A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation

MYC is a key player in tumor development, but unfortunately no specific MYC-targeting drugs are clinically available. MYC is strictly dependent on heterodimerization with MAX for transcription activation. Aiming at targeting this interaction, we identified MYCMI-6 in a cell-based protein interaction screen for small inhibitory molecules. MYCMI-6 exhibits strong selective inhibition of MYC:MAX interaction in cells and in vitro at single-digit micromolar concentrations, as validated by split Gaussia luciferase, in situ proximity ligation, microscale thermophoresis and surface plasmon resonance (SPR) assays. Further, MYCMI-6 blocks MYC-driven transcription and binds selectively to the MYC bHLHZip domain with a KD of 1.6 ± 0.5 μM as demonstrated by SPR. MYCMI-6 inhibits tumor cell growth in a MYC-dependent manner with IC50 concentrations as low as 0.5 μM, while sparing normal cells. The response to MYCMI-6 correlates with MYC expression based on data from 60 human tumor cell lines and is abrogated by MYC depletion. Further, it inhibits MYC:MAX interaction, reduces proliferation and induces massive apoptosis in tumor tissue from a MYC-driven xenograft tumor model without severe side effects. Since MYCMI-6 does not affect MYC expression, it is a unique molecular tool to specifically target MYC:MAX pharmacologically and it has good potential for drug development.

(Qiagen) affinity bench column, or using a HisTrap HP column (5 mL) with an ÄKTA system.
The purifications were carried out according to manufacturer instructions. Other MYC proteins were purified in denaturing conditions as described above. All the proteins were dialyzed against PBS, pH 7, at 5 °C overnight. The purity of the proteins was confirmed by SDS-PAGE analysis and Mass Spectrometry analysis. Lyophilized YFP and CFP were purchased from Medical and Biological Laboratories Co and Bovine serum albumin (BSA) was included in the MST kit from NanoTemper. BCL-X L was purchased from Abcam, GST-MAD bHLHZip was purchased from Novus Biologicals. Proteins were dissolved in PBS, with or without 0.05% Triton-X-114 (Sigma). Truncated p53 core protein was a kind gift from Klas Wiman, CCK, Karolinska Institutet.
Cell proliferation assays. Cell growth and viability was estimated in triplicates with WST-1 (Roche) or Resazurin sodium salt (Sigma-Aldrich) assays in medium at 37°C and 5% CO 2 for 2 hours after which absorbance or fluorescence, respectively, was measured with an Omega Fluostar (BMG Labtech) in a 96 well plate format. Cell counting was done in 96 well plates with CellTracker Green (ThermoFisher) for 30 min in the incubator, after which DAPI was added for 5 min. Images were taken in the green (FITC) and blue (DAPI) channels using a fluorescence microscope (ImageXpress Micro, Molecular Devices). For anchorageindependent growth assay, cells were suspended in 250 µl 0.35% SeaPlaque agarose (InVitro) and seeded into 24 well plates which had previously been coated with a bottom layer of 250 µl 0.7% agarose. After 16 days, the colonies were stained with 100 µg/ml MTT (Sigma) overnight and colonies were counted.
Immunohistochemistry. Apoptosis was visualized at the single-cell level on tumor cryosections using the TUNEL method using the In Situ Cell Death Detection Fluorescein-Roche kit and analyzed by fluorescence microscopy. Briefly, tumor cryosections were fixed in 4% paraformaldyde (PFA) and the assay was performed following the instructions from the manufacturer. Negative controls were run using the reagent without the TdT enzyme. Samples were mounted using Vectashield mounting medium (Vector) and DAPI was used as nuclear counterstaining. Cell proliferation and microvascular density (MVD) were evaluated through Ki67 and CD31staining, respectively, on tumor cryosections and detected by immunofluoresence. Visualization and image acquisition was done in a Zeiss microscope, and for panoramic views by a Vectra imaging system. For quantification, areas with positive TUNEL and Ki67 staining were measured using Image J software. For the analysis of MVD, the number of vessel structures per microscopic field was calculated.  and GI50 response data to MYCMI-6 (y-axis) were extracted from the NIH database CellMiner™, see Supplemental Table S1, and plotted in a log graph. Red dots represent cell lines with elevated MYC protein levels. Orange dots represent cell lines with lower MYC protein levels. Black dots represent cell lines that have ABCB transporter mutations, which for this reason possibly have a reduced response to MYCMI-6. A general linear model was used to calculate correlation between MYC mRNA levels and response to MYCMI-6. P=0.1606761.

Supplemental References
B) Data was extracted as in A and supplemented with MYC protein data from the Novartis proteome scout SymAtlas Project and literature as described in Table 1. Cell lines were divided into higher or lower total levels of MYC (mRNA/protein) than average (x-axis) and their response to MYCMI-6 (y-axis) and plotted in a bar graph. A general linear model was used to test for the relationship between MYC total levels and response to MYCMI-6 (P=0.0008924, X 2 =11.0385).