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Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour xenograft

growth

L C Lustig, D Dingar, W B Tu, C Lourenco, M Kalkat, I Inamoto, R Ponzielli, W C W Chan, J A Shin & L Z Penn

Version Post-print/accepted manuscript

Citation

(published version)

Lustig, L. C., D. Dingar, W. B. Tu, C. Lourenco, M. Kalkat, I. Inamoto,

R. Ponzielli, W. C. W. Chan, J. A. Shin, and L. Z. Penn. "Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour

xenograft growth." Oncogene 36, no. 49 (2017): 6830. Doi: 10.1038/onc.2017.275

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Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour xenograft growth.

1Lustig L.C., 1Dingar D., 1,2Tu W.B., 1,2Lourenco C., 1,2Kalkat M., 4Inamoto I., 1Ponzielli R., 3Chan W.C.W., 4Shin J. A., 1,2Penn L.Z.* 1Princess Margaret Cancer Centre, University Health Network

2Department of Medical Biophysics, University of Toronto 3Institute of Biomaterials and Biomedical Engineering, University of Toronto, Donnelly Centre for Cellular and Biomolecular Research, Department of Materials Science and Engineering, Department of Chemical Engineering 4Department of Chemistry, University of Toronto *Corresponding author: Linda Z. Penn Princess Margaret Cancer Centre 101 College Street Toronto, ON, M5G 1L7, Canada Tel: (416)-634-8770 Email: lpenn@uhnres.utoronto.ca Sources of Funding: Operating grant support from the Collaborative Health Research Program (JS, WC, LZP). Salary and stipend support from the Canadian Research Chairs Program (LZP), Natural Sciences and Engineering Research Council (LCL), Canadian Breast Cancer Foundation Ontario Region Doctoral Fellowship (WBT and MK) Running Title: Blocking MYC:E-box binding inhibits tumour growth

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ABSTRACT Developing therapeutics to effectively inhibit the MYC oncoprotein would mark a

key advance towards cancer patient care, as MYC is deregulated in over 50% of human cancers. MYC deregulation is correlated with aggressive disease and poor patient outcome. Despite strong evidence in mouse models that inhibiting MYC would significantly impact tumour cell growth and patient survival, traditional approaches have not yet yielded the urgently needed therapeutic agents that directly target MYC. MYC functions through its interaction with MAX to regulate gene transcription by binding to E-box DNA response elements of MYC target genes. Here we used a structure-based strategy to design ME47, a small minimalist hybrid protein (MHP) able to disrupt the MAX:E-box interaction/binding and block transcriptional MYC activity. We show that inducing ME47 expression in established tumour xenografts inhibits tumour growth and decreases cellular proliferation. Mechanistically, we show by chromatin immunoprecipitation that ME47 binds to E-box binding sites of MYC target genes. Moreover, ME47 occupancy decreases MYC:DNA interaction at its cognate E-box binding sites. Taken together, ME47 is a prototypic MHP inhibitor that antagonizes tumour cell growth in vitro and in vivo and inhibits the interaction of MYC with DNA E-box elements. These results support ME47’s role as a MYC inhibitor and suggest that MHPs provide an alternative therapeutic targeting system that can be used to target transcription factors important in human diseases, including cancer. Descriptive Keywords: Inhibiting protein:DNA interaction; anti-cancer agent; small protein inhibitor, Max-E47, MYC inhibitor

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INTRODUCTION The MYC oncoprotein is estimated to be involved in the etiology of over 50% of human cancers and is associated with aggressive disease and poor patient outcome1-4. MYC is considered a hub protein as it integrates multiple signalling pathways to function as a master regulator with transcriptional control over biological processes, such as cellular proliferation and transformation. Mechanistically, MYC interacts with MAX and activates gene transcription by binding to E-box DNA elements, such as the canonical sequence CACGTG, in the regulatory regions of target genes5. MYC expression and activity are highly regulated in normal cells; however, MYC is often deregulated and constitutively active in tumour cells2, 6. Studies show that when MYC activity is impaired systemically in cells of tumour-bearing transgenic mice, tumour cell proliferation is blocked, whereas normal cells remain functionally intact7-9. These results provide proof-of-concept that a therapeutic index can be achieved by targeting MYC despite its widespread expression. Evidence shows that tumour cells are dependent on the MYC oncogene, which provides a vulnerability for us to target10.

Therapeutically restraining MYC activity would significantly impact cancer patient care, yet targeting MYC directly appears to be an intractable challenge due to the lack of structural features traditionally associated with the design of small-molecule inhibitors (SMIs). New strategies to inhibit MYC are therefore required and are presently under development. SMIs blocking transcriptional activators of the MYC gene, such as the bromodomain and extra-terminal (BET) proteins11-13, can lead to down-regulation of MYC transcription in addition to other MYC-independent effects14. SMIs have garnered much excitement with some having been fast-tracked to Phase I clinical trials12. Unfortunately, resistance mechanisms to these inhibitors have already been described15-

17. Additional SMIs have been designed to perturb the MYC:MAX protein-protein interaction; however, the target specificity of these inhibitors remains unclear and suggests additional strategies are needed18-22. Dominant negative proteins have been designed to interfere with MYC:MAX interaction, such as ectopic expression of the MAX basic helix-loop-helix leucine zipper (B-HLH-LZ)23, 24. Similarly, Omomyc (92 amino acids) was designed to heterodimerize with MYC and preclude interaction with MAX 7, 8, 25, 26. Both chemical and protein inhibitors are presently under investigation for their potential as novel anti-MYC inhibitors. Developing an arsenal of MYC inhibitors with unique mechanisms of action would significantly impact patient care. We have undertaken a structure-based strategy to design small protein inhibitors that competitively block native transcription factors from binding to their DNA recognition motifs. These minimalist hybrid protein (MHP) inhibitors are small fusion proteins comprised of functional domains from two or more transcription factors. We have previously demonstrated in vitro evidence that the MHP ME47 (previously called MAX-E47) has potential to block E-box sequence-specific transcription factor interaction with DNA27, 28. This MHP is comprised of the DNA-binding basic region of MAX and the HLH dimerization domain of the E47 transcription factor27 (Figure 1A). At 66 amino acids, ME47 has a simplified structure compared to the larger native MAX protein (160 amino acids), yet binds to E-box elements with low nanomolar affinity comparable to that of MAX as determined by electrophoretic mobility shift assays (EMSA)27. Structural and yeast one-hybrid analyses show ME47 exists as a

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dimer and can outcompete MAX for binding to E-box elements, further supporting its utility as a MHP inhibitor of transcription factor binding in cells27, 28.

Here we evaluate the mechanism of action and efficacy of ME47 in transformed human cell lines, in both cell culture and in vivo tumour xenograft settings. We demonstrate that this small protein inhibitor decreases tumour cell proliferation and transformation, including tumour xenograft growth. We further show that ME47 binds to E-box-containing promoters of MYC target genes and decreases MYC binding at these regulatory regions. Our study highlights the prototypic MHP, ME47, as a new class of transcription factor inhibitor designed to target the interaction of MYC:MAX with E-box DNA. Moreover, this study reinforces the concept of MHPs as a therapeutic platform to target transcription factors. RESULTS and DISCUSSION ME47 decreases cellular proliferation

To evaluate the biological and molecular effect of ME47 expression, we first evaluated ME47 in comparison with Omomyc. ME47 and Omomyc are designed to block MYC:MAX activity by distinct mechanisms of action. ME47 targets MYC:MAX binding to the E-box motif, whereas Omomyc was designed to block MYC and MAX interaction25. ME47 and Omomyc were tagged with an in-frame N-terminal FLAG and nuclear localization signal (FLAG-NLS) to visualize ectopic expression and facilitate nuclear localization, respectively29, 30. We used two MYC-sensitive transformed cell lines to evaluate the effects of ME47: MDA-MB-231 is a triple negative basal breast tumour-derived cell line and HEK293Tv is an embryonic kidney cell line transformed with SV40 large T-antigen. Both systems showed MYC sensitivity in that they exhibited significantly decreased cellular proliferation and transformation upon MYC knockdown by shRNA (Supplementary Figures 1 and 2). We generated MDA-MB-231 and HEK293Tv cells to stably express 4-hydroxytamoxifen (4OHT) inducible FLAG-NLS-Vector, FLAG-NLS-Omomyc, or FLAG-NLS-ME47. Conditional expression of ME47 and Omomyc was evident in response to 4OHT exposure in the MDA-MB-231 (Figure 1B) and HEK293Tv (Supplementary Figure 3A) cells, but not with Vector and ethanol (EtOH) controls. Omomyc showed consistently higher expression relative to ME47 for reasons that remain unclear. We next evaluated the effect of ME47 and Omomyc expression on cellular proliferation using a variety of assays. We measured cellular proliferation by assaying EdU (5’-ethynyl-2’-deoxyuridine) incorporation as a measure of active DNA synthesis (Figure 1C). Expression of Omomyc (p<0.05) or ME47 (p<0.01) significantly decreased EdU incorporation compared to Vector control. We also assessed whether ME47 exerts an anti-proliferative effect at the level of population doubling time. Following 24 hours incubation in EtOH control or 4OHT-containing media, MDA-MB-231 cells expressing inducible Vector, Omomyc, or ME47 were analyzed for relative DNA content over five consecutive days (Figure 1D). ME47 expression significantly decreased DNA content compared to Vector control at days four and five (p<0.05). By extension, the doubling time of cells expressing ME47 was increased to 26.8 hours compared to Vector control cells at 23.9 hours (Figure 1D, inset). Previous studies of Omomyc in Rat1 fibroblasts and SH-SY5Y neuroblastoma demonstrated a significant inhibitory effect on cell proliferation31. In MDA-MB-231 cells, we observed a similar trend with Omomyc;

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however, the results did not reach significance at experimental endpoint (p=0.077). These data indicate that ME47 negatively impacts the growth of MDA-MB-231 breast cancer cells and demonstrates an anti-proliferative phenotype that is comparable to Omomyc. Inhibiting anchorage-independent cell growth with ME47 expression As ME47 demonstrated a decrease in cell proliferation, we tested its effect on anchorage-independent growth in soft agar, a well-established surrogate measure of cellular transformation32, 33. Induction of ME47 resulted in a significant decrease in colony number (p<0.05) relative to the Vector control (Figure 1E). Omomyc did not significantly decrease colony number in this assay. Both Omomyc and ME47 expression led to a decreasing trend in anchorage-independent growth of HEK293Tv cells (Supplementary Figure 3). These results show that ME47 antagonizes MYC-dependent transformation more strongly than does Omomyc, despite consistently lower ME47 expression. Omomyc has been shown to form homodimers as well as interact with MYC, MAX, and other MYC interactors25, 31, 34. By comparison, structural analysis reveals that ME47 forms stable homodimers28. ME47 has no activation domain and shows no interaction with the B-HLH-LZ region of MYC27. Further comparisons between these two protein inhibitors with different mechanisms of action will help address relative mechanisms of action and biological activities. ME47 decreases xenograft tumour growth We further evaluated the anti-proliferative activity of ME47 by assaying the effect of ME47 on tumour xenograft growth. Animal work was carried out in accordance with the regulations of the Canadian Council on Animal Care. MDA-MB-231 cells with inducible ME47 or Vector control were injected into the flanks of 10 week old female NOD/SCID mice and allowed to grow until tumours were approximately 200 mm3 in volume. Mice with tumours at 200mm3 were switched onto food that was infused with tamoxifen to induce ME47 expression35. Expression of ME47 delayed tumour growth compared to Vector control mice. Tumour volumes after day 22 were not considered as animals began reaching experimental end-point on day 22 (Figure 2A). This is consistent with the decreased colony growth due to ME47 expression in MDA-MB-231 soft agar assays (Figure 1E). A significant decrease in tumour volume (p<0.0001; Figure 2B) was evident 22 days after inducing ME47 expression. Moreover, ME47 significantly increased experimental survival (p=0.0003; Figure 2C). Thus, ME47 demonstrates a potent anti-proliferative effect on tumour xenograft growth leading to a significant impact on animal survival. To interrogate the mechanism of ME47, the experiment was repeated with tumours expressing Vector control and ME47 harvested on the same day while tumours were in growth phase. At endpoint, the average tumour weight of the ME47 xenografts was decreased in size relative to Vector controls, as expected (Figure 2D). This was further evident in side-by-side comparison of tumour images (Figure 2D, inset). Evidence of ME47 expression was shown by immunoblot using anti-FLAG to detect ME47 expression in tumour xenograft extracts prepared at experimental end-point from the Vector control and ME47-treated animals (Figure 2E). Immunohistochemical analyses were performed on the excised tumours. Based on the tissue culture results demonstrating the anti-proliferative effect of ME47, we assayed tumour sections for Ki67 and TUNEL

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staining to evaluate the proliferative and apoptotic index, respectively (Figure 2F and 2G). Consistent with the results in culture, Ki67 staining was significantly lower (p<0.05) in the ME47-expressing xenografts compared to control (Figure 2F). Levels of apoptosis did not change as a result of ME47 induction (Figure 2G). This suggests that the ME47-mediated decrease on cellular transformation and xenograft tumour weight is due to an anti-proliferative effect. ME47 binds to the DNA E-box and antagonizes MYC binding to target gene promoters

To evaluate mechanism and determine whether ME47 binds to the E-box as designed27, we assessed whether ME47 is localized to the nuclear compartment and whether induction of ME47 affected endogenous MYC or MAX expression in MDA-MB-231 cells. Immunoblot of extracts harvested following exposure to vehicle control or 4OHT showed robust induction of ME47 and no change in endogenous MYC or MAX expression, as expected (Supplementary Figure 4). Cellular fractionation followed by immunoblot analysis was conducted on MDA-MB-231 cells exposed to 4OHT or EtOH control (Figure 3A). The results show that ME47 (detected by FLAG antibody) is expressed exclusively in the nuclear fraction and not in the cytoplasmic fraction as indicated by acetylated histone H3 (AcH3) and tubulin expression, respectively (Figure 3A).

ME47 was designed to bind DNA E-box motifs through its MAX basic region. We compared the binding specificity of ME47 to MAX on a series of E-box and non-E-box sequences (Supplementary Figure 5). ME47 and the B-HLH-LZ domain of MAX bound to the MAX E-box sequence with comparable affinities (dissociation constant (Kd) of 15 nM). ME47 and MAX B-HLH-LZ also showed similar affinities for the ARNT E-box sequence (Kd of 30 and 60 nM, respectively). These two E-box sequences featured the central CACGTG hexamer, but differed in their flanking base pairs. Both proteins had decreased affinity for the XRE sequence (Kd of 40 and 70 nM, respectively), which contained a half E-box motif. The binding of both proteins was further diminished with sequences unrelated to the E-box motif (C/EBP, AP-1, and non-specific DNA sequences; ME47 Kd of 60 for all three sequences and MAX B-HLH-LZ Kd of 100 nM for all three). Against a series of different DNA sequences, ME47 and MAX exhibit similar preferential binding affinities to E-box motifs.

To determine whether ME47 is bound to chromatin at E-box-containing regulatory regions of MYC-activated genes in cells, ChIP-qPCR was performed. MDA-MB-231 cells with inducible ME47 or Vector control were treated with 4OHT, and then ChIP-qPCR was conducted using antibody to FLAG to detect ME47 or IgG as a negative control (Figure 3B). These analyses reveal significant binding in the ME47-expressing cells compared to Vector control, as measured by the percent input enriched at the E-box-containing promoters of CAD (p<0.001), CCND1 (p<0.0001), CDK4 (p<0.05), and NCL (p<0.0001), but not the established non-E-box negative control region on chromosome 6 (chr6)36, 37. We also determined MYC occupancy on these promoter regions by conducting ChIP-qPCR using an anti-MYC antibody, N262 (Figure 3C). MYC binding was readily detected in Vector control cells at MYC-induced gene promoters (CAD, CCND1, CDK4, NCL), but was significantly decreased in cells expressing ME47. IgG controls demonstrated negligible binding to any of the regions under interrogation under

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all cellular conditions. Together, these data strongly suggest that ME47 binds to E-box DNA and antagonizes MYC binding to target gene promoters.

To establish the potential utility of ME47 as a therapeutic inhibitor, we sought to determine the amount of ME47 relative to endogenous MAX when reduced MYC binding and decreased proliferation were observed upon ME47 induction. Quantifying the amount of ME47 induced in MDA-MB-231 cells after 24 hours against a standard curve of purified ME47, we calculated that ME47 protein is expressed at 0.117 nmol/µg lysate or 1.36 ng/µg lysate (Supplementary Figure 6A, C). Using a similar approach, a standard curve with purified MAX was generated and MAX was quantified at 0.00273 nmol/µg lysate or 0.0527 ng/µg lysate (Supplementary Figure 6B, C). At a time point when ME47 displayed molecular and cellular inhibitory effects, ME47 was present at approximately 42.9 times the molality of endogenous MAX (Supplementary Figure 6C). This suggests that inducible expression of the ME47 protein inhibitor has the capacity to target endogenous MYC:MAX in tumour cells. IMPACT AND CONCLUSIONS This study explores the efficacy of our MHP strategy to target transcription factor binding to DNA in human cells. We use ME47 as the prototypic MHP to disrupt MYC:MAX interaction with the E-box DNA element. We demonstrate that ME47 decreases cancer cell proliferation and transformation in MDA-MB-231 breast cancer cells in cell culture, colony growth, and tumour xenograft settings. Mechanistically, ME47 is expressed exclusively in the nuclear compartment of the cell, binds to the DNA E-box-containing promoter regions of MYC-activated target genes, and antagonizes MYC binding to these regions (Figure 4). In contrast to other agents, such as those generated to interfere with MYC expression or the MYC:MAX protein-protein interaction, ME47 is specifically designed to perturb MYC:MAX at the level of its DNA-binding activity. Given MYC:MAX exhibits genome-wide chromatin binding that becomes widespread in oncogenic conditions38, 39, this class of MYC inhibitor has enormous potential for further development as anti-cancer agents. Our work also shows that the MHP inhibitor platform may be extended to target other sequence-specific transcription factors that have implications in human diseases40, 41. CONFLICT OF INTEREST The authors declare no competing financial interests. ACKNOWLEDGEMENTS We would like to thank the Penn, Shin, and Chen labs for their helpful reviews and contributions to this manuscript. Special thanks to Dr. Peter Mullen, Dr. Sam Sathiamoorthy and Peter Tang. SUPPLEMENTARY INFORMATION Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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FIGURE LEGENDS Figure 1. ME47 decreases cellular proliferation and transformation in MDA-MB-231 breast cancer cells. (A) ME47 is a minimalist hybrid protein (MHP) inhibitor designed to homodimerize and inhibit the interaction of the MYC:MAX heterodimer with DNA E-box sequences. This 66-amino acid hybrid protein consists of the DNA-binding basic region (B) of MAX and the helix-loop-helix (HLH) dimerization domain of E47. The N-terminal FLAG-tag and nuclear localization signal (FLAG-NLS) were included for experimental and subcellular localization purposes, respectively. Schematics of MYC, MAX, E47, and ME47 are shown with relevant regions and amino acid numbers. MB: MYC homology box, LZ: leucine zipper. (B) MDA-MB-231 cells expressing 4-hydroxytamoxifen (4OHT)-inducible Vector, Omomyc, or ME47 were generated by lentiviral infection with the two-plasmid GEV16 system.29,30 ME47, Omomyc (a kind gift from Dr. Laura Soucek), and Vector were cloned into this system with the FLAG-NLS tag. In this system, the constitutively expressed GEV16 transcription factor is translocated to the nucleus only in the presence of 4OHT, which enables the GAL4-DNA binding domain (DBD) fusion to induce the inserted gene of interest. To evaluate protein expression, cells were treated for 24 hours with 12.5 nM 4OHT (+) or EtOH (-) control, harvested in boiling SDS lysis buffer, and evaluated by immunoblot with FLAG (F3165, Sigma) and ACTIN (A2066, Sigma) antibodies42. Immunoblots are representative of three independent experiments. (C) The MDA-MB-231 panel was treated with 10 �g/mL EdU for 10 hours and percent EdU incorporation was evaluated using the Click-It EdU Alexa Fluor 488 Imaging Kit (Life Technologies). Individual and mean values from three independent experiments are shown, *p<0.05, **p<0.01, one-way ANOVA with Bonferroni post-test. (D) Cell proliferation was assessed using the CyQUANT® NF Cell Proliferation Assay Kit (Invitrogen). The panel of MDA-MB-231 cells was seeded at 1.0x103 cells per well in 96-well plates, treated for 24 hours with 12.5 nM 4OHT (+) or EtOH control (-) and subsequently measured for DNA content over five days. Mean ± standard deviation (s.d.) for three independent experiments are shown, *p<0.05, one-way ANOVA with Bonferroni post-test for multiple testing. Blue and red asterisks indicate significance of Vector (+) compared to Omomyc (+) or ME47 (+), respectively. Population doubling times (inset) were calculated using Prism Graphpad v5.0. (E) The panel of MDA-MB-231 cells was treated for 24 hours with 12.5 nM 4OHT or EtOH control, then seeded in triplicate at 5.0x103 cells per dish to assess anchorage-independent colony growth in soft agar43. Plates were imaged after three weeks on a Leica MZ FLIII stereomicroscope, with representative images shown (left; scale bars = 2mm). Colony numbers were quantified using ImageJ software (NIH). Individual and mean values from three independent experiments are shown, *p<0.05, one-way ANOVA with Bonferroni post-test for multiple testing. Normal distribution and equal variances were confirmed for all datasets using the D'Agostino & Pearson omnibus normality test and Brown-Forsythe tests, respectively. Figure 2. ME47 inhibits MDA-MB-231 xenograft tumour growth. (A) MDA-MB-231 cells with 4OHT-inducible Vector or ME47 were subcutaneously injected with 2.0x106 cells in 0.2 mL phosphate-buffer saline (PBS) into the right flank of 10 female NOD/SCID mice per group. 10 animals per group was chosen based off of previous

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experience with this model and to ensure enough animals for statistical analysis in the event of animals becoming sick. Once tumours reached approximately 200 mm3 in volume, mice were assigned to standard chow alone or standard chow supplemented with 400 mg tamoxifen citrate per kg (Harlan Laboratories) and measured every 2 days. The first tumour to reach 200 mm3 was put on tamoxifen chow and the second animal was given standard chow with subsequent animals continuing this pattern until all animals were assigned a group. Measurement of the animals was blinded, with one researcher providing a blinded cage to a second researcher for measurement. Animals were sacrificed after tumour volume reached humane (experimental) endpoint of approximately 1000 mm3. Only experimental time points having all animals per group were considered for statistical testing. Tumour volumes are shown at each corresponding day post tamoxifen treatment. *p<0.05, ****p<0.0001, Student’s unpaired, two-tailed, t-test. (B) Individual and mean tumour volumes are shown after 22 days of treatment, the time point at which the first control animal reached humane endpoint (bearing a 1000 mm3 tumour), ****p<0.0001, Student’s unpaired, two-tailed, t-test. (C) Percent survival of tamoxifen-treated Vector or ME47 mice. Survival curve comparison was performed using Log-rank (Mantel-Cox) test (p=0.0003). (D) An independent MDA-MB-231 xenograft experiment was performed as described in (A), using 4 animals for vector tumours and 5 animals for ME47. All animals were sacrificed 33 days post treatment, and tumours were excised and weighed. Individual and mean tumour weights are shown, p=0.0635, Student’s unpaired, two-tailed, t-test. Representative tumour images are shown (scale bars =5mm). (E) Expression of ME47 was detected by immunoblot using antibody to FLAG and ACTIN as loading control. (F and G) Tumours were formalin-fixed, paraffin-embedded and cross-sectioned for IHC analysis of Ki67 (F) and TUNEL staining (G) to measure proliferation and apoptosis, respectively. Scale bars = 500 µm. Slides were scanned by the Advanced Optical Microscopy Facility at the Princess Margaret Cancer Centre using ScanScope XT (Aperio Technologies) and quantified using the Aperio Positive Pixel Count Algorithm. Individual and mean values are shown, *p<0.05, Student’s unpaired, two-tailed, t-test. Normal distribution and equal variances were confirmed for all datasets using the D'Agostino & Pearson omnibus normality test and F-test test respectively. Figure 3. ME47 binds to MYC target gene promoters and interferes with MYC binding to DNA. (A) MDA-MB-231 cells with inducible ME47 were treated with 12.5 nM 4OHT or EtOH control for 24 hours. Cellular fractionation was performed as previously described42 and immunoblotted with antibodies to FLAG for ME47 detection, TUBULIN (CP06, Millipore) for cytoplasmic fraction (C) control, and Acetylated Histone H3 (AcH3) (sc-8655, Santa Cruz) for nuclear fraction (N) control. (B) MDA-MB-231 cells with inducible ME47 or Vector control were induced with 12.5 nM 4OHT for 24 hours. Chromatin immunoprecipitation followed by quantitative real-time PCR (ChIP-qPCR) was performed as previously described44, with a FLAG antibody (F1804, Sigma) or mouse IgG control (sc-2025, Santa Cruz) for E-box-containing promoter regions of MYC target genes (CAD, CCND1, CDK4, NCL) and a non-E-box negative control (chr6). (C) ChIP-qPCR was performed as in (B), with a MYC antibody (N262, homemade) or rabbit IgG control (sc-2027, Santa Cruz). Mean ± s.d. for three independent experiments are shown in (B) and (C), *p<0.05, **p<0.01, ***p<0.001,

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****p<0.0001, ns: not significant, one-way ANOVA with Bonferroni post-test. CAD: carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; CCND1: cyclin D1; CDK4: cyclin-dependent kinase 4; NCL: nucleolin. Figure 4. Working model of ME47 minimalist hybrid protein inhibitor mechanism of action. In the absence of ME47, the MYC:MAX complex binds to the E-box DNA sequences and activates transcriptional programs leading to cancer cell growth (left). Introducing ME47 out-competes MYC:MAX for E-box binding, which inhibits the transcriptional activity of MYC:MAX, leading to anti-proliferative effects on cancer cells (right).

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Supplementary Figure 1

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ARNT E-box 30 60

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ME47 1.36 0.117

MAX 0.0527 0.00273

ME47/MAX Ratio 26.2 42.9

Supplementary Figure 6

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1

SUPPLEMENTARY INFORMATION Supplementary Figure 1. MDA-MB-231 breast cancer cells are MYC sensitive for proliferation and anchorage-independent growth. (A) MDA-MB-231 cells were infected with lentivirus generated in HEK293Tv cells transfected with shRNAs targeting MYC or luciferase control (shLUC); shMYC #1 was designed by the Penn lab (5’GATGAGGAAGAAATCGATG3’), shMYC #2 was obtained from the RNAi Consortium (TRC; TRCN0000039640). After puromycin selection, cells were harvested to confirm MYC knockdown by immunoblotting with a MYC antibody (9E10, homemade) and ACTIN as loading control (left). These cells were plated in soft agar, imaged, and quantified (Supplementary Figure 1A, right) as described in Figure 1E. Individual and mean colony numbers relative to shLUC control from three independent experiments are shown with corresponding representative images below, ****p<0.0001, one-way ANOVA with Bonferroni post-test. Scale bars = 2 mm. (B) Immunoblot analysis (left) demonstrates that MYC knockdown persists from day 1 through day 6 (experimental endpoint for proliferation, right). Cells were assayed for proliferation over six days as described in Figure 1D. Mean relative DNA content ± s.d. from three independent experiments is shown, *p<0.05, **p<0.01, Student’s unpaired, two-tailed, t-test. Normal distribution and equal variances were confirmed using the D'Agostino & Pearson omnibus normality test and the F-test respectively. Doubling time in hours is indicated in the inset.

Supplementary Figure 2. HEK293Tv cells are MYC sensitive for proliferation and anchorage-independent growth. (A) HEK293Tv cells infected with shLUC or shMYC #1 were generated as described in Supplementary Figure 1A. MYC knockdown was confirmed by immunoblot analysis (left). Cells were seeded in soft agar and quantified as described in Figure 1E (right). Individual and mean colony numbers relative to shLUC control from three independent experiments are shown with corresponding representative images below, **p<0.01, Student’s unpaired, two-tailed, t-test. Scale bars = 2 mm. (B) MYC knockdown is assessed at day 1 and day 6 post-seeding by immunoblotting (left). HEK293Tv cells were seeded at 2.5x102 cells in 96-well plates to measure proliferation over six consecutive days, as described in Supplementary Figure 1B. Mean relative DNA content ± s.d. from three independent experiments is shown, *p<0.05, Student’s unpaired, two-tailed, t-test. Doubling time is indicated in the inset. Normal distribution and equal variances were confirmed for all datasets using the D'Agostino & Pearson omnibus normality test and F-test respectively.

Supplementary Figure 3. Effect of ME47 and Omomyc on anchorage-independent growth of HEK293Tv cells. (A) HEK293Tv cells stably infected with 4OHT-inducible FLAG-NLS-Vector, -Omomyc or -ME47 were treated for 24 hours with 12.5 nM 4OHT (+) or EtOH control (-). Expression is confirmed by immunoblotting using FLAG to detect ME47 or Omomyc and ACTIN as loading control. (B) HEK293Tv cells were seeded for anchorage-independent growth in soft agar as described in Figure 1E and quantified following 10 days of incubation. Mean colony numbers relative to Vector control are shown ± s.d. for three independent experiments, *p<0.05, one-way ANOVA with Bonferroni post test for multiple testing. Normal distribution and equal variances were confirmed for all datasets using the D'Agostino & Pearson omnibus normality test

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and Brown-Forsythe test respectively. Individual and mean values from three independent experiments are shown. (C) Representative images of HEK293Tv colonies grown in soft agar. Scale bars = 2 mm.

Supplementary Figure 4. Endogenous MYC and MAX expression are not affected by ME47 expression. (A) MDA-MB-231 cells with inducible ME47 were treated with 12.5 nM 4OHT or EtOH control for 24 hours, harvested in boiling SDS lysis buffer, and immunoblotted for FLAG, MYC (9E10, homemade), MAX (sc-765, Santa Cruz), and ACTIN as loading control.

Supplementary Figure 5. ME47 and MAX B-HLH-LZ proteins possess similar DNA-binding affinities and sequence specificities. (A) Interactions of ME47 and MAX B-HLH-LZ against six different DNA sequences were tested by electrophoretic mobility shift assay (EMSA). Proteins were purified as previously described1, and sequences of the 24-base pair 6-FAM-labeled target DNA duplexes are shown. Core motifs are underlined and E-box sequences are in red. Purified proteins were titrated against 2 ng DNA duplexes at the concentrations indicated and analyzed by non-denaturing 10% PAGE2. NS: non-specific. (B) Protein:DNA dissociation constants (Kd) were derived from the EMSA titrations shown in (A). Supplementary Figure 6. Quantification of ME47 and endogenous MAX protein expression in cells. (A and B) Three biological replicates of MDA-MB-231 cells with inducible ME47 were treated with 12.5 nM 4OHT or EtOH control for 24 hours, and harvested in boiling SDS lysis buffer. Lysates were quantified using Pierce™ 660nm Protein Assay Reagent with Ionic Detergent Compatibility Reagent (Thermo Fisher). To quantify ME47 (A), 15 μg of lysate was loaded alongside 1.56 to 25 ng of recombinant FLAG-NLS-ME47 protein1, 2. For MAX (B), 30 μg of lysate was loaded alongside 0.4 to 6.24 ng of recombinant MAX protein (ab95309, Abcam). Blots were developed using LICOR OdysseyTM Infrared Imaging System and quantified using ImageJ. A standard curve was constructed from the ME47 and MAX recombinant controls. (C) The amount of ME47 and MAX (ng), relative to the amount of total lysate loaded (μg), was calculated using the standard curves to give a normalized ng/μg value. To calculate a molar ratio, kDa values of MAX (19.3 g/mol) and FLAG-NLS-ME47 (11.7 g/mol) were used to convert the amount of protein into moles. REFERENCES

1 Xu J, Chen G, De Jong AT, Shahravan SH, Shin JA. Max-E47, a designed minimalist protein that targets the E-box DNA site in vivo and in vitro. Journal of the American Chemical Society 2009; 131: 7839-7848.

2 Inamoto I, Chen G, Shin JA. The DNA target determines the dimerization partner selected by bHLHZ-like hybrid proteins AhRJun and ArntFos. Molecular bioSystems 2017; 13: 476-488.