Intrinsic cell-penetrating activity propels Omomyc from proof of concept to viable anti-MYC therapy

Abstract

Inhibiting MYC has long been considered unfeasible, although its key role in human cancers makes it a desirable target for therapeutic intervention. One reason for its perceived undruggability was the fear of catastrophic side effects in normal tissues. However, we previously designed a dominant-negative form of MYC called Omomyc and used its conditional transgenic expression to inhibit MYC function both in vitro and in vivo. MYC inhibition by Omomyc exerted a potent therapeutic impact in various mouse models of cancer, causing only mild, well-tolerated, and reversible side effects. Nevertheless, Omomyc has been so far considered only a proof of principle. In contrast with that preconceived notion, here, we show that the purified Omomyc mini-protein itself spontaneously penetrates into cancer cells and effectively interferes with MYC transcriptional activity therein. Efficacy of the Omomyc mini-protein in various experimental models of non-small cell lung cancer harboring different oncogenic mutation profiles establishes its therapeutic potential after both direct tissue delivery and systemic administration, providing evidence that the Omomyc mini-protein is an effective MYC inhibitor worthy of clinical development.

Fig. 1. The Omomyc mini-protein forms highly stable DNA binding homodimers and heterodimers with MAX in solution.

(A) Overlay of the ¹H-¹⁵N-HSQC of ¹⁵N-Myc° in the absence (red) and in the presence (black) of Omomyc. (B) Far–ultraviolet (UV) CD spectra of c-Myc° (red, 8 μM monomer units), Omomyc (green, 8 μM monomer units), the arithmetic sum of both red and green spectra (gray), and the spectrum of an equimolar mix of c-Myc° and Omomyc at a total concentration of 16 μM in monomer units (black) recorded at 25°C. (C) Thermal denaturation of the solutions described in (B). (D) Thermal denaturation of the solutions described in (B) to which equimolar amounts (in dimer units) of an E-box DNA duplex were added. Contribution of the DNA to the denaturation curves was removed. (E) Overlay of the 1H-¹⁵N-HSQC of ¹⁵N-Max° in the absence (blue) and in the presence (black) of Omomyc. (F) Far-UV CD spectra of Max° (blue, 8 μM monomer units), Omomyc (green, 8 μM monomer units), arithmetic sum of both blue and green spectra (gray), and experimental equimolar mix of Max° and Omomyc at a total concentration of 16 μM in monomer units (black) recorded at 25°C. (G) Thermal denaturation of the solutions described in (F). (H) Thermal denaturation of the solutions described in (F) to which equimolar amounts (in dimer units) of E-box DNA duplex were added. All thermal denaturations were monitored at 222 nm.

Fig. 2. The Omomyc mini-protein spontaneously penetrates into human NSCLC cells.

(A) Lung adenocarcinoma H1299, H1975, and A549 cell lines were treated with 0.32, 0.64, 3.2, or 12.8 μM Omomyc-AF488 for 15 min in serum-free medium, trypsinized, and analyzed by flow cytometry. (B) H1299, H1975, and A549 cells were treated with 3.2 μM Omomyc-AF488 for 4 hours, stained with Hoechst 3342, washed, mounted, and analyzed by confocal microscopy. Scale bar, 10 μm. (C) NSCLC cell lines were preincubated at 4° or 37°C and treated with 0.64 μM Omomyc-AF488 for 15 min at the same temperature, trypsinized, and immediately analyzed by flow cytometry or pretreated with inhibitors (Blebb, blebbistatin; Chlor, chlorpromazine; Cyt D, cytochalasin D; EIPA, 5-ethylisopropylamiloride; Mβ methyl-β-cyclodextrin) of endocytosis or lipid raft-mediated macropinocytosis (M, macropinocytosis; Cav, caveolin-mediated; Clat, clathrin-mediated) and then treated with 0.64 μM Omomyc-AF488 for 15 min in the presence of the inhibitor followed by trypsinization and analysis by flow cytometry. Inhibition of entrance (%) compared to vehicle-treated cells at 37°C is shown for each cell line. (D) Dose response of the NSCLC panel of cells and of MYC-independent SH-EP cells to increasing concentrations of Omomyc as measured by resazurin dye colorimetric assay. The median inhibitory concentration (IC₅₀) was 5.9 μM for H1299, 8.2 μM for H1975, 11.4 μM for A549, and 25.6 μM for SH-EP cells. (E) Quantification of cell cycle phase populations from flow cytometric analysis of PI incorporation after 3 days of treatment with 12.8 μM Omomyc (OMO) compared to vehicle control (CTRL). All experiments were performed at least twice for each condition. Mean and SD are shown in (D) and (E), and statistical significance was calculated by a two-tailed unpaired Student’s t test.

Fig. 3. Omomyc disrupts MYC transcriptional regulation and binding to promoters of its target genes.

(A and B) GSEA comparing gene expression of vehicle-treated versus Omomyc-treated NSCLC cells. Representative plots of MYC signatures enriched in the vehicle samples compared to the treated samples are shown in (A). Normalized enrichment scores (NES) and q values of well-characterized MYC signature gene sets are listed in (B) and were calculated from technical triplicates. (C to E) MYC chromatin immunoprecipitation (ChIP)–quantitative polymerase chain reaction (qPCR) from H1299 (C), H1975 (D), and A549 (E) cells treated for 72 hours with 12.8 μM Omomyc (red) or vehicle (black) is shown for typical MYC target proximal promoter regions [Chromosome 8 “gene desert” region (Chr8), nucleolin (Ncl), nucleophosmin (Npm1), interferon-related developmental regulator 2 (Ifrd2), and MYB proto-oncogene–like 2 (Mybl2); vehicle-treated (black) and Omomyc-treated (red)]. (C to E) Mean and SD are shown, and statistical significance was calculated by a two-tailed unpaired Student’s t test. ***P < 0.005 and ****P < 0.001. (F and G) MYC ChIP sequencing (ChIP-seq) from A549 cells treated for 48 hours with 12.8 μM Omomyc or vehicle (CTRL). (F) MYC occupancy at selected MYC target proximal promoter regions: ribosomal protein L23 (RPL23), chromobox protein homolog 5 (CBX5), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), and eukaryotic translation initiation factor 4B (EIF4B). The ChIP-seq enrichment is displayed as reads per million (RPM). (G) Global analysis: MYC ChIP-seq RPM were calculated in the region (±1 kb) around the transcriptional start site (TSS) of all active promoters (n = 32292), in 50–base pair bins. The promoters are sorted by the RPM in the full 2-kb promoter region in the vehicle sample. The panel on the right shows promoters with a MYC ChIP-seq peak in the vehicle condition (n = 3014).

Fig. 4. Omomyc displays efficacy in a lung adenocarcinoma mouse model upon intranasal administration.

(A) Quantification of Omomyc-DFO-⁸⁹Zr detected in the lungs of healthy mice as a function of time is represented as %ID. Mean, SD, and number of animals are shown. (B) Immunofluorescence of lung tissue from mice treated with the Omomyc mini-protein. A specific anti-Omomyc antibody confirms the presence of Omomyc in the lung cells 4 hours after administration. Arrowheads indicate positively stained nuclei. Scale bars, 10 μm. Higher magnification of the area surrounded by a white dashed line is shown in the right panel. (C) 3D rendering of mPET/mCT imaging of lungs of a tumor-bearing mouse 24 hours after intranasal administration of Omomyc-DFO-⁸⁹Zr (2.37 mg/kg). CT data are displayed in gray scale and Omomyc-DFO-⁸⁹Zr mPET data in color scale (n = 2 mice were analyzed). The color scale is expressed as %ID/g for Omomyc-DFO-⁸⁹Zr uptake. See the accompanying supplementary movie for a rotating representation (Movie S1). (D) Graphical representation of total cells scored as proliferating (Ki67-positive) cells in the lung of mice treated for 3 days or 1 week with Omomyc (2.37 mg/kg) or vehicle (VEH). Median, interquartile range (IQR), and number of animals are shown. Two-tailed unpaired Mann-Whitney test was used to analyze statistical differences between the groups. (E) GSEA comparing gene expression in lung tumors from vehicle-treated versus Omomyc-treated mice. NES and false discovery rate (FDR) q values of MYC signatures and other relevant gene sets are shown. Four representative plots are shown.

Fig. 5. Omomyc reduces tumor burden in a KRas^G12D-driven lung adenocarcinoma mouse model.

Mice bearing KRas^LSL-G12D/+-induced lung tumors were treated for 4 weeks with Omomyc (2.37 mg/kg) or vehicle administered intranasally. (A) Representative transverse planar CT images from each experimental group taken at treatment onset and endpoint with tumors circled by yellow dotted lines. (B) Relative tumor volumes (RTVs) of vehicle-treated (open circles) and Omomyc-treated (black dots) mice were measured weekly. Mean normalized to size at treatment onset and SEM are shown. Longitudinal growth within a group was analyzed by Kruskal-Wallis test. In contrast to the vehicle-treated group (P = 0.0002), the Omomyc-treated tumors did not show significant growth throughout treatment (P = 0.1579). For analysis at endpoint, two-tailed unpaired Mann-Whitney test was used to assess statistical significance of the difference between the groups (**P < 0.01). (C) Tumor grade was blindly assessed by histological and pathological analysis of hematoxylin and eosin–stained slides of lungs of treated mice and is represented as a pie chart for each group. (D to G) Ki67 (D), CC3 (E), and intratumoral CD3-positive cells (F) of representative animals were quantified by immunostaining at endpoint. Median, IQR, and number of mice are shown. Statistical significance was calculated using a two-tailed Mann-Whitney test. (G) Representative images are shown. Scale bars, 20 μm for Ki67 and CC3 panels. Scale bars, 50 μm for CD3 panel.

Fig. 6. Intravenous treatment with Omomyc and combination with paclitaxel display superiority to chemotherapy in a lung adenocarcinoma xenograft model.

(A) RTV is reduced in mice with established disease (H1975 xenografts) treated with Omomyc (60 mg/kg) four times per week (OMO, green dots) (n = 14 per group). (B) RTV of vehicle-treated (open circles), paclitaxel (PTX)–treated (open squares), Omomyc-treated (120 mg/kg; green dots), and combination-treated (OMO + PTX; red squares) mice measured twice per week (n = 20 per group). Mean and 95% confidence interval are shown, and statistical significance compared to vehicle-treated group at each time point was calculated by a two-tailed unpaired Student’s t test (A and B). *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001. (C) Tumor volume at experimental endpoint (30 days). Mean and SD are shown. (D) A Kaplan-Meier survival curve is shown, and the statistical significance was determined by a log-rank test. (E and F) Ki67 (E) and CC3 (F) positivity was quantified by immunostaining at endpoint for seven representative animals per group. Median and IQR are shown.