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

Castell, Alina; Yan, Qinzi; Fawkner, Karin; Hydbring, Per; Zhang, Fan; Verschut, Vasiliki; Franco, Marcela; Zakaria, Siti Mariam; Bazzar, Wesam; Goodwin, Jacob; Zinzalla, Giovanna; Larsson, Lars-Gunnar

doi:10.1038/s41598-018-28107-4

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Published: 03 July 2018

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

Alina Castell¹^na1,
Qinzi Yan¹^na1,
Karin Fawkner¹^na1^nAff2,
Per Hydbring¹^nAff3,
Fan Zhang¹,
Vasiliki Verschut¹,
Marcela Franco¹,
Siti Mariam Zakaria¹,
Wesam Bazzar¹,
Jacob Goodwin¹,
Giovanna Zinzalla¹ &
…
Lars-Gunnar Larsson¹

Scientific Reports volume 8, Article number: 10064 (2018) Cite this article

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Abstract

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 K_D 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.

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Introduction

The MYC family of oncogenes (MYC, MYCN and MYCL, here collectively referred to as “MYC”), encodes basic helix-loop-helix leucine zipper (bHLHZip) transcription factors. Through the HLHZip domain, MYC heterodimerizes with the bHLHZip protein MAX, which enables the MYC:MAX complex to bind E-box regulatory DNA elements throughout the genome, thereby controlling transcription of a large group of specific genes^1,2,3,4,5,6. The direct target gene products in turn influences global RNA and protein synthesis, thereby coordinating multiple fundamental cellular processes, including cell cycle progression, cell growth, apoptosis, senescence, metabolism and stem cell functions^{2,7,8,9,10,11}. Deregulation of expression of MYC family genes/proteins occurs in over half of all human tumors, and is often correlated with aggressive disease, resistance to therapy and poor prognosis^2,4,5,12,13. MYC is therefore considered as one of the most important drivers of tumor development and has been highlighted as a key therapeutic target for cancer therapy for a number of tumor types. However, so far there are no specific anti-MYC drugs available in the clinic. A number of attempts have been made to target MYC indirectly by interfering with transcription of the MYC gene, translation or turnover of the MYC protein or by inhibiting downstream effectors of MYC^14,15,16. Due to the diversity of signals regulating the MYC genes/proteins and the pleiotropic functions of MYC, tumor cells have multiple ways of escaping these pathways to maintain MYC-family expression and activity. The most reliable strategy is therefore probably to target the MYC proteins directly. Since MYC is strictly dependent on MAX for binding E-boxes, targeting MYC:MAX interaction is a conceivable approach to target MYC. Several examples of successful targeting of protein-protein interactions (PPIs) with small molecules, including Nutlin-3a (targeting p53:MDM2)¹⁷, BET inhibitors such as JQ1¹⁸ (bromodomains:histones) and the BH3 mimetic compound Navitoclax/ABT-263 (BCL-2 family interactions)¹⁹ have been reported recently. These compounds, or improved versions thereof, are now in clinical trials^20,21, which have encouraged further research on PPIs as drug targets. Several groups have attempted to find compounds targeting the MYC:MAX interaction by screening small-molecule libraries using FRET²², fluorescence polarization²³, or yeast-two-hybrid (Y2H)²⁴. As a result, a number of small molecules have been reported to target the MYC:MAX or MYC:MAX:DNA interaction^{15,16,22,24,25,26,27,28,29,30,31,32,33}. However, none of these compounds have made their way for clinical studies due to a number of limitations including low potency in vitro or in cells, poor specificity or inadequate bioavailability in vivo^15,16,26,34. Development of more potent and selective MYC:MAX inhibitors is therefore much warranted. The aim of this work was to identify bioactive molecules that (1) efficiently and selectively inhibit the MYC:MAX interaction in vitro and in cells, that (2) bind MYC directly with high affinity, that (3) inhibit MYC-dependent tumor cell growth with high efficacy, that (4) do not affect MYC expression, and that (5) are active in vivo. In a cellular MYC:MAX PPI inhibitor screen, we identified the compound MYCMI-6 that fulfills all these criteria.

Results

Establishment of a cell-based Bimolecular Fluorescence Complementation (BiFC) assay for screening for small molecules interfering with MYC:MAX protein interactions

To screen for inhibitors of the MYC:MAX protein interaction, we utilized BiFC^35,36,37 to monitor the interaction in living cells. Two constructs encoding complementary BiFC fragments of yellow fluorescent protein (YFP) fused to full length MYC (MYC-YFP-C) and MAX (MAX-YFP-N)³⁸, respectively (Figs 1A and S1A), were cotransfected into cells, resulting in a nuclear BiFC signal as a result of MYC:MAX interaction bringing the YFP fragments in close proximity³⁸ (Fig. 1B, left panel). To optimize the system for screening purposes, MYC-YFP-C and MAX-YFP-N were transiently coexpressed in HEK293 cells together with a full-length cyan fluorescent protein (CFP) vector as internal control (Fig. 1C, upper panels), after which the ratio of BiFC/CFP fluorescence intensities was calculated. Replacing wt MYC-YFP with a MYC mutant lacking the bHLHZip domain (ΔbHLHZip) required for interaction with MAX, did not produce BiFC signals, thus demonstrating that the MYC:MAX BiFC signal is highly specific (Fig. 1C, lower panels). A strictly standardized mean difference (SSMD) of −5 was calculated from cells expressing CFP together with MYC:MAX BiFC and MYCΔbHLHZip:MAX BiFC, respectively, suggesting a strong threshold for hit selection. We concluded that this system was suitable for screening small molecule libraries to identify inhibitors of MYC:MAX interaction in cells.

Identification and validation of small molecules selectively targeting the MYC:MAX interaction in cells

We next utilized a diversity set library from the NCI/DTP Open Chemical Repository (http://dtp.cancer.gov) consisting of 1990 compounds to screen for small molecules reducing the MYC:MAX BiFC signal relative to the CFP signal as an indication of potential MYC:MAX inhibition in cells. Compounds (25 μM) decreasing the BiFC/CFP fluorescence ratio by more than the arbitrary threshold of 40% relative to the vehicle (DMSO) control after 24 hours treatment were considered as positive hits. Six compounds fulfilling this criterion (Fig. 1E), while not affecting the interaction between the bZip transcription factors FOS and JUN (Suppl. Fig. S2A), were chosen for further studies and were dubbed MYCMIs (MYC:MAX Inhibitors).

For validation, we established another cellular protein interaction assay based on the use of complementary fragments of the Gaussia luciferase (GLuc)³⁹ fused to full length MYC (MYC-GLuc-C) and MAX (MAX-GLuc-N), respectively (Suppl. Fig. S1B). Cotransfection of HEK293 cells with these constructs together with Firefly luciferase in a dual luciferase assay resulted in a high relative GLuc activity, while a mutant MYC-GLuc-C protein lacking the Zip interaction domain (MYCΔZip) gave only background activity, thus demonstrating the specificity of the system (Yan et al., manuscript in prep.). Treatment of cotransfected cells with the hit molecules (25 μM) showed that all six compounds significantly inhibited MYC:MAX GLuc activities (Fig. 1F), most of them more efficiently than the previously reported MYC:MAX inhibitor 10058-F4²⁴, which was used at a concentration of 64 μM. The most potent among the molecules were MYCMI-6 and MYCMI-7. All compounds inhibited MYCN:MAX GLuc activity to a similar extent as MYC:MAX, but did not reduce GLuc activity of the homodimeric bZIP transcription factor GCN4, thus showing selectivity for MYC family protein:MAX interactions (Fig. 1F).

Next, the effect of the compounds on endogenous MYC protein levels was examined. Two of the compounds, MYCMI-7 and MYCMI-9, reduced MYC protein expression as determined by western blot analysis (Fig. 1G). We focus this paper on three compounds, MYCMI-6 (NSC354961), MYCMI-11 (NSC11656) and MYCMI-14 (NSC49689) (Fig. 2A) that did not affect MYC protein levels to any greater extent, with the aim to identify compounds specifically targeting MYC:MAX interaction without interfering with other MYC activities.

MYCMI-6 selectively inhibits endogenous MYC:MAX interaction and MYC-induced gene expression

To validate the inhibitory effects of the selected MYCMIs on endogenous MYC:MAX interaction in cells, in situ proximity ligation assay (isPLA)⁴⁰ was performed using MYC and MAX antibodies. The interactions were visualized as fluorescent dots mainly localized in the cell nucleus by fluorescence microscopy (Fig. 2B) as previously reported⁴⁰. Treatment of breast cancer cells with the MYCMI-6, MYCMI-11 and MYCMI-14 for 24 hours significantly decreased MYC:MAX isPLA signals to 7%, 23% and 23% of DMSO-treated controls, respectively (Fig. 2B and C). Titration showed an IC₅₀ for inhibition of MYC:MAX of less than 1.5 μM for MYCMI-6 and of approximately 6 μM for MYCMI-11 and MYCMI-14 by isPLA (Fig. 2D). Further, coimmunoprecipitation of endogenous MYC:MAX proteins showed that MYCMI-6 reduced the MYC:MAX protein interaction already at 3 hours post treatment (Fig. 2E). In contrast, the three compounds did not significantly affect the interaction between the bZip transcription factors FRA1 and JUN (Fig. 2B and C) or the interaction between MAX and the bHLHZip protein MXD1(MAD1) – an intracellular competitor of MYC for MAX^41,42 (Suppl. Fig. S2B), thus further supporting MYC:MAX selectivity of the compounds.

To investigate the effect of the compounds on MYC-driven transcription, we utilized U2OS cells expressing a MYC-estrogen receptor (MYC-ER) fusion protein, the activity of which is regulated by the ligand 4-hydroxytamoxifen (HOT)⁴³. Treatment with MYCMI-6 significantly reduced HOT-induced expression of three previously described direct MYC target genes, ODC1, RSG16 and CR2^10,11, while MYCMI-11 and -14 in general reduced expression of the genes to a lesser extent, being significant only for RSG16 (Fig. 2F). Taken together, these results highlight MYCMI-6 as the most potent and selective inhibitor of endogenous MYC:MAX protein interactions and of MYC-driven transcription among the three MYCMIs.

MYCMI-6 is a potent and selective inhibitor of the MYC:MAX bHLHZip interaction in vitro

We next investigated the capacity of the small molecule inhibitors to directly target the MYC:MAX interaction in vitro. Two biophysical interaction assays, microscale thermophoresis (MST)⁴⁴ and surface plasmon resonance (SPR)⁴⁵, were developed using recombinant MYC and MAX proteins (Suppl. Fig. S1C) (manuscript in prep.). MST is based on the shift of fluorescent molecules moving in a temperature gradient (thermophoresis), which can be perturbed by interactions. In the MST MYC:MAX interaction assay, fluorescently labeled MAXbHLHZip was combined with MYCbHLHZip pre-mixed with compound. All three MYCMIs, and in particular MYCMI-6, shifted MAX thermophoresis relative to DMSO, indicating that the compounds affected the MYC:MAX conformation (Fig. 3A). Titration of MYCMI-6 in mixture with MYC and labeled MAX resulted in a thermophoresis shift with a K_d of 4.3 +/− 2.9 μM, while having minor effects on labeled MAX when pre-mixed with unlabeled MAX (Fig. 3B), suggesting that MYCMI-6 discriminates well between MYC:MAX and MAX:MAX interactions.

The SPR method is a highly sensitive assay to determine the affinity between protein and ligand and to measure the kinetics of the interaction⁴⁵. In the SPR MYC:MAX interaction assay, MAXbHLHZip was covalently attached onto a sensor chip and MYCbHLHZip pre-mixed with compound was injected over the surface and thereafter allowed to dissociate. MYCMI-6 inhibited the MYC:MAX heterodimer formation with an IC₅₀ of 3.8 + /− 1.2 μM (Fig. 3C and D), whereas the experimental MYC inhibitor 10058-F4 was less efficient (Figs 3D and S3A). The previously identified MYC:MAX interaction inhibitor KJ-Pyr-9²⁷ had no effect on MYC:MAX formation up to 10 μM (Supp. Fig. S3B).

MYCMI-6 binds directly and selectively to the MYC bHLHZip domain with high affinity

To determine whether MYCMI-6 binds directly to MYC (or MAX), MST and SPR were applied. The bHLHZip domains of MYC or MAX, respectively (Suppl. Fig. S1C), were titrated and mixed with 3 μM of MYCMI-6. There were clear changes in thermophoresis at 1–15 μM of MYC while no obvious changes were observed for MAX, indicating binding of MYCMI-6 to MYC but not to MAX at these concentrations (Fig. 4A). Further, MYCMI-6 did not bind the control proteins BCL-X_L (Suppl. Fig. S4A) and bovine serum albumin (BSA) (Suppl. Fig. S4B). Similar results were obtained in a reverse MST experiment, keeping labeled MYC at a fixed concentration while titrating MYCMI-6, showing relative changes in thermophoresis at concentrations above 100 nM, supporting that MYCMI-6 binds MYC with affinity in the low micromolar range (Suppl. Fig. S4C).

Direct binding of MYCMI-6 to MYC was also analyzed by SPR, where MYC bHLHZip was immobilized on the chip and different concentrations of MYCMI-6 were injected in kinetic experiments. Association and dissociation rates of the compound were obtained from a 1:1 Langmuir model after subtraction of reference cell values, suggesting a K_D value of 1.6 ± 0.5 μM (Fig. 4B). From equilibrium binding experiments (Suppl. Fig. S5A), a K_D value of approximately 1.5–2 μM was determined by an equilibrium binding plot (Fig. 4C). The previously identified MYC:MAX interaction inhibitors 10074-G5 and #474 (an analogue of 10058-F4)³² used as reference compounds were found to bind to MYC with K_Ds of 28 and 15 μM, respectively (data not shown). We could, however, not detect any binding of KJ-Pyr-9 up to 8 μM in this assay (Suppl. Fig. S5B). Further, MYCMI-6 binding to MAX, MXD1 (MAD1) (Figs 4C and S5C and D), p53 (Fig. 4D), BSA (Suppl. Fig. S5E) and YFP (data not shown) were negligible. Binding to BCL-X_L was only 10% of the theoretical maximum at 2 μM MYCMI-6 (Suppl. Fig. S5F). In summary, the MST and SPR experiments suggest that MYCMI-6 binds the bHLHZip domain of MYC directly, selectively and with affinity in the low micromolar range.

MYCMI-6 selectively suppresses MYC-driven tumor cell growth with high efficacy

We next addressed if treatment with MYCMI-6, -11 and -14 inhibits tumor cell growth and if this correlates with the MYC status of the cells. For this purpose, we first utilized a panel of neuroblastoma cell lines with or without MYCN-amplification. Since a subset of MYCN-non-amplified neuroblastomas have been shown to exhibit deregulated expression of (c)-MYC⁴⁶, we investigated the expression of MYCN and MYC as well as total level of MYC-family proteins in the six cell lines using antibodies specific for MYCN, MYC or pan-MYC. While the three cell lines with MYCN-amplification all expressed high levels of MYCN protein, two out of three of the MYCN-non-amplified cell lines expressed MYC, although the total level of MYC-family proteins were much lower in these cells than in the MYCN-amplified cell lines (Fig. 5A). The cell lines were exposed to MYCMI-6 (6.25 μM), MYCMI-11 or MYCMI-14 (25 μM) or the reference 10058-F4 (64 μM) for 48 hours. All four compounds reduced growth of the MYCN-amplified cell lines significantly stronger than the MYCN-non-amplified cell lines (Fig. 5A). Titration experiments confirmed that MYCMI-6 discriminates between MYCN-amplified and MYCN-non-amplified neuroblastoma cell lines cells, with average growth inhibition of 50% (GI₅₀) values of 2.5–6 μM for MYCN-amplified cell lines and around 20 μM or higher for MYCN-non-amplified cell lines (Fig. 5B). Notably, in the SK-N-F1 cell line, which essentially did not respond at all to MYCMI-6, MYCN or MYC were hardly detectable. These results demonstrate that the response to MYCMI-6 and the other MYCMIs in general correlated well with the total level of MYC-family proteins. Further, the MYCMI-6, MYCMI-11 and MYCMI-14 efficiently inhibited anchorage-independent growth of MYCN-amplified neuroblastoma cells with GI₅₀ values of <0.4, 5 and 0.75 μM, respectively (Fig. 5C and D). These results demonstrate that MYCMI-6 is clearly the more potent and selective of the MYCMIs, and we therefore focused the remainder of this work on MYCMI-6.

To verify that MYCMI-6 inhibited MYCN:MAX interaction and the transcriptional output of MYCN in MYCN-amplified neuroblastoma cells, we performed MYCN:MAX isPLA and measured the expression of a panel of verified MYC family target genes in neuroblastoma⁴⁷ in response to MYCMI-6. Figure 5E–G shows that MYCMI-6 significantly inhibited MYCN:MAX interaction and the expression of MYC/MYCN target genes already at a concentration of 2.5 μM, while maintaining the expression of MYCN and MAX (Suppl. Fig. S6). This is consistent with the results from MYCN:MAX GLuc interaction assays after MYCMI-6 treatment (Fig. 1F).

Moreover, MYCMI-6 significantly inhibited growth of Burkitt’s lymphoma (BL) cells - another classical example of a MYC-driven tumor, having translocations of MYC to one of the immunoglobulin loci - in a dose-dependent manner with an average GI₅₀ of 0.5 μM (Fig. 6A).

To further investigate whether the levels of MYC expression in tumor cells correlate with the growth inhibitory response, we utilized GI₅₀ data from the NCI-60 diverse human tumor cell line panel available for MYCMI-6 by the Developmental Therapeutics Program of the U.S. National Cancer Institute (DTP-NCI), extracted from the NIH-supported CellMiner™ version 2.1 (https://discover.nci.nih.gov/cellminer). Since deregulated MYC expression is frequently manifested at the protein level, for instance through protein stabilization or deregulated mRNA translation^14,48,49, we complemented the analysis with MYC protein data obtained from Novartis proteome scout SymAtlas Project (https://proteomescout.wustl.edu/proteins/52581/expression) or elsewhere in the literature (for details, see Supplementary Table S1), and separated cell lines with high or low MYC mRNA and/or high protein level into the categories “higher MYC” and “lower MYC” cell lines, respectively, as indicated in Supplementary Table S1 and Suppl. Fig. S7A and B. There was a significant correlation between the response to MYCMI-6 and the MYC mRNA/protein levels among the 60 tumor cell lines, whereas an analysis based on MYC mRNA alone did not reach significance (Suppl. Fig. S7A and B). Categorizing the cell lines as “responsive” or “less responsive” to MYCMI-6 based on average logarithmic GI₅₀ values, we found that 79% of the cell lines with higher MYC expression showed a good growth inhibitory response to MYCMI-6, while 70% of the cell lines with lower MYC expression were less responsive (Fig. 6B). Statistical analysis showed that the probability of a “higher MYC” cell line being responsive was significantly higher than not being responsive, while the probability of a “lower MYC” cell line to be less responsive was significantly higher than being responsive (Fig. 6B), thus demonstrating a strong correlation between MYC expression and growth inhibitory response to MYCMI-6 in human cancer cell lines.

MYCMI-6 treatment reduces cell growth and survival in a MYC-dependent manner but is not cytotoxic to normal human cells

To further explore the MYC-dependence of MYCMI-6 cell growth inhibitory responses, we utilized immortal H015.19 MYC null rat fibroblasts derived from Tgr1 (parental cells) and H0Myc3 cells, which has been reconstituted with MYC⁵⁰. MYCMI-6 strongly inhibited growth of wt and reconstituted cells, but did not significantly affect growth of the MYC null cells, thus showing a clear difference in response between MYC expressing and MYC-deficient cells (Fig. 6C), thus further supporting the conclusion that the cellular response after MYCMI-6 treatment is MYC-dependent.

We next addressed the question whether cellular responses to MYCMI-6 treatment differentiate between MYC-dependent tumor cells and normal primary human cells. As shown in Fig. 6D, MYCMI-6 (12.5 μM) dramatically increased cell death and reduced cell number in MYCN-amplified SK-N-DZ neuroblastoma cells after 24 hours treatment but had only marginal effects on the number of viable cells and the percentage of dead cells in normal lung (IMR-90) and foreskin (BJ) human fibroblasts. We concluded that MYCMI-6 was well tolerated by the normal human cells at a concentration that was highly toxic to MYC-dependent tumors cells, showing good discrimination between cancer cells and normal cells in response to MYCMI-6 resulting in a good therapeutic window for this compound.

MYCMI-6 induces massive apoptosis and reduces tumor cell proliferation, tumor microvasculature density and MYC:MAX interaction in a MYC-dependent xenograft tumor model in vivo

To analyze the effects of MYCMI-6 on tumor physiopathology in vivo, we utilized a mouse xenograft tumor model based on human MYCN-amplified SK-N-DZ neuroblastoma cells. Tumor cells were injected into the flank of athymic nude mice and allowed to form tumors after which MYCMI-6 or vehicle were administered by daily intraperitoneal injection at a dose of 20 mg/kg body weight for 1–2 weeks. TUNEL-staining of tumor sections revealed a dramatic increase in the extension of apoptotic areas in the tumors (Fig. 7A and B) and a significant increase in non-proliferative areas as determined by Ki67 staining (Fig. 7C and D) in tumors from MYCMI-6-treated mice compared with vehicle-treated mice. CD31 staining of endothelial cells revealed a significantly reduced microvascular density (MVD) in MYCMI-6-treated mice compared with vehicle-treated mice (Fig. 7C,E and F). Further, tumors from MYCMI-6-treated mice displayed signs of necrosis and hemorrhage and exhibited scar tissue to a larger extent than vehicle-treated mice (Suppl. Fig. S8A). Importantly, isPLA analysis showed a significant reduction in MYCN:MAX interaction in tumors from MYCMI-6- compared to vehicle-treated mice (Fig. 7G and H), indicating that MYCMI-6 reaches, and is active against, its target in vivo. MYCMI-6 administration was well tolerated in mice with only slight and temporal effects on body weight (Suppl. Fig. S8B).

In summary, administration of MYCMI-6 to MYCN-dependent tumor-bearing mice resulted in the reduction of MYCN:MAX interaction, tumor cell proliferation and MVD and in induction of apoptosis in the tumor tissue without causing severe side effects.

Discussion

Targeting the interaction between MYC and its obligatory heterodimerization partner MAX, which is required for specific DNA-binding of the MYC:MAX complex, has been a desired goal ever since the discovery of MAX¹. A number of efforts from several laboratories has been made to identify small molecule inhibitors targeting this interaction^15,16,26,34, but many of these suffer from either poor efficacy, weak affinity for MYC, poor selectivity (or the lack of information about selectivity) and/or discrepancy between activity in vitro and in vivo. While these compounds were all identified through biochemical in vitro protein interaction assays or on the yeast2hybrid system^{22,24,25,27,28,30}, we utilized a cell-based BiFC assay^35,36,37 to screen for MYC:MAX interaction inhibitors. BiFC has previously been used successfully to identify inhibitors of p65:p50 NFkB subunit interactions in cells⁵¹. Cell-based screens have the advantage that hit compounds exhibit cell compatible features and interact with targets in their physiological state, thereby increasing the probability of finding hit molecules with higher bioactivity already at the screening level.

Here we describe three “hits” from our BiFC screening approach using a diverse library of small molecules from DTP/NIH (http://dtp.cancer.gov), MYCMI-6, MYCMI-11 and MYCMI-14. We could not find any obvious structural resemblance between the identified compounds, nor between these and previously described MYC:MAX inhibitors. The three MYCMIs were validated using a number of cell based and biophysical in vitro validation assays with the aim to identify molecules fulfilling the following criteria: (1) inhibition of MYC:MAX interaction in cells and in vitro with high efficacy and selectivity; (2) direct binding to MYC with high affinity and selectivity; (3) inhibition of tumor cell growth with high efficacy in a MYC-dependent manner; (4) MYC:MAX inhibition occurring under maintained MYC expression, thereby enabling specific pharmacological studies of the role of MYC:MAX in MYC biology; and (5) MYC:MAX inhibitory activity in tumor tissue in vivo. To our knowledge, no previously reported MYC:MAX inhibitor has fulfilled all of these five criteria.

Regarding the first criterion, MYCMI-6, MYCMI-11 and MYCMI-14 significantly inhibited MYC:MAX and MYCN:MAX interactions in human cells as verified by GLuc and isPLA assays (Figs 2 and 3), showing IC₅₀s for inhibition of endogenous interactions of 1.5, 6 and 6 μM, respectively. Importantly, at active concentrations, the three compounds did not significantly inhibit cellular interactions between MAX and the intracellular bHLHZip MYC competitor MXD1 (MAD1), or hetero- or homodimer interactions between the bZip transcription factors FOS:JUN, GCN4:GCN4 and FRA1:JUN, which form similar dimeric α-helical structures as interacting bHLHZip proteins⁵² (Figs 2 and 3 and S2). As comparison, the IC₅₀s of MYC:MAX inhibition in cells for previously reported compounds range from 10–100 μM in different cellular MYC:MAX interaction assays^{25,27,32,53,54,55}. Apart from the MYCMIs, only 10058-F4 has been shown to be selective for MYC:MAX vs. MXD:MAX (in Y2H assays in yeast cells)²⁴, and for most inhibitors data on selectivity in general in cells have been poor or are lacking.

In the biophysical PPI validation assays based on MST and SPR, MYCMI-6 inhibited the interaction between the recombinant bHLHZip domains of MYC and MAX with a K_d of 4.3 and 3.8 μM, respectively (Fig. 3), which is well in line with the IC₅₀ of 1.5 μM in the cellular isPLA assay. In contrast, MYCMI-11 and MYCMI-14 were less effective at inhibiting the MYC:MAX interaction in the MST assay as well as in the isPLA. Further, MYCMI-6 did not inhibit MAX:MAX homodimerization at active concentrations, thus showing selectivity for MYC:MAX. As comparison, the reference 10058-F4 inhibited the MYC:MAX interaction less well, with an IC₅₀ of >25 μM in the SPR assay. KJ-Pyr-9 has been shown to inhibit recombinant MYC:MAX:DNA and MAX:MAX:DNA interaction with an IC₅₀ of approximately 10 and 30 μM, respectively, in fluorescence polarization assays^25,27. In our hands, KJ-Pyr-9 did, however, not affect the MYC:MAX interaction in the SPR assay at concentrations up to 10 μM. The reason for this discrepancy between the results obtained by these two methods is unclear. Using a GLuc screening assay in vitro, Choi et al.²⁵ identified the molecule sAJM589, which inhibited MYC:MAX interaction with an IC₅₀ of 1.8 μM while not affecting MAX:MAX and other interactions at 20 μM, which is comparable with our data for MYCMI-6. However, inhibition of MYC:MAX in cells by sAJM589 seemed to require higher concentrations (10–30 μM), which coincided with downregulation of MYC expression²⁵.

Regarding the second criterion, direct binding of MYCMIs to the recombinant bHLHZip domain of MYC in vitro, MST and SPR analyses showed that MYCMI-6 binds to MYC with a K_D of 1.6 ± 0.5 μM (Figs 4 and S4 and S5), again in good agreement with both MYC:MAX in vitro MST, SPR and cellular isPLA data. MYCMI-6 did not bind to MAX in vitro in the MST or SPR assays. Further, binding of MYCMI-6 to the bHLHZip domain of MAD1, the p53 core domain, BSA and YFP were negligible by SPR (Figs 4 and S5), thus demonstrating selectivity for MYC bHLHZip domain. By comparison, 10074-G5, 10058-F4 and the 10058-F4 analogue #474 were reported to have affinities for MYC with a K_D around 32, 40 and 17 μM as monitored by SPR, respectively⁵⁴. The selectivity of these compounds with respect to direct binding to reference proteins by SPR has, however, not been described. KJ-Pyr-9, was reported to bind MYC, but not MAX, with a K_d of 6.5 nM in a Backscattering Interferometry (BSI) assay²⁷. We could not detect any binding of KJ-Pyr-9 to MYC up to 8 μM in our SPR assay (Suppl. Fig. S5B). The reason for this discrepancy is unclear at present.

Park and coworkers previously published work describing MYCMI-6 (BXI-61) as an inhibitor of BCL-X_L:BAK interactions by binding BCL-X_L at a concentration of 14 nM based on an in vitro fluorescence polarization assay⁵⁶. We did not observe any binding of MYCMI-6 to BCL-X_L in the MST assay up to 10 μM, but detected some binding in the SPR assay (Suppl. Figs S4A and S5F), indicating that MYCMI-6 has at least a 5-fold lower affinity for BCL-X_L than for MYC. In the light of our findings, MYC:MAX inhibition is likely to contribute to the reported inhibition of lung tumor cell growth by MYCMI-6 in a mouse xenograft model⁵⁶. MYCMI-6 (NSC354961) was also reported as a hit in in vitro screens for inhibitors of hTERT, S-adenosylmethionine decarboxylase (AdoMetDC) and of HDM2, in all these cases with approximately 10-fold higher IC₅₀s than what we found for inhibition of MYC:MAX in vitro^57,58,59. Notably, the hTERT inhibitory activity was found to be artificial due to unspecific binding to DNA in vitro at these concentrations⁵⁸. While the activity towards AdoMetDC was not validated in cells⁵⁷, MYCMI-6/NSC354961 did cause increased expression of p53 in cells at concentrations from 5 μM⁵⁹. However, in the light of our findings, inhibition of MYC:MAX by MYCMI-6 is likely to contribute to p53 activation since inhibition of MYC by the dominant negative OmoMYC protein, which interferes with the MYC:MAX complex, is known to activate the p53 pathway⁶⁰. One should also point out that in none of these studies direct binding of MYCMI-6 to the proposed target was demonstrated.

Regarding the third criterion, MYC-dependent tumor cell inhibition, MYCMI-6, MYCMI-11 and MYCMI-14 inhibited growth of neuroblastoma cells with MYCN-amplification to a significantly larger extent than MYCN-non-amplified cell lines, which correlated with the much higher expression of MYCN in the MYCN-amplified cell lines than of MYC in the non-amplified cell lines as determined by western blot analysis using pan-MYC antibodies (Fig. 5). MYCMI-6 was again clearly the most potent and selective of the MYCMIs. MYCMI-6 has GI₅₀s as low as 0.5 μM in neuroblastoma and Burkitt’s lymphoma cells with deregulated MYCN/MYC, which is much more potent in comparison to most of the previously reported MYC:MAX inhibitors, such as 10058-F4, 10058-F4 analogues and 10074-G5. MYCMI-6 GI₅₀s are well in agreement with the isPLA IC₅₀ data and the effect on MYC target gene expression (Fig. 2). The cell growth inhibitory activities of certain analogues to 10074-G5, KJ-Pyr-9 and sAJM589 have been reported to be in a similar range as MYCMI-6 for some tumor cell lines^25,26,27, but it is not clear if these effects are primarily linked to MYC:MAX inhibition or reduced MYC expression (or other effects). The MYC-dependent effects of MYCMI-6 was further supported by the significant correlation between the levels of MYC mRNA/protein and the response to MYCMI-6 within the NCI-60 tumor cell line panel, and the differential response to MYCMI-6 between MYC knockout and wt/reconstituted Rat1 cells (Fig. 6 and Suppl. Fig. 7). The former result also underscores that measuring MYC protein, not only mRNA, levels in tumors is important for predicting response to MYC inhibitors. Importantly, MYCMI-6 was not cytotoxic to primary normal human fibroblasts at concentrations that killed almost 100% of MYC-dependent tumor cells, thus demonstrating a promising therapeutic window for MYCMI-6.

Regarding the 4^th criterion, effects on MYC expression, it is important to keep in mind that many MYC:MAX inhibitors, including 10058-F4, 10074-G5 and sAJM589 down-regulate MYC-family protein levels, while MYCMI-6, MYCMI-11, MYCM-14 (Figs 2 and S6) and #474 do not^25,54,61. For the former category, it is therefore difficult to distinguish whether the effects on MYC-dependent tumor cells is due to down-regulation of MYC expression, which will affect all MYC functions, or inhibition of MYC:MAX interaction, which will only affect MYC functions controlled by the MYC:MAX complex.

Inhibition of tumor cell growth in vivo using mouse tumor models has been reported previously for the MYC:MAX inhibitors 10058-F4, KJ-Pyr-9, KSI-3716 and Mycro3^27,28,61,62. In these studies it was not demonstrated whether the compounds inhibited MYC:MAX interaction in vivo, but Mycro3 was shown to reduce expression of MYC. Utilizing a MYCN-amplified neuroblastoma xenograft tumor model, we could show that MYCMI-6 treatment significantly reduced MYCN:MAX interaction in tumor tissue, suggesting that MYCMI-6 reached and was active against its target in vivo (Fig. 7). This correlated significantly with massive induction of apoptosis and reduction of tumor cell proliferation in large areas of the tumors, which is typically observed after blocking MYC function in MYC ON/OFF and in the dominant negative Omomyc mouse tumor models^63,64,65,66, as well as by 10008-F4 and by Mycro3 in neuroblastoma⁶¹ and pancreatic⁶² tumor models, respectively. Other features of MYCMI-6 treatment were reduction in microvascularity and signs of increased hemorrhage and necrosis, which might reflect collapse of tumor vasculature previously observed in the Omomyc tumor model⁶⁵. The effects of MYCMI-6 treatment therefore resemble the effects of MYC depletion by genetic means in mouse tumor models. Importantly, MYCMI-6 treatment was well tolerated by the mice and did not result in severe side effects. As mentioned above, MYCMI-6 has previously been shown to inhibit lung tumor growth in a mouse xenograft model⁵⁶. Further validation of the efficacy of MYCMI-6 in MYC-dependent mouse tumor models is needed in the future.

In conclusion, we have identified three new MYC:MAX inhibitors in a cell-based protein interaction screen. Among these, MYCMI-6 fulfills all the criteria we set up for a useful MYC:MAX inhibitor: (1) Potent and selective inhibition of MYC:MAX interaction in cells and in vitro; (2) selective direct binding to MYC; (3) inhibition of tumor cell growth in a MYC-dependent manner, all at similar single-digit micromolar concentrations, while sparing normal cells; (4) In contrast to many other MYC:MAX inhibitors, MYCMI-6 did not affect MYC or MYCN expression, and thus could be used as a specific molecular tool to explore role of the MYC:MAX and MYCN:MAX interactions in normal and tumor cells; and (5) Exhibiting MYC:MAX inhibitory activity in tumor tissue in vivo. For future research, it will be important to determine the precise mechanism of action of MYCMI-6 to enable the development of more potent and selective molecules towards clinically relevant direct MYC inhibitors.

Methods

All experiments were performed in accordance with the guidelines and regulations of the Karolinska Institutet.

Cell lines

HEK293A, MCF7, MDA-MB231, SK-N-F1, SK-N-RA, SK-N-AS, COS-7, HeLa, BJ cells, and Rat1 fibroblasts TGR-1 (wt), HO15.19 (MYC knockout) and HOmyc3 (MYC reconstituted HO15.19) were maintained in DMEM. The neuroblastoma cell lines Kelly, IMR-32 and SK-N-DZ, and the Burkitt’s lymphoma cell lines Mutu, Daudi and ST486 were kept in RPMI. SK-N-BE(2) cells were maintained in a 1:1 mixture of EMEM and F12 media, supplemented with 1% non-essential amino acid solution (NEAA) and 0.5% Glutamine. U2OS-MYC-ER cell lines were cultured in phenol-red free DMEM and treated with 100 nM 4-hydroxytamoxifen (HOT) (Sigma-Aldrich) to activate MYC-ER. IMR-90 was maintained in MEM. All media were supplemented with 10% FBS and 1% penicillin and streptomycin and kept at 37 °C and 5% CO₂. All cell lines were mycoplasma free. Cell proliferation assays were performed as described in Supplementary Information.

Compounds

A screening library of small molecules, 10 mM of each compound in DMSO delivered in a 96 well plate format, from the NCI/DTP Open Chemical Repository Diversity set library (http://dtp.nci.nih.gov) was used. After hit selection, three independent compound batches were obtained from NCI/DTP to confirm activity. In addition, compound MYCMI-6 was synthesized in larger quantities by Latvian Institute of Organic Synthesis, Riga, Latvia. All compounds were dissolved in DMSO (Sigma-Aldrich) to a final concentration of 10 mM, verified by mass spectrometry (LC-MS) and stored in −80 ^oC for further use. DMSO, JQ1, 10058-F4 and 10074-G5 were purchased from Sigma-Aldrich. KJ-Pyr-9 was purchased from Cayman Chemical and was verified by LC-MS.

Bimolecular fluorescence complementation assay (BiFC)

The BiFC assay using MYC fused to the C-terminal fragment of YFP and MAX fused to the N-terminal fragment of YFP has been described previously³⁸. Fluorescence intensity was analyzed using an automated Axiovert 200M inverted microscope (Zeiss). Images were captured and processed using a Hamamatsu ORCA-ER camera together with software from Improvision (OpenLab 4.0.1). CMV-CFP was cotransfected as an internal standard and the ratio between YFP and CFP fluorescence intensity was calculated and normalized to the value of the DMSO treated cells.

Gaussia luciferase protein fragment complementation assay (GLuc)

The protein fragment complementation assay using the Gaussia luciferase has been described³⁹. 0.2–0.4 μg of each GLuc-construct (see Supplementary Information) together with 0.05–0.2 μg pCMV-Luc (Firefly luciferase) were transiently transfected into HEK293 or COS-7 cells in 6-well plates. 24 hours later cells were treated with compound (10–25 μM) or DMSO. After another 17 hours, the cells were harvested and lyzed in passive lysis buffer (Promega) supplemented with complete protease inhibitor (Roche). After 60 min incubation at room temperature 20 μM D-luciferin was added (substrate of Firefly luciferase) and luminescence was measured in a Lumat LB9501 (Berthold) or OmegaFluostar (BMG Labtech) luminometer. Directly after, Gaussia luciferase substrate Coelenterazine (Promega) was added to a final concentration of 20 μM in a mixture with “Stop n’ Glow” (Promega) and the luciferase activity was measured. The ratio between Gaussia and Firefly luciferase values were calculated and normalized to DMSO-treated control cells.

isPLA

The isPLA assay has been described⁴⁰. Antibodies used are listed in Supplementary Information. Briefly, cells were grown on collagen-coated chamber slides (Falcon), and treated with 1–10 μM of compounds, respectively, for 16 hours, then washed twice with PBS and fixed in ice cold methanol for 5–15 min at room temperature. Slides were washed in PBS with 0.05% Tween 20 and incubated in blocking buffer after which isPLA was performed using the Duolink® in situ PLA kit (Sigma-Aldrich) according to the manufacturer’s protocol. DNA was stained with DAPI. Incubation with primary antibodies were performed at +4 °C overnight. Images were taken using an Axiovert 200M inverted microscope (Zeiss) and fluorescent dots were quantified using semi-automated analysis in ImageJ (http://imagej.net) and averaged to number of dots per cell.

isPLA for tumor tissue was performed using 4% formaldehyde fixed paraffin embedded material. The tissue slides were deparaffinized at 65 °C for an hour, rehydrated in 1X Aqua De Par (Biocare Medical) and incubated at 80 °C for 10 min in a pressure chamber (Decloaking Chamber™ Biocare Medical). Slides were soaked in antigen retrieval buffer (10 mM sodium citrate, pH 6.0) and heated in the pressure chamber to 100 °C for 20 minutes, after which the temperature was lowered to 65 °C. Slides were removed from the chamber and cooled down to room temperature. The antigen retrieval buffer was replaced gradually by deionized H₂O for 15 min. Slides were placed in blocking buffer (Duolink® isPLA kit) for 1.5 hours at 37 °C followed by an extra blocking step using the Mouse on Mouse (M.O.M.™) basic kit (Vector Laboratories) according to manufacturers’ protocol. isPLA was performed as above.

Coimmunoprecipitation, western blot, immunohistochemistry and RT-qPCR analyses

These assays were performed essentially as described in^67,68. For further information on immunohistochemistry, and on primers and antibodies, see Supplementary Information.

Plasmids and recombinant proteins

For information about plasmids and production and purification of recombinant proteins see Supplementary Information.

Microscale thermophoresis (MST)

MST was carried out on a Monolith NT.115 with blue/green filters, kindly provided by Luca Jovine, Karolinska Institutet Huddinge, according to manufacturer’s protocol (NanoTemper). MYCbHLHZip, MAXbHLHZip, BSA and BCL-X_L recombinant proteins and compounds were diluted and titrated in PBS supplemented with 0.05% Triton-X-114 or 0.05% Tween-20. Titration of molecules was carried out in 16 PCR tubes into which a fixed concentration of fluorescent molecule was added. The mixture was applied to capillaries (standard treated, NanoTemper), and scanned in the Monolith NT.115 to measure initial fluorescence of molecule. MST was induced and fluorescence was measured during 40 seconds. Double measurements were carried out (MST power of 20% and 40%, or 40% and 60%) for each sample. Delta fluorescence or relative fluorescence of fluorescent molecule normalized to control vehicle was plotted against titrated molecule concentration. The protein labeling kit GREEN-NHS (NanoTemper) was used to label primary amines of proteins according to manufacture’s protocol.

Surface plasmon resonance (SPR)

The SPR experiments were performed at 25 °C using a Biacore T200 (GE Healthcare) instrument kindly provided by SciLifeLab Solna. An amino coupling procedure was used to immobilize protein on a CM5 sensor chip (GE Healthcare). Sensorgrams were generated by subtraction of the reference (blank immobilized) surface. All details will be described in Castell et al. (manuscript in preparation). In general, for the MYC:MAX interaction assay, 200–500 RU of MAXbHLHZip was immobilized and 100 nM MYCbHLHZip pre-incubated with compound in PBS supplemented with 0.05% Tween-20 and 1% DMSO was injected with a flow rate of 30 µl/min and allowed to dissociate. MAX was regenerated by injection of PBS buffer supplemented with Urea.

The MYC SPR assay was carried out as described above with MYCbHLHZip immobilized to a level of 800–1000 RU. For kinetic binding experiments, a Langmuir 1:1 binding event was applied using the Biacore T200 Evaluation Software 2.0 (GE Healthcare) to determine association (k_a) and dissociation (k_d) constants of the compound and to calculate affinity (K_D) by the formula; K_D = k_d/k_a. Binding responses from equilibrium binding experiments were plotted against compound concentration and K_D values were determined at 50% of the theoretical Rmax with the formula Rmax = (MW analyte/MW ligand) × immobilized ligand level on the chip (RU) × stoichiometry (1:1). Same coupling method described above was used to immobilize the control proteins. MAX was immobilized to approximately 2000 RU, MAD to 600 RU, BSA to 1000 RU, p53 to 3000 RU and BCL-X_L, to 500 RU.

Tumor xenograft experiments

All animal protocols in this study were approved by the ethical committee for animal experiments of northern Stockholm (N241/15). Mice were maintained under pathogen-free conditions according to guidelines of the animal facility at MTC, Karolinska Institutet. 6–8 weeks old athymic nude mice (NMRI nude (NMRINU)) (female) (Taconic) were injected s.c. with 5 × 10⁶ MYCN-amplified SK-N-DZ neuroblastoma cells. When tumors reached a size of 100–200 mm³, mice started receiving treatment with MYCMI-6 or vehicle (10% Kolliphore, 5% Tween 80, 5% DMSO) administered daily via i.p. (200 μl). The last dose was administered 3 hrs before termination. Tumors were collected and frozen in OCT (Cryomount, Histolab) or fixed in buffered 4% formaldehyde solution (Histolab) and paraffin-embedded.

Statistical analyses

Proportional data corresponding to GLuc, isPLA in cells for MYC:MAX and qPCR experiments in U2OS cell line were analyzed with a non-parametric Kruskal-Wallis test, while comparisons across treatments were performed with Bonferroni post hoc or Wilcoxon tests, to account for heteroscedasticity. Proportional data corresponding to MYCN:MAX isPLA and RT-qPCR experiments in neuroblastoma cell lines were initially transformed (square root and arcsine) and analyzed with generalized linear models (GLMs) with a normal distribution. Pairwise comparisons were performed with Bonferroni post hoc tests. Proportional data corresponding to isPLA in vivo experiments were log-transformed and analyzed with a GLM. The effect of MYC mRNA and protein levels on the response of the NCI-60 cancer cell lines to MYCMI-6 were analyzed with GLM assuming a normal distribution, while the hypothesis on the probability of a cell line with “high MYC” or “low MYC” protein levels to respond to MYCMI-6 was tested with the binomial exact test. The rest of the data were analyzed with the Student’s t-test. All analyses were carried out in R (v. 3.3.3; R Foundation for Statistical Computing, Vienna, AT), at a level of significance α = 0.05, with packages car, agricolae, multcomp and MASS.

References

Blackwood, E. M. & Eisenman, R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 251, 1211–1217 (1991).
Article PubMed ADS CAS Google Scholar
Dang, C. V. MYC on the path to cancer. Cell 149, 22–35, https://doi.org/10.1016/j.cell.2012.03.003 (2012).
Article PubMed PubMed Central CAS Google Scholar
Eilers, M. & Eisenman, R. N. Myc’s broad reach. Genes Dev 22, 2755–2766, https://doi.org/10.1101/gad.1712408 (2008).
Article PubMed PubMed Central CAS Google Scholar
Larsson, L. G. & Henriksson, M. A. The Yin and Yang functions of the Myc oncoprotein in cancer development and as targets for therapy. Exp Cell Res 316, 1429–1437, https://doi.org/10.1016/j.yexcr.2010.03.025 (2010).
Article PubMed CAS Google Scholar
Meyer, N. & Penn, L. Z. Reflecting on 25 years with MYC. Nat Rev Cancer 8, 976–990, https://doi.org/10.1038/nrc2231 (2008).
Article PubMed CAS Google Scholar
Nair, S. K. & Burley, S. K. X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors. Cell 112, 193–205, doi:S0092867402012849 [pii] (2003).
Kress, T. R., Sabo, A. & Amati, B. MYC: connecting selective transcriptional control to global RNA production. Nat Rev Cancer 15, 593–607, https://doi.org/10.1038/nrc3984 (2015).
Article PubMed CAS Google Scholar
Lin, C. Y. et al. Transcriptional amplification in tumor cells with elevated c-Myc. Cell 151, 56–67, https://doi.org/10.1016/j.cell.2012.08.026 (2012).
Article PubMed PubMed Central CAS Google Scholar
Nie, Z. et al. c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells. Cell 151, 68–79, https://doi.org/10.1016/j.cell.2012.08.033 (2012).
Article PubMed PubMed Central CAS Google Scholar
Sabo, A. et al. Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis. Nature 511, 488–492, https://doi.org/10.1038/nature13537 (2014).
Article PubMed PubMed Central ADS CAS Google Scholar
Walz, S. et al. Activation and repression by oncogenic MYC shape tumour-specific gene expression profiles. Nature 511, 483–487, https://doi.org/10.1038/nature13473 (2014).
Article PubMed ADS CAS Google Scholar
Gabay, M., Li, Y. & Felsher, D. W. MYC activation is a hallmark of cancer initiation and maintenance. Cold Spring Harb Perspect Med 4, https://doi.org/10.1101/cshperspect.a014241 (2014).
Sodir, N. M. & Evan, G. I. Finding cancer’s weakest link. Oncotarget 2, 1307–1313, https://doi.org/10.18632/oncotarget.396 (2011).
Article PubMed PubMed Central Google Scholar
Castell, A. & Larsson, L. G. Targeting MYC Translation in Colorectal Cancer. Cancer Discov 5, 701–703, https://doi.org/10.1158/2159-8290.CD-15-0660 (2015).
Article PubMed CAS Google Scholar
McKeown, M. R. & Bradner, J. E. Therapeutic strategies to inhibit MYC. Cold Spring Harb Perspect Med 4, https://doi.org/10.1101/cshperspect.a014266 (2014).
Whitfield, J. R., Beaulieu, M. E. & Soucek, L. Strategies to Inhibit Myc and Their Clinical Applicability. Front Cell Dev Biol 5, 10, https://doi.org/10.3389/fcell.2017.00010 (2017).
Article PubMed PubMed Central Google Scholar
Vassilev, L. T. et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844–848, https://doi.org/10.1126/science.1092472 (2004).
Article PubMed ADS CAS Google Scholar
Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073, https://doi.org/10.1038/nature09504 (2010).
Article PubMed PubMed Central ADS CAS Google Scholar
Tse, C. et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res 68, 3421–3428, https://doi.org/10.1158/0008-5472.CAN-07-5836 (2008).
Article PubMed CAS Google Scholar
Arkin, M. R., Tang, Y. & Wells, J. A. Small-molecule inhibitors of protein-protein interactions: progressing toward the reality. Chemistry & biology 21, 1102–1114, https://doi.org/10.1016/j.chembiol.2014.09.001 (2014).
Article CAS Google Scholar
Nero, T. L., Morton, C. J., Holien, J. K., Wielens, J. & Parker, M. W. Oncogenic protein interfaces: small molecules, big challenges. Nature reviews. Cancer 14, 248–262, https://doi.org/10.1038/nrc3690 (2014).
Article PubMed CAS Google Scholar
Berg, T. et al. Small-molecule antagonists of Myc/Max dimerization inhibit Myc-induced transformation of chicken embryo fibroblasts. Proc Natl Acad Sci USA 99, 3830–3835, https://doi.org/10.1073/pnas.062036999 (2002).
Article PubMed PubMed Central ADS CAS Google Scholar
Kiessling, A., Sperl, B., Hollis, A., Eick, D. & Berg, T. Selective inhibition of c-Myc/Max dimerization and DNA binding by small molecules. Chem Biol 13, 745–751, https://doi.org/10.1016/j.chembiol.2006.05.011 (2006).
Article PubMed CAS Google Scholar
Yin, X., Giap, C., Lazo, J. S. & Prochownik, E. V. Low molecular weight inhibitors of Myc-Max interaction and function. Oncogene 22, 6151–6159, https://doi.org/10.1038/sj.onc.1206641 (2003).
Article PubMed CAS Google Scholar
Choi, S. H. et al. Targeted Disruption of Myc-Max Oncoprotein Complex by a Small Molecule. ACS Chem Biol 12, 2715–2719, https://doi.org/10.1021/acschembio.7b00799 (2017).
Article PubMed CAS Google Scholar
Fletcher, S. & Prochownik, E. V. Small-molecule inhibitors of the Myc oncoprotein. Biochim Biophys Acta 1849, 525–543, https://doi.org/10.1016/j.bbagrm.2014.03.005 (2015).
Article PubMed CAS Google Scholar
Hart, J. R. et al. Inhibitor of MYC identified in a Krohnke pyridine library. Proc Natl Acad Sci USA 111, 12556–12561, https://doi.org/10.1073/pnas.1319488111 (2014).
Article PubMed PubMed Central ADS CAS Google Scholar
Jeong, K. C. et al. Intravesical instillation of c-MYC inhibitor KSI-3716 suppresses orthotopic bladder tumor growth. J Urol 191, 510–518, https://doi.org/10.1016/j.juro.2013.07.019 (2014).
Article PubMed CAS Google Scholar
Jung, K. Y. et al. Perturbation of the c-Myc-Max protein-protein interaction via synthetic alpha-helix mimetics. J Med Chem 58, 3002–3024, https://doi.org/10.1021/jm501440q (2015).
Article PubMed PubMed Central CAS Google Scholar
Kiessling, A., Wiesinger, R., Sperl, B. & Berg, T. Selective inhibition of c-Myc/Max dimerization by a pyrazolo[1,5-a]pyrimidine. ChemMedChem 2, 627–630, https://doi.org/10.1002/cmdc.200600294 (2007).
Article PubMed CAS Google Scholar
Mo, H. & Henriksson, M. Identification of small molecules that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation. Proc Natl Acad Sci USA 103, 6344–6349, https://doi.org/10.1073/pnas.0601418103 (2006).
Article PubMed PubMed Central ADS CAS Google Scholar
Wang, H. et al. Improved low molecular weight Myc-Max inhibitors. Mol Cancer Ther 6, 2399–2408, https://doi.org/10.1158/1535-7163.MCT-07-0005 (2007).
Article PubMed CAS Google Scholar
Wang, H. et al. Direct inhibition of c-Myc-Max heterodimers by celastrol and celastrol-inspired triterpenoids. Oncotarget 6, 32380–32395, https://doi.org/10.18632/oncotarget.6116 (2015).
Article PubMed PubMed Central Google Scholar
Prochownik, E. V. & Vogt, P. K. Therapeutic Targeting of Myc. Genes Cancer 1, 650–659, https://doi.org/10.1177/1947601910377494 (2010).
Article PubMed PubMed Central CAS Google Scholar
Hu, C. D., Chinenov, Y. & Kerppola, T. K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9, 789–798 (2002).
Article PubMed CAS Google Scholar
Kerppola, T. K. Complementary methods for studies of protein interactions in living cells. Nat Methods 3, 969–971, https://doi.org/10.1038/nmeth1206-969 (2006).
Article PubMed PubMed Central CAS Google Scholar
von der Lehr, N. et al. The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription. Mol Cell 11, 1189–1200, doi:S109727650300193X [pii] (2003).
Grinberg, A. V., Hu, C. D. & Kerppola, T. K. Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol Cell Biol 24, 4294–4308 (2004).
Article PubMed PubMed Central CAS Google Scholar
Remy, I. & Michnick, S. W. A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat Methods 3, 977–979, https://doi.org/10.1038/nmeth979 (2006).
Article PubMed CAS Google Scholar
Soderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3, 995–1000, https://doi.org/10.1038/nmeth947 (2006).
Article PubMed CAS Google Scholar
Ayer, D. E., Kretzner, L. & Eisenman, R. N. Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell 72, 211–222 (1993).
Article PubMed CAS Google Scholar
Larsson, L. G., Pettersson, M., Oberg, F., Nilsson, K. & Luscher, B. Expression of mad, mxi1, max and c-myc during induced differentiation of hematopoietic cells: opposite regulation of mad and c-myc. Oncogene 9, 1247–1252 (1994).
PubMed CAS Google Scholar
Adhikary, S. & Eilers, M. Transcriptional regulation and transformation by Myc proteins. Nat Rev Mol Cell Biol 6, 635–645 (2005).
Article PubMed CAS Google Scholar
Seidel, S. A. et al. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59, 301–315, https://doi.org/10.1016/j.ymeth.2012.12.005 (2013).
Article PubMed CAS Google Scholar
Schasfoort, R. B. M. & Tudos, A. J. Handbook of surface plasmon resonance (RSC Pub., 2008).
Zimmerman, M. W. et al. MYC Drives a Subset of High-Risk Pediatric Neuroblastomas and Is Activated through Mechanisms Including Enhancer Hijacking and Focal Enhancer Amplification. Cancer Discov 8, 320–335, https://doi.org/10.1158/2159-8290.CD-17-0993 (2018).
Article PubMed CAS Google Scholar
Jung, M. et al. A Myc Activity Signature Predicts Poor Clinical Outcomes in Myc-Associated Cancers. Cancer Res 77, 971–981, https://doi.org/10.1158/0008-5472.CAN-15-2906 (2017).
Article PubMed CAS Google Scholar
Bhat, M. et al. Targeting the translation machinery in cancer. Nat Rev Drug Discov 14, 261–278, https://doi.org/10.1038/nrd4505 (2015).
Article PubMed CAS Google Scholar
Farrell, A. S. & Sears, R. C. MYC degradation. Cold Spring Harb Perspect Med 4, https://doi.org/10.1101/cshperspect.a014365 (2014).
Mateyak, M. K., Obaya, A. J., Adachi, S. & Sedivy, J. M. Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination. Cell Growth Differ 8, 1039–1048 (1997).
PubMed CAS Google Scholar
Yu, H. et al. Measuring drug action in the cellular context using protein-fragment complementation assays. Assay Drug Dev Technol 1, 811–822, https://doi.org/10.1089/154065803772613444 (2003).
Article PubMed CAS Google Scholar
Baxevanis, A. D. & Vinson, C. R. Interactions of coiled coils in transcription factors: where is the specificity? Curr Opin Genet Dev 3, 278–285 (1993).
Article PubMed CAS Google Scholar
Clausen, D. M. et al. In vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of 10074-G5, a novel small-molecule inhibitor of c-Myc/Max dimerization. J Pharmacol Exp Ther 335, 715–727, https://doi.org/10.1124/jpet.110.170555 (2010).
Article PubMed PubMed Central CAS Google Scholar
Muller, I. et al. Targeting of the MYCN protein with small molecule c-MYC inhibitors. Plos One 9, e97285, https://doi.org/10.1371/journal.pone.0097285 (2014).
Article PubMed PubMed Central ADS CAS Google Scholar
Raffeiner, P. et al. In vivo quantification and perturbation of Myc-Max interactions and the impact on oncogenic potential. Oncotarget 5, 8869–8878, https://doi.org/10.18632/oncotarget.2588 (2014).
Article PubMed PubMed Central Google Scholar
Park, D. et al. Novel small-molecule inhibitors of Bcl-XL to treat lung cancer. Cancer Res 73, 5485–5496, https://doi.org/10.1158/0008-5472.CAN-12-2272 (2013).
Article PubMed PubMed Central CAS Google Scholar
Brooks, W. H. et al. In silico chemical library screening and experimental validation of a novel 9-aminoacridine based lead-inhibitor of human S-adenosylmethionine decarboxylase. J Chem Inf Model 47, 1897–1905, https://doi.org/10.1021/ci700005t (2007).
Article PubMed CAS Google Scholar
Sassano, M. F., Schlesinger, A. P. & Jarstfer, M. B. Identification of G-Quadruplex Inducers Usinga Simple, Inexpensiveand Rapid High Throughput Assay, and Their Inhibition of Human Telomerase. Open Med Chem J 6, 20–28, https://doi.org/10.2174/1874104501206010020 (2012).
Article PubMed PubMed Central CAS Google Scholar
Weissman, A. M. et al. Inhibiting Hdm2 and ubiquitin-activating enzyme: targeting the ubiquitin conjugating system in cancer. Ernst Schering Found Symp Proc, 171–190 (2008).
Soucek, L. et al. Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice. Genes Dev 27, 504–513, https://doi.org/10.1101/gad.205542.112 (2013).
Article PubMed PubMed Central CAS Google Scholar
Zirath, H. et al. MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells. Proc Natl Acad Sci USA 110, 10258–10263, https://doi.org/10.1073/pnas.1222404110 (2013).
Article PubMed PubMed Central ADS Google Scholar
Stellas, D. et al. Therapeutic effects of an anti-Myc drug on mouse pancreatic cancer. J Natl Cancer Inst 106, https://doi.org/10.1093/jnci/dju320 (2014).
Felsher, D. W. & Bishop, J. M. Reversible tumorigenesis by MYC in hematopoietic lineages. Mol Cell 4, 199–207, doi:S1097-2765(00)80367-6 [pii] (1999).
Jain, M. et al. Sustained loss of a neoplastic phenotype by brief inactivation of MYC. Science 297, 102–104, https://doi.org/10.1126/science.1071489 297/5578/102 [pii] (2002).
Sodir, N. M. et al. Endogenous Myc maintains the tumor microenvironment. Genes Dev 25, 907–916, https://doi.org/10.1101/gad.2038411 (2011).
Article PubMed PubMed Central CAS Google Scholar
Soucek, L. et al. Modelling Myc inhibition as a cancer therapy. Nature 455, 679–683, https://doi.org/10.1038/nature07260 (2008).
Article PubMed PubMed Central ADS CAS Google Scholar
Bahram, F. et al. Interferon-gamma-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation. Oncotarget 7, 2837–2854, https://doi.org/10.18632/oncotarget.6693 (2016).
Article PubMed Google Scholar
Tabor, V., Bocci, M., Alikhani, N., Kuiper, R. & Larsson, L. G. MYC synergizes with activated BRAFV600E in mouse lung tumor development by suppressing senescence. Cancer Res 74, 4222–4229, https://doi.org/10.1158/0008-5472.CAN-13-3234 (2014).
Article PubMed CAS Google Scholar

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Acknowledgements

We would like to acknowledge National Cancer Institute (NCI)/Division of Cancer Treatment and Diagnosis (DCTD)/Developmental Therapeutics Program (DTP) NCI/DTP for compounds. We specially thank Tom Kerppola, Stephen Michnick, Bernhard Lüscher, Klas Wiman, Marie Arsenian Henriksson, Martin Eilers and GE Healthcare for protocols, cells and/or reagents and valuable discussions. We also thank SciLife Lab Solna and Luca Jovine, Karolinska Huddinge for the use of instruments. We are grateful to Laboratory of Chemical Biology at Karolinska Institutet (LCBKI), KI-Innovations and Innovationskontoret for valuable discussions and services. We also thank Camilla Mannich Uggla, Katalin Benedek and Kenth Andersson for technical support and Raoul Kuiper for sectioning and staining of tumor tissue as well as for histopathological analysis. We also thank Johan Heldin, Axel Klaesson and Ola Söderberg for advice regarding the isPLA technique and Nyosha Alikhani for discussions concerning the animal work. This work was supported by the Swedish Cancer Society, Swedish Childhood Cancer Foundation, the Swedish Research Council, Olle Engkvists Foundation, Knut and Alice Wallenberg and KI Foundations to LGL, Bayer’s Grants4targets to A.C., L.G.L. and G.Z., Swedish Childhood Cancer Foundation, Felix Mindus Leukemia Research Grant, Mary Bevé’s Childhood Cancer Research Grant to A.C. and KID. Grants (KI) to Q.Y., F.Z., S.M.Z. and W.B.

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Karin Fawkner
Present address: TLV, Box 225 20, 104 22, Stockholm, Sweden
Per Hydbring
Present address: Department of Oncology-Pathology, Karolinska Institutet, SE-17176, Stockholm, Sweden
Alina Castell, Qinzi Yan and Karin Fawkner contributed equally to this work.

Authors and Affiliations

Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 65, Stockholm, Sweden
Alina Castell, Qinzi Yan, Karin Fawkner, Per Hydbring, Fan Zhang, Vasiliki Verschut, Marcela Franco, Siti Mariam Zakaria, Wesam Bazzar, Jacob Goodwin, Giovanna Zinzalla & Lars-Gunnar Larsson

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Alina Castell
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Qinzi Yan
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Karin Fawkner
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Per Hydbring
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Fan Zhang
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Vasiliki Verschut
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Marcela Franco
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Siti Mariam Zakaria
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Jacob Goodwin
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Contributions

A.C., Q.Y., K.F., P.H., F.Z., V.V., M.F., S.M.Z., W.B., J.G., G.Z. and L.G.L. contributed to acquisition, analysis and interpretation of data. A.C., Q.Y., K.F., P.H., F.Z., V.V., M.F., S.M.Z., G.Z. and L.G.L. conceived and designed the experiments, as well as developed methodology. A.C., Q.Y., K.F., P.H., F.Z., V.V., M.F. and L.G.L. wrote the manuscript.

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Correspondence to Lars-Gunnar Larsson.

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Castell, A., Yan, Q., Fawkner, K. et al. A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation. Sci Rep 8, 10064 (2018). https://doi.org/10.1038/s41598-018-28107-4

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Received: 29 January 2018
Accepted: 22 May 2018
Published: 03 July 2018
DOI: https://doi.org/10.1038/s41598-018-28107-4

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