipitated with either anti-Myc, anti-Max, or antibiotin antibodies; pulled down with magnetic beads; run on an SDS-PAGE gel; and then blotted with antibodies to Myc, Max, or biotin. (B) Myc, Omomyc, and Max associate with translating ribosomes. GFP and GFP-Rpl10 plasmids were transfected into HCT116 cells for 24 h and then treated with ProteoJuice with and without 10 μM Omomyc for 6 h. The cells were lysed, and ribosomes were affinity purified with anti-GFP antibody. Purified ribosome complexes were washed and then blotted for the presence of Myc, Max, and biotinylated Omomyc. (C) RNA immunoprecipitation (RIP) assay demonstrating that Myc, Max, and Omomyc can bind MAX RNA. HCT116 cells were plated overnight, treated with ProteoJuice with and without 10 μM Omomyc for 6 h, lysed, and then immunoprecipitated with antibodies to Myc, Max, and biotin. After immunoprecipitation, RNA was isolated from the immunoprecipitates, cDNA was made, and RT-PCR was performed for the primer/probe sets indicated. (D) Myc, Max, and Omomyc interact with MAX RNA in a complex involving ribosomes. HCT116 cells were treated as described above for panel B. Two hours before lysis, 200 μg/ml puromycin was added to cells to inhibit protein translation. At 6 h, cells were lysed, and some lysates had 10 mM EDTA added to dissociate polysomes. Samples were treated as described above for panel B, and RT-PCR was performed with the primer/probe sets indicated.

FIG 8

Omomyc displays poor pharmacodynamic properties. (A) Plasma concentration of DyLight 650-labeled Omomyc after administration to mice. A total of 5.22 mg/kg DyLight 650-Omomyc was administered to mice in a single i.v. dose. Terminal plasma samples were collected over time, and the amount of the compound was determined. (B) Tissue distribution of DyLight 650-Omomyc. Tissue samples were collected at the indicated times and analyzed for the presence of the compound. (C) CAFÉ analysis of plasma from non-tumor-bearing mice treated with Omomyc. Shown are examples of capillary electrophoresis and quantitation of the area under the curve for the peaks from the electropherograms for plasma from mice treated with DyLight 650-Omomyc. (D) CAFÉ analysis of tissues from non-tumor-bearing mice treated with Omomyc. Shown are examples of capillary electrophoresis and quantitation of the area under the curve for the peaks from the electropherograms for mouse plasma liver, kidney, spleen, and lung tissue from mice treated with DyLight 650-Omomyc.

FIG 8

Omomyc displays poor pharmacodynamic properties. (A) Plasma concentration of DyLight 650-labeled Omomyc after administration to mice. A total of 5.22 mg/kg DyLight 650-Omomyc was administered to mice in a single i.v. dose. Terminal plasma samples were collected over time, and the amount of the compound was determined. (B) Tissue distribution of DyLight 650-Omomyc. Tissue samples were collected at the indicated times and analyzed for the presence of the compound. (C) CAFÉ analysis of plasma from non-tumor-bearing mice treated with Omomyc. Shown are examples of capillary electrophoresis and quantitation of the area under the curve for the peaks from the electropherograms for plasma from mice treated with DyLight 650-Omomyc. (D) CAFÉ analysis of tissues from non-tumor-bearing mice treated with Omomyc. Shown are examples of capillary electrophoresis and quantitation of the area under the curve for the peaks from the electropherograms for mouse plasma liver, kidney, spleen, and lung tissue from mice treated with DyLight 650-Omomyc.

FIG 9

Models for Max cotranslational association with Myc and Omomyc inhibition of MYC transcriptional activity. (A) Cotranslational binding of Myc to Max when Myc levels are low (normal cells) and when Myc levels are high (transformed cells). (B) Two models of how Omomyc inhibits Myc-mediated transcriptional activity in the cell. (Left) Cotranslational binding of Omomyc to Max, blocking Myc binding to Max, leading to Myc degradation. (Right) Direct binding of Omomyc to E boxes in promoters of Myc-regulated genes as proposed previously by Jung et al. (23).