Undruggable oncoproteins cMyc and NMyc bind to mediator of transcription with superior high affinity

The overexpression of MYC genes is frequently found in many human cancers including adult and pediatric malignant brain tumors. Targeting MYC genes continues to be challenging due to their undruggable nature. The nine-amino-acid activation domain (9aaTAD) has been identified using our prediction algorithm in all four Yamanaka factors including c-Myc and showed to activate transcription as short peptides. We generated a set of c-Myc constructs (1-108, 69-108 and 98-108) in the N-terminal regions and tested their ability to initiate transcription. We discovered strong interactions in nanomolar scale between the 9aaTAD of c-Myc and N-Myc proteins with the KIX domain of CBP coactivator. The c-Myc 9aaTAD (region 98-108) was not overlapping with the MBII (region 128-143) and therefore represents TRRAP independent activation region. Next, we showed the 9aaTADs in human c-Myc and N-Myc conservation within the MYC family. Interestingly, the loss of the 9aaTAD in L-Myc paralogs was identified in higher metazoans suggesting the deletions had occurred in early tetrapod evolution. In summary, as c-Myc is larg ely intrinsically disordered protein and therefore difficult to target by small molecule inhibitors, our finding of the c-Myc 9aaTAD in complex with the KIX domain represents a promising druggable target for development of new peptide inhibitors in MYC-driven tumors.

myc, MycN, KIX, MLL .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint

The MYC (also known as c-Myc) oncogene, located on chromosome 8q24.21, encodes a transcription factor that normally regulates the expression of approximately 15% of human genes implicated in proliferation, growth, differentiation, metabolism, and stemness [1–3]. Myc is one of the four Yamanaka factors (c-Myc, Oct4, Sox2, and Klf4) that could reprogram differentiated somatic cells into pluripotent stem cells. Aberrant c- Myc expression has been observed in ~70% of human cancers by driving autonomous cell cycle progression, recruitment of inflammatory cells, blocking differentiation, extensive stromal remodeling, invasion, and angiogenesis. Thus, MYC is thought to be a central driver of initiation of tumorigenesis [4–7]. Myc regulates broad transcriptional networks that controls developmental and homeostatic processes through direct activation of gene transcription, chromosomal translocation, genomic amplification, mRNA upregulation, and retroviral integration [8–11]. Structurally, MYC contains a basic helix-loop-helix leucine-zipper (bHLH- LZ) motif, mediating dimerization with its obligate partner, the Myc-associated protein x (MAX), and DNA binding [12–14]. The c-Myc protein harbors conserved regions MB0 (10-32), MBI (44-63), MBII (128-143), MBIIIa (188-199), MBIIIb (259-270), MBIV (304-324) and C-terminal DNA binding domain bHLHZip [15–17]. The molecular dynamic simulation for c-Myc (protein conformation combined with NMR experiments), provided the first insights into intrinsically disordered N-terminal region including MB0, MBI, MBII and activation domain regions [17]. The MBII region is responsible for interaction with general transcriptional coactivator TRRAP [18], which is component of human SAGA complex [19]. Noteworthy, EZH2 protein, a subunit of Polycomb repressive complex /i1 2, is essential for oncogenesis of MLL rearranged leukaemias. The c-Myc (central region 144-349) build a complex with EZH2 and mediated activation of non-canonical EZH2 targets [20]. Despite four decades of re search and drug development, c-Myc has been recognized as highly undruggable oncotarget [21,22]. Apart from the DNA binding domain, the c-Myc protein is largely unstructured and intrinsically disordered protein [23], and is linked with difficulty to design small molecule inhibitors. One of the most extensively investigated MYC inhibitors is a peptide OmoMYC, which acts by blocking of the c-Myc binding [15,24,25]. The OmoMYC peptide (OMO-103) is in the first-in-human phase I/II clinical trial in patients with advanced solid tumors (VHIO-born spin-off Peptomyc S.L., Spanish Agency of Medicines and Medical Devices, EU-funded project SYST-iMYC), (https://ClinicalTrials.gov/show/NCT04808362). Recent study shows that the coactivator EZH2 promotes c-Myc-driven oncogenesis by binding to the c-Myc protein. Both EZH2 and c-Myc are efficiently destroyed by the small drug MS177, which targets the EZH2 protein [20]. It was discovered that the small drugs JQ1 and dBET6, which target the BET4 protein, also inhibit MYC expression [26–28]. Mitotic Aurora kinase A (AURKA) functions as a transcription factor stabilizes and binds c-Myc and N-Myc [29,30]. Previously, we had identified the nine-amino-acid activation domains (9aaTAD) using our prediction algorithm (www.med.muni.cz/9aaTAD) in numerous transcription factors including members of the Gal4-p53-E2A-MLL- SREBP-NF-SP-KLF-SOX families [31–39]. Recently, we have identified the 9aaTAD activation domain in MYCN and also in all Yamanaka transcription activators and showed the 9aaTADs activating transcription as short peptides [40]. In this study, we sought to investigate the activation of c-Myc transcription in constructs with and without the 9aaTAD. We discovered a strong interaction at nano-molar scale between the KIX domain of general coactivator CPB and the 9aaTAD activation domains of c-Myc and N-Myc proteins. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint

Expression construct and peptides The GST-His6x-TEV-KIX construct was generated by PCR resulting in fusion product (GSTseq) - G S (linker) - H H H H H H (His6x tag) - G (linker) - E N L Y F Q / G (TEV tag) - (KIX region 586–672) and cloned into pGEX-2T vector. Custom peptide synthesis services were provided by Apeptide Shanghai Chutide Biotechnology Co., Ltd. Peptides have 95% purity and 2 mg were delivered #MLL: Biotin - GS-NILP-SDIM D FVLKNT, #c-Myc: Biotin - GSSS-TQLEMVTELLG, #N-Myc: Biotin - GSSS-EPP-SWVTEMLLEN, #SpQ: Biotin - GSS- GQ VSWQ T LQLQ NLQ, #SpD: Biotin - GSS- GD VSWD T LDLD DLD. Sample Preparation The non-labelled KIX domain was expressed as described [38]. In brief, the uniformly labelled 13C, 15N labelled KIX domain used for assignment experiments and 15N labelled KIX domain used for titration experiments were expressed in M9 minimal media containing 15N-ammonium sulfate with and without 13C6- D-glucose, respectively. The cell pellet obtained from 1 L culture was re-suspended in 40 mL buffer (50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 40 mM imidazole, 3 mM NaN3, pH 7.5). Lysozyme was added to the pellet to the final concentration of 0.3 mg/mL and sonicated at 40 amplitude 1s on, 5s off while kept on ice. The lysate was centrifuged at 21,000 g for 1 hour at 4 °C. The supernatant was loaded on the His trap HP 5 mL column packed with Nickel (GE Healthcare). FPLC was used for elution of the GST-His6x-TEV-KIX protein with 300 mM imidazole in 50 mM Tris-HCl buffer (150 mM NaCl, 3 mM NaN3, pH 7.5). The GST-His6x-tag was removed by TEV protease overnight at 4 °C while dialyzing in 50 mM Tris-HCl buffer (150 mM NaCl, 3 mM NaN3, pH 7.5). Cleaved and dialyzed protein was loaded on the His trap HP 5 mL column packed with Nickel resin equilibrated with 50 mM Tris-HCl buffer (150 mM NaCl, 3 mM NaN3, pH 7.5) and the KIX domain was eluted with 150 mM imidazole in 50 mM Tris-HCl buffer (150 mM NaCl, 3 mM NaN3, pH 7.5). Eluted KIX domain was loaded on HiLoad S30 120 mL equilibrated with 20 mM NaPi buffer (50 mM NaCl, 1 mM NaN3, pH 6.0). The purity of protein samples between purification steps was verified by SDS PAGE and MALDI-TOF mass spectrometer and the quantity of KIX domain was determined by 280 nm absorbance. The quality of the protein was determined using Differential Scanning Fluorimetry (Prometheus NT.48, NanoTemper Technologies GmbH). The uniform labelling of KIX domain was confirmed by MALDI-TOF MS. Assessment of enzyme activities The β -galactosidase activity was determined in the yeast strain L40 [34]. The strain L40 has integrated the lacZ reporter driven by the lexA operator. In all hybrid assays, we used 2 μ vector pBTM116 for generation of the LexA hybrids. The yeast strain L40, the Saccharomyces cerevisiae Genotype: MATa ade2 his3 leu2 trp1 LYS::lexA-HIS3 URA3::lexA-LacZ, is deposited at ATCC (#MYA-3332). For β -galactosidase assays, overnight cultures propagated in YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose) were diluted to an A600 of 0.3 and further cultivated for two hours and collected by centrifugation. The crude extracts were prepared by vortexing with glass beads for 3 minutes. The assay was done with 10 ul crude extract in 1ml of 100 mM phosphate buffer pH7 with 10 mM KCl, 1 mM MgSO4 and 0.2% 2-Mercaptoethanol; reaction was started by 200 ul 0.4% ONPG and stopped by 500 ul 1 M CaCO3. The average value of the β -galactosidase activities from two independent transformants is presented as a percentage of the reference with the standard deviation (means and plusmn; SD; n = 2). We standardized all results to previously reported Gal4 construct HaY including merely the activation domain 9aaTAD with the activity set to 100% [34]. Bio-layer interferometry .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint Bio-layer interferometry on Octet RED96e (ForteBio) was used for the analysis of interaction between KIX domain and proposed activation domains. All experiments were performed at 30°C at 1000 rpm shaking. Streptavidin-bearing SA biosensors (ForteBio) were immersed for 300 sec into the assay buffer (20mM sodium phosphate pH 6.0, 50mM NaCl) containing 60 μ M biotinylated MLL, MYC or NMY peptide, respectively. After subsequent wash in the assay buffer, the final steady response of ligand reached 0.6-0.8 nm. Parallel blank sensor was treated the same way with pure assay buffer being used instead of biotinylated peptide solution in the immobilization step. For the binding assay, all sensors were used in parallel applying the following procedure: 120 sec baseline in the assay buffer, 180 sec association in the assay buffer containing increasing concentration of KIX domain for each cycle (0.31, 0.63, 1.25, 2.5 and 5.0 μ M), 240 sec dissociation in the assay buffer and 3 repetitions of 30 sec regeneration in 50mM NaOH followed by 30 sec assay buffer wash. Each binding experiment was performed in pentaplicate and the data were processed using Data Analysis 11.1 evaluation SW (ForteBio). Obtained binding curves were blank-subtracted and fitted by a 1:1 binding model using steady state analysis. Final KD (apparent) values were calculated as an average of all measurements. Western Blot Analysis The desaturated yeast total protein samples were prepared by heating cells in lysis buffer at 94°C for 5 minutes (lysis buffer: 10% SDS, 500 mM Tris-HCl pH 6.8, 500 mM DTT, 50% glycerol v/v, 0.025% bromophenol blue dye). Proteins from samples were spread out by molecular size during SDS-PAGE and blotted to nitrocellulose. The immuno-detection of proteins was carried out using mouse anti-HA monoclonal antibody (2-2.2.14, #26183, ThermoFisher Sci) and secondary anti-mouse IgG antibodies conjugated with horseradish peroxidase (#A9044, Sigma Aldrich). The proteins were visualized using Pierce ECL (#32106, ThermoFisher Sci) according to the manufacturer’s instructions. Noteworthy, we observed higher abundance of c-Myc protein generated from both shorter constructs (69-103 and 69-108), which expressions were significantly higher than the longer c-Myc constructs (1-103 and 1-108).

The 9aaTAD is well conserved in evolution of the MYC family Previously, we have identified the 9aaTADs in human c-Myc and N-Myc and also in all three Myc animal paralogs in lobe-finned bony fish coelacanth (including functional L-Myc) and experimentally determined the 9aaTADs activating transcription as short peptides [37]. In higher metazoans, including humans, the 9aaTAD activation domains is completely absent in L-Myc paralogs (Fig 1a), what suggests that the loss of 9aaTAD in L-Myc paralogs has occurred already in early tetrapod evolution. In lower metazoans, including all fishes, we found the conservation of the 9aaTAD in all members of the Myc family and that to the last unicellular ancestor of animals, choanoflagellates, represented here by Monosiga brevicollis (Fig 2). Activation of c-Myc transcription is linked with the 9aaTAD Next, we tested the N-terminal regions of c-Myc as activators of transcription. The alone-standing c-Myc 9aaTAD activation domain (region 98-108), the c-Myc N-terminal regions with 9aaTAD (1-108, 69-108) and corresponding c-Myc regions without 9aaTAD (1-103, 69-103). All generated constructs were tested for activation of transcription in one hybrid assay (Fig. 1b). The constructs without 9aaTAD have lost the majority of their transcriptional capacity in comparison with corresponding constructs including the 9aaTAD. Furthermore, we observed higher abundance of c-Myc protein generated from both shorter constructs (69-103 and 69-108), which expressions were significantly higher than the longer c-Myc constructs (1-103 and 1-108) and their expression were below our immuno-detection threshold (Supplementary Figure S1). The naturally disordered prediction for c-Myc region 1-108 was generated by PONDR algorithm [41] (Fig. 1c). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint The KIX domain interactions with c-Myc and N-Myc Next, we sought to investigate the KIX interactions with the 9aaTADs by bio-layer interferometry (BLI). As MLL and other members of the 9aaTAD family bind to KIX domain of CBP [42], we used well-studied MLL peptide as a positive control here and previously [38]. The MLL 9aaTADs occupied the same space on the KIX domain as E2A and p53. Furthermore, they induced the KIX intramolecular re-formation realized by the two- point interaction involving 9aaTAD positions p3-4 and p6-7 [38]. For all three tested 9aaTADs, a clear binding to the KIX was observed (Fig. 3a). Through steady-state analysis of the BLI-binding assay, the equilibrium dissociation constant (Kd) values in the micromolar to sub-micromolar range were discovered. The N-Myc peptide displayed Kd(apparent) of 0.88±0.11 μ M and c-Myc peptide Kd(apparent)= 2.16±1.17 μ M. The BLI measurements for immobilized MLL peptide and KIX protein (Kd(apparent)= 1.17±0.21 μ M) are in good agreement with our prior reference's measurement using isothermal titration calorimetry for free MLL peptide and KIX protein (Kd= 1.4±0.1 μ M) [38]. Final KD (apparent) values were calculated as an average of all measurements (for more details see Methods). In conclusion, both Myc peptides displayed strong binding to the KIX comparable to MLL peptide. Each binding experiment was performed in pentaplicate and the data were processed using Data Analysis 11.1 evaluation SW-ForteBio. Obtained binding curves were blank-subtracted (including Streptavidin-bearing SA- biosensors and KIX but excluding peptide) and fitted by a 1:1 binding model using steady state analysis (Fig. 3b). Glutamines prevent Sp1 from KIX binding To further confirm the above results and demonstrate the unbiased BLI-assay outcomes, we carried out additional experiment with activation domain that does not bind to KIX. Previously, we addressed the importance of over-represented glutamines in Sp1 activation region for ability to activated transcription (37). In this study, we substituted the over-represented glutamines for aspartic acid residues (construct Sp1-Ac, acidic form of Sp1 9aaTAD) (Fig. 3c). We tested native Sp1-9aaTAD and the acidic modification for KIX binding by BLI-assay. The result from BLI- measurement for Sp1-Ac peptide and KIX (Kd(apparent)= 9.77±1.57 μ M) was comparable to MLL (Kd(apparent)= 1.17±0.21 μ M) but the native Sp1 did not bind to KIX in the 3-16 μ M range (Kd not determined) and served as the negative control for all BLI-measurements (Fig. 3c, d). These results confirmed that the overabundance of glutamine residues prevents binding to the KIX domain.

The ongoing efforts to identify molecules able to directly bind, interfere and/or suppress MYC activity have been the scope of intense research in numerous laboratories. The Omomyc, a 90-residue peptide, was shown to bind Myc by interfering with dimerization with its partner MAX and thus suppressing cell proliferation [24]. Besides the attempts to target Myc directly, another approach was to suppress MYC activity by targeting factors regulating MYC degradation [43], alternative strategies target MYC stability or blocking its interaction between acetylated histones and the bromodomain protein BRD4 [44]. The JQ1, a small molecule inhibitor of BET proteins, has been shown to suppress Myc expression and cancer cell growth in vitro and in vivo in preclinical models of multiple myeloma, AML and CML [26–28]. Ongoing studies among others led by Dr Laura Soucek [45] and future studies including the focus on the 9aaTADs c-Myc and the KIX interaction will aim to translate .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint new findings into development of small drug inhibitors and can guide therapies targeting Myc-dependent tumors.

In this study, we tested the human c-Myc constructs (1-108, 69-108 and 98-108) in N-terminal regions for their ability to activate transcription. We discovered a very strong interaction at nano-molar scale between the KIX domain of general coactivator CPB and the 9aaTADs of c-Myc and N-Myc proteins. The close-fitting Myc and KIX binary interaction represents new promising druggable target, which could initiate further studies. Abbreviations 9aaTAD: nine-amino-acid activation domain AML: acute myeloid leukemia BLI: bio-layer interferometry CML: chronic myeloid leukemia MB: conserved regions in Myc proteins, Myc boxs Kd: equilibrium dissociation constant KIX: binding domain in CBP a P300 proteins Declarations Ethics approval and consent to participate Not relevant for this study! Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have no competing interests to declare. Funding This work was supported by Ministry of Health, Czech Republic (AZV NV19-05-00410 to AK) and by Ministry of Health, Czech Republic Conceptual Development of research organization (FNBr, 65269705). All rights reserved. The Czech Infrastructure for Integrative Struct ural Biology (CIISB) research infrastructure project LM2018127 funded by MEYS CR is gratefully acknowledged for financial support at the Josef Dadok National NMR Centre, Biomolecular Interactions, Crystallizatio n and Proteomics Core Facilities, CEITEC-Masaryk University. Author's Contributions MP and AK conceived and designed the project. JH and MP performed the experiments. VJ, VV and TO discussed the data. JH, MP and AK wrote the manuscript. All authors have contributed critically to intellectual content and have approved the final manuscript.

.CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint We thank Alena Hofrova for technical support and Apeptide Shanghai Chutide Biotechnology for excellent quality of supplied peptides. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint

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It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint Figure Legends Figure 1. Conservation of the 9aaTAD in MYC family. A, Conservation of the 9aaTAD domain in MYC family. We found conservation of the activation domains in all three Myc paralogs in lobe-finned bony fish coelacanth (Latimeria chalumnae, fin-to-limb transition, related to lungfishes and tetrapods), which also conserved in human c-Myc and N-Myc but lost in L-Myc. The 9aaTADs activation domains are colored for faster orientation. The 9aaTAD deletion in L-Myc is in grey. B, Transactivation Assay. The activation of transcription by identified 9aaTAD alone (region 98-108) is comparable with entire N-terminal region of c-Myc (region 98-108). The regions with the c-Myc 9aaTAD activation domains were tested in a reporter assay with hybrid LexA DNA binding domain for the capacity to activate transcription. The average value of the β - galactosidase activities from two independent transformants is presented as a percentage of the reference with standard deviation (means and plusmn; SD; n = 3). We st andardized the results to positive control p53 construct 6p53, which was set to 100% (raw value 273 +/-9) and control empty vector Hdd (raw value 1,5 +/-1). The 9aaTADs activation domains are colored for faster orienta tion. Deuterated 9aaTADs or their partial deletion are in grey. C, Predictor of Natural Disordered Protein Regions (PONDR). Prediction result for c-myc region 1-108 is shown. PONDR® is copyright ©1999 by the WSU Research Foundation. Figure 2. Alignment of the MYC family. The N-terminal regions of MYC proteins were aligned by sequence similarities and their predicted 9aaTAD activation domains are shown. The 9aaTADs activation domains are colored for faster orientation. The MYC clades with conservation MYB box O, I and II are shown. The deletion of activation domain in L clade are in grey box. Conservation of threonine in position 55 is highlighted in red. Figure 3. Bio-layer interferometry assay (BLI). A single experiment of pentaplicate is shown for each of the tested peptides (MLL, N-Myc, c-Myc, Sp1-Ac, Sp1). A and C , blank-subtracted association (180 sec) and dissociation (240 sec) curves for 5 concentrations of KIX domain interaction with the immobilized peptide. B and D, steady-state analysis of corresponding m easurement in panel A. The re sponse for each concentration shown as red circle, fitted curve for 1:1 binding model shown in grey. The goodness of each curve fit is given by the coefficient of determination R2. Values close to 1.0 indicate proximity to 100% fit. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint c-MYC Transactivation Assay c-Myc regions tested Activation Domain 9aaTAD Reporter activation 1-103 MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQL EM VT 9 ±01 1-108 MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQL EM VT E LL GG 49 ±03 69-103 MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAP-EDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQL EM VT 29 ±02 69-108 MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAP-EDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQL EM VT E LL GG 90 ±03 98-108 MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAP-EDIWKKFELLPTPPLSPSRRS-LCSPSYVAVTPFSLRGDNDGGGGSFSTAD QL EM VT E LL GG 67 ±04 Control constructs 6p53 set to 100 ±03 Empty vector 1 ±01 MYC proteins MYC Box II MBII Activation domain 9aaTADHuman C-Myc STA TQL EM VT E LL GG DMVNQS FI CD PDDETFI KNIIIQDCMWSG N-Myc HSS EPP SW VT E ML LE NE LW GSP AEEDAF GLGGLGGLTP NPVILQDCMWSG L-Myc (with 9aaTAD deletion) GIG PPE PW P GG CT GDEAESR GHSKGWGRNY ASIIRRDCMWSG Latimeria chalumnae C-Myc STA DQL SV MD E IL GV SPEE KTKP SSVTLHDDGFSG N-Myc WSG DPL DW AS E LL LL PE GD LW SG CE GEEDQFEL GS GSRLEQGNL NAIILQDCMWSG L-Myc SSG DKL EW VS E FL GA DEE QF KI NP GEIWGNL SS IIIHDCMWSG 9aaTAD conservation in MYC family 1 0,5 0 Predictor of Natural Disordered Protein Regions PONDR Score for c-Myc region 1-108 0 10 20 30 40 50 60 70 80 90 100 108 PONDR Score c-Myc amino acids A B C Figure 1 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint Human and viral Myc group hsa L-Myc inactive myc paralog M DYD SYQHYFY DYD CGED FY RST APSEDI WKKFELVPSPPTSPPW GLGPGAGDPAPGIGP PE PW P GG C TGDEAESRGHSKGWGRN YASIIRRD CMWSG hsa N-Myc active myc parapog MPSCSTSTMPGMICKNPDL EFD SLQPCFY PDEDDFYFGGPD STPPGEDI WKKFELLPTPPLSPSR GFAEHSSE PP SWVT EMLL E NE LWG SPAEEDAFGLGGLGGLT PNPVILQD CMWSG hsa C-Myc active c-myc MPLN VSF TNRNYDL DYD SVQPYFY CDEEENFYQQQQQ SELQPPAPSEDI WKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTAD QL EMVT ELLG G DMVN QS FIC DPDDETF IKNIIIQD CMWSG Myc T17 Q67004 inactive viral v-myc MPLN VSF ANRNYDL DYD SVQPYFY CDEEENFYQQQQQ SELQPPAPSED AN HKNIIIQDCMWSG MYC_AVIM2 P10395 active viral v-myc MPLS ASL PSKNYDY DYD SVQPYFY FEEEEENFYLAAQQRGSELQPPAPSEDI WKKFELLPTPPLSPSRR SSLAAASCFPSTAD QL EMVT ELLG G DMVN QS FIC DPDDESF VKSIIIQD CMWSG Myc deletion 106-143 active mutant c-myc MPLN VSF TNRNYDL DYD SVQPYFY CD EEENFYQQQQQSELQPP APSEDI WKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTAD QL EMVT ELLV S EKLASYQAARKDSGSPN... C-Myc group hsa-C Homo sapiens MPLN VSF TNRNYDL DYD SVQPYFY CDEEENFYQQQQQSELQPP APSEDI WKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTAD QL EMVT ELLG G DMVN QS FIC DPDDETF IKNIIIQD CMWSG lch-C XP005992710 lobe-finned bony fish Latimeria chalumnae MPLS SSF PSKNYDY DYD SVQPYFY CDYEDEHFYHHQQNQ LQPS APSEDI WKKFELLPTPPLSPSRRSS FSSIYPSTAD QL EMVS EFLG D DVVN QT FIC DPEDDSF LKSIIIHDCMWSG cmi-C XP007901659 jawed cartilaginous fish Callorhinchus milii MGRG...NFLC KNYNYDY DYD SFQPVFY DEDENFYQQQ LPX APSEDI WKKFELLPTPPLSPSRRPS FSLFPSNAD QL EMVT EFLG D DLVN QS FIC DSDSESV LKSIIIQDCMWSG rty-C XP020371961 jawed cartilaginous fish Rhincodon typus MPLTASATFLG KSYNYDY DYD SFQPVFY DEEENFYQQQ LP APSEDI WKKFELLPTPPLSPSRRPSFS LFPSNAD QL EMVT EFLG D DLVN QS FIC DADSESV LKSIIIQDCMWSG bbe-C BAD93381 Cephalochordata Branchiostoma belcheri MPGINAHHAVS VPSSKHY DFE SLQPYFY...PAREDDFYSTP SPSV PPSEDI WKKFELLPTPPRSPSHPAPK SFIPTVAE KL EMVS ELLD E DVVN QS FIF PLDTQS LKSKLIQD CMWSG bfl-C C3Y235 Cephalochordata Branchiostoma floridae MPGINAHHAVT VPSSKHY DFE SLQPYFY...PAREDDFYST PSPSV PPSEDI WKKFELLPTPPRSPSHPAPK SFIPTVAE KL EMVS ELLD E DAVN QS FIF PLDTQS LKSKLIQD CMWSG aca-C XP003219450 Reptilia Anolis carolinensis MPLT GPSF PSRTYDY DYD SVQPYFY FEDEEENFYLSAHNRGCELQPPAPSEDI WKKFELLPTPPLSPSRRGGC YYPSGAD QL EMVT ELLG S DAVN QS FIC DPDDDAF VKSIIIQD CMWSG pbi-C XP007438325 Reptilia Python bivittatus MPLT VPSY PSRAYDY DYD SVQPYFY YEDEEENFYLSAQHRGCELQPPAPSEDI WKKFELLPTPPLSPSRRSGS FPSSVD QL EMVT ELLG S EVVN QS FIC DPDDDAF VKSIIIQD CMWSG L-Myc group hsa-L inactive myc paralog, Homo sapiens M DYD SYQHYFY DYD CGED FY RST APSEDI WKKFELVPSPPTSPPW GLGPGAGDPAPGIGP PE PW PGGCTG DEAESRGH SKGW GRN YASIIRRD CMWSG lch-L XP005993959 lobe-finned bony fish Latimeria chalumnae M EYD TSQHYFY DDD NEED FY RSI APSEDI WKKFELVPTPPASPAWITGDK NFYPSSGD KL EWVS EFLG A DEEQFKINP GEIW GN LSSIIIHD CMWSG rty-L XP020379445 jawed cartilaginous fish Rhincodon typus MYEIATKANPAAHVTGGILMQILTSEGGFATNNS..... LLEEEDFYQTCEEF L KTVEMLPASPQSPLPKSN ICDSLSSLIPSKSD QL ELMS EFLL DDEEFIS QSLI CDLE ASLKMEQD CMWSN xtr-L NP001011144 Amphibia Xenopus tropicalis M DFGSCNNHYFY DVD MKED FY RCI APSEDI WKKFELVPGFPLSPGGCP GGG GT DWGA ELMD L GWESPMKLT GL SSVVLLRD CMWSG cmy-L M7B8F5 Reptilia Chelonia mydas M EFD SYQHYFY DHD SEED FY RST APSEDI WKKFELVPTPPTSPLCSAAGK ACCPGAEE RS DWLS HCCLA GEEPEYLIGT GEIF GN LSAFVLQD CMWSG psi-L XP006139201 Reptilia Pelodiscus sinensis M EFD SHQHYFY DHD SEED FY RST APSEDI WKKFELVATPRKAPPCPAGGK ACCPGAGE RS DRLS HCCLP GEEPEYLLGP GAIL GN LSAFMLQD CMWSG asi-L A0A1U7RG17 Reptilia Alligator sinensis M ELD SYQHYFY DYD SEED FY RST APSEDI WKKFELVPTPPTSPACGPADK ACCSGAAGE LG DWL PSCCLA GEEP GEIF AN LSAFILQD CMWSG gja-L XP015273252 Reptilia Gekko japonicus M ELDPPHQPYFF EAD PQAEDFH PSS APSEDI WKKFELVPPPPLLGAGPARD DA DYLL EP GGLL GN LSAFVLRD CMWSG afo-L XP009274523 Aves Aptenodytes forsteri M EFD SYQHYFY DHD AQED FH RST APSEDI WKKFELVPTPPLSPLGTPGEK ACCSGAED RS GWLS RYCLA GEEPEYLIGT GEIF GN LSAFILKD CMWSG gga-L F1NXY9 Aves Gallus gallus M ERD AYQHYLY DYD AGED FH RST APSEDI WKKFEFVPTPPLSPLGERP GCSAAEE RG CPP RCCLP DEPEYLIGT GQLF GN LSAFILRD CMWSG sha-L G3X282 Marsupialia Sarcophilus harrisii M DFD SYQHYFY DCD YEED FY RST APSEDI WKKFELVPSPPMSPPW GSGSSPGACCSSAANGP PE PW PGGCGV DEAEGRGY SKAL ARN YASIIRSD CMWSG N-Myc group hsa-N Homo sapiens MPSC...MPGM ICKNPDL EFD SLQPCFY PDEDDFYFGGPDS TPPGEDI WKKFELLPTPPLSPSR GFAEHSSE PP SWVT EMLL E NE LWG SPAEE DAFGL GGLGGLT PNPVILQD CMWSG lch-N XP006009573 lobe-finned bony fish Latimeria chalumnae MPGI ISKNSDL EFD SLQPCFY PDEDDFYFCGPDS APPGEDI WKKFELLPTPPLSPSRA GLQGDPAAGSP...LLGWGYRGGPRWSGD PL DWAS ELLL LPE GD LWS GCEGEE DQFEL GSGSRLEQGN LNAIILQD CMWSG rty-N XP020371801 jawed cartilaginous fish Rhincodon typus MPGV... VGS SCKDYDL EYD SYQPYFY PDD YGPEADF YPPSEDI WKKFELLPTPPLSPSRA XEMPS PX GWVS EMLL LEEDGD GEEPG... GAGI RSQSGVSRSQ ANPIVLQD CMWSG psi-N K7G066 Reptilia Pelodiscus sinensis MPGM VSKNPDL EFD SLQPCFY PDEDDFYLCGPDS APPGEDI WKKFELLPTPPLSPSRA GLQENPPGGA PL PWGGVALGGFRTRD PL DWAS ELLL LPPEAD LWG STD GGDLF ETGFEEGNN LNSIIIQD CMWSC gga-N E1C3E1 Reptilia Gekko japonicus MPGM ISKNPDL EFD SLQPCFY PDEDDFYLCGPDS APPGEDI WKKFELLPTPPLSPSRA GLQEHPPGGA PL PWGGAALGSCRPAD PL DWAS ELLL LPPEAE LWG STD GADFF ETGLGASNN LNSIIIQD CMWSG Distal Myc orthologs in early branched metazoans hsa-C Homo sapiens MPLN V SFT NRNYDL DYD SVQPYFY CD EEENFYQQQQQSELQ PP APSEDI WKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTAD QL EMVT ELLG GDMVN QS FIC DPDDETF IKNIIIQDCMWSG mbr A9V5B4 unicellular Choanoflagellate Monosiga brevicollis M S SFC AMSMSL DHLSPGMKNSF E SL SPGAQH FLRSLLETTTNSHTDRDRLR PSPVDEDDDPEFGARISASLRE LN SVMD EILG VS PEE KTK PSSVTLHDDGFSG aqu XP003390966 Porifera Amphimedon queenslandica MASL V ESG AE LL PHLDEEKMQYM AFTEAIS FAEQFE SPLYDLHEET FRELYLRPSPPLSPDDCEPSCSQSPVEETP...QARVQDNISTDAM AD SLIS NIME YEQIMDALQ EAEF ALSSGSSQP AEDLLIQD CMWSA tad B3S238 Placozoa Trichoplax adhaerens MA V HAE AFSNKL DFE PYGSYYM GED SEDDN I WSCLDIMPTPPLSPARQ QYITDTSSNYLA DKLLQVT EN LDFD NALI DMVGDT NSIF NGGSK LRSSLIQD CMWNA nve A7RIE4 Cnidaria Nematostella vectensis MTL VAEHL LMDTFGS DFD SLPPSLF KDF PEDGFN M KKKSMTSIEEDIMSDYSFPPTPPISPGCSSIAS EIGDPE RI QPVC DELE DDFNFAA EEKSLYF QEND FKDILIKD CMWNG hec AEG66931 Cnidaria Hydractinia echinata MI ITSA NRDLSVWS NID ESDNEEIM NLAKPMMDA S NPSDDI WRKFALPLTPPSSPSR TYGETSEDRRTDIAE RL HDVC ESLD STFDLT SYNVS KLAGVLN LRSKLISD CMWSG hvu D2KBP8 Cnidaria Hydra attenuata MT GSNWC THD ILPTDEIL LHKSILD TS SPSDDV WKKF LTPPSSPQSIGESS ESEETTDSFDTCA RL QYVC DNLD IGLELT TRCTNSFN LGSKLISDCMWSG lak A0A1S3JL92 Protostomia brachiopoda Lingula unguis MPR VQMKKMAHSLVKD DYE TFQPIYF QDDNEDHLHA GTP APSEDI WKKFELLPTPPRSPRR DREPPNITIPSDINTTE CL YKVS ELLD DDFL TQTQIILF PGST EQGSESQCHCPPP cgi XP011441199 Protostomia mollusca Crassostrea gigas ML IGRKNE KTAIM DYE MFQPCFY ENEPETI SPSS LPNEDM WKKFELLPTPPLSPRR DDDYDM NS LDLE DFNL IFDEFFE KESAI PKIMTLSETPPN LNSNLIQD CMWSG pfu AGS18763 Protostomia mollusca - Pinctada fucata M DYD TFQPCFY ENE TELSPVGSY PNEDM WKKFELLPTPPLSPQR DDQFD ST LDLE NLTL PLTDYIL NDDDDASLF EKMTFPESPPSPS LHSKLIQD CMWSG mye XP021367795 - Mizuhopecten yessoensis MV VRTAKAHSHQM ENE MHKCQT CDNGT TLCMN I WNKFDLGPPTPPGSPPH ETMS DF PELD GFNL SDNDVEFF DSREEDV LATMKHIFAEGTPLN LQSKLIQD CMWSG cte ELT88315 Protostomia annelida Capitella teleta MNR... TKHSLQSNTPLM EFD EVREGASAWSLSEVEFYGDM SSPNMDI WNKFEMGQPTPPASPER DSSTDGDDFS SQ SMWD SLLD SQLNF AHLNEP LNTKLIQDCMWSA pdu AGS55451 Protostomia annelida Platynereis dumerilii MDEK MCSLHQRPVSE DYD SFNPYFF EVGTEDEFYDV P APSEDI WKKFELLPTPPRSPKH ETSYPSSTASTGLMNSTLE KL QMVS DILE PVSLTSDFSMFPEDCALCSRDTLFS... ELNQS LKSNLIQD CMWSG MB0 MBI MBII9aaTAD T58 MB0 Figure 2 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 15, 2024. ; https://doi.org/10.1101/2024.03.15.585150doi: bioRxiv preprint