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. 2024 Nov 13;30:211. doi: 10.1186/s10020-024-00896-7

Myc 9aaTAD activation domain binds to mediator of transcription with superior high affinity

Andrea Knight 1,2,3,✉,#, Josef Houser 4,5,6,#, Tomas Otasevic 2, Vilem Juran 3, Vaclav Vybihal 3, Martin Smrcka 3, Martin Piskacek 2,✉,#
PMCID: PMC11558822  PMID: 39538178

Abstract

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. Using our prediction algorithm, the nine-amino-acid activation domain (9aaTAD) has been identified in all four Yamanaka factors, including c-Myc. The predicted activation function was experimentally demonstrated for all these short peptides in transactivation assay. 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 in one hybrid assay. The presence and absence of 9aaTAD (region 100–108) in the constructs strongly correlated with their activation functions (5-, 3- and 67-times respectively). Surprisingly, we observed co-activation function of the myc region 69–103, called here acetyl-TAD, previously described by Faiola et al. (Mol Cell Biol 25:10220–10234, 2005) and characterized in this study as a new domain collaborating with the 9aaTAD. We discovered strong interactions on a nanomolar scale between the Myc-9aaTAD activation domains and the KIX domain of CBP coactivator. We showed conservation of the 9aaTADs in the MYC family. In summary for the c-Myc oncogene, the acetyl-TAD and the 9aaTAD domains jointly mediated activation function. The c-Myc protein is largely intrinsically disordered and therefore difficult to target with small-molecule inhibitors. For the c-Myc driven tumors, the strong c-Myc interaction with the KIX domain represents a promising druggable target.

Supplementary Information

The online version contains supplementary material available at 10.1186/s10020-024-00896-7.

Keywords: 9aaTAD, Myc, MycN, KIX, CBP

Introduction

The MYC (also known as c-Myc) oncogene encodes a transcription factor that normally regulates the expression of approximately 15% of human genes implicated in proliferation, growth, differentiation, metabolism, and stemness (Dang 2012; Lin et al. 2012; Nie et al. 2012). c-Myc is one of the four Yamanaka factors (c-Myc, Oct4, Sox2, and Klf4) that could reprogram already 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, c-Myc is thought to be a central driver of tumorigenesis (Beroukhim et al. 2010; Trakala et al. 2021; Walz et al. 2014; Zeller et al. 2006).

c-Myc regulates broad transcriptional networks that controls developmental and homeostatic processes through gene activation, chromosomal translocation, genomic amplification, mRNA upregulation, and retroviral integration (Casey et al. 2018; Kortlever et al. 2017; Kress et al. 2015; Sodir et al. 2011). Structurally, c-Myc contains a basic helix-loop-helix leucine-zipper (bHLH-LZ) motif, mediating dimerization with its obligate partner Myc-associated protein x (MAX) what enables DNA binding (Bédard et al. 2017; Patel et al. 2004; Vita and Henriksson 2006).

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 (Cowling and Cole 2006; Madden et al. 2021; Sullivan and Weinzierl 2020). 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 (Sullivan and Weinzierl 2020). The MBII region (128–143) is responsible for interaction with general transcriptional coactivator TRRAP (Feris et al. 2019), which is component of human SAGA complex (Herbst et al. 2021). Noteworthy, EZH2 protein, a subunit of Polycomb repressive complex 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 (Wang et al. 2022). Mediator transcription p300 associates with Myc TAD residues 1 to 110 and acetylates Myc protein at several lysine residues located between the TAD and DNA-binding domain (Faiola et al. 2005).

Despite four decades of research and drug development, c-Myc has been recognized as highly undruggable oncotarget (Carabet et al. 2018; Wang et al. 2021). Apart from the DNA binding domain, the c-Myc protein is largely unstructured and intrinsically disordered protein (Andresen et al. 2012), 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 (Duffy et al. 2021; Madden et al. 2021; Soucek et al. 1998). The OmoMYC peptide (OMO-103) is in the first-in-human phase I/II clinical trial in patients with advanced solid tumors (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 (Wang et al. 2022). It was discovered that the small drugs JQ1 and dBET6, which target the BET4 protein, also inhibit MYC expression (Muhar et al. 2018; Peter et al. 2022; Zuber et al. 2011). Mitotic Aurora kinase A (AURKA) functions as a transcription factor stabilizes and binds c-Myc and N-Myc (Dauch et al. 2016; Zheng et al. 2016).

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 (Hofrova et al. 2021; Knight and Piskacek 2022; Piskacek et al. 2015, 2016, 2017, 2018, 2019, 2007; Sandholzer et al. 2007). Recently, we have identified the 9aaTAD activation domain (small peptide sequence) in all Yamanaka transcription factors (c-Myc, Oct4, Sox2, and Klf4). The predicted activation function was experimentally demonstrated (transactivation assay with short predicted peptides within one hybrid constructs) (Piskacek et al. 2021).

In this study, we sought to investigate the activation of c-Myc transcription in constructs with and without the 9aaTAD domain. 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.

Materials and methods

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-2 T 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 (Hofrova et al. 2021). 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 1 s on, 5 s off while kept on ice. The lysate was centrifuged at 21,000 g for 1 h 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 (Piskacek et al. 2017). 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 min. The assay was done with 10 μl crude extract in 1 ml of 100 mM phosphate buffer pH7 with 10 mM KCl, 1 mM MgSO4 and 0.2% 2-Mercaptoethanol; reaction was started by 200 μl 0.4% ONPG and stopped by 500 μl 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% (Piskacek et al. 2017).

Bio-layer interferometry

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 s into the assay buffer (20 mM sodium phosphate pH 6.0, 50 mM NaCl) containing 60 μM 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 s baseline in the assay buffer, 180 s 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 μM), 240 s dissociation in the assay buffer and 3 repetitions of 30 s regeneration in 50 mM NaOH followed by 30 s 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 min (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).

Results

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 demonstrated activating function for these short 9aaTAD peptides (Piskacek et al. 2019). In higher metazoans, including humans, the 9aaTAD activation domains are completely absent in L-Myc paralogs (Fig. 1a), which suggests that the loss of 9aaTAD in L-Myc paralogs has occurred already in early tetrapod evolution and furthermore underpins the 9aaTAD activation function. 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).

Fig. 1.

Fig. 1

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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 standardized 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 orientation. 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

Fig. 2.

Fig. 2

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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

Activation of c-Myc transcription is linked with the 9aaTAD

We have analysed several previously reported mutants with surprising phenotypes. Reported insertion of glutamic acid into c-Myc (EMVT EL/E/LVS, region 100–105/inserted E/144–146) (Stone et al. 1987) diminished the 9aaTAD pattern and its function. The activation domain 9aaTAD was also destroyed in mutants with deletions 3–103 and 106–143. Nevertheless, partial activity was observed in mutants with deleted regions 41–103, 56–103, 93–103 (including also small inserts due to DNA manipulation), which seem to have a partial repair/restoration of the universal 9aaTAD motif (QSEL/E LLGG, ELLD/E LLGG and GSSI/E LLGG).

To identify the 9aaTAD activation domain, we ran our online 9aaTAD prediction service for trans-activating region 55–120 (localised between MYC boxes MBI and MBII) and revealed a 92% hit (sequence EMVT E LLGG, region 100–120) (Fig. 1). Similarly, we revealed a 100% hit for the c-Myc mutant, which has an intact activation potential despite the internal deletion (fusion result for the modified trans-activation region with the sequence EMVT E LLVS, which is the fusion of regions 100–105 to 144–146).

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).

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 (Romero et al. 2001) (Fig. 1c).

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 (Goto et al. 2002), we used well-studied MLL peptide as a positive control here and previously (Hofrova et al. 2021). 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 (Hofrova et al. 2021). For all three tested 9aaTADs, a clear binding to the KIX was observed (Fig. 3a).

Fig. 3.

Fig. 3

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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 s) and dissociation (240 s) curves for 5 concentrations of KIX domain interaction with the immobilized peptide. B and D, steady-state analysis of corresponding measurement in panel A. The response 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

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 μM and c-Myc peptide Kd(apparent) = 2.16 ± 1.17 μM. The BLI measurements for immobilized MLL peptide and KIX protein (Kd(apparent) = 1.17 ± 0.21 μM) 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 μM) (Hofrova et al. 2021). 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 μM) was comparable to MLL (Kd(apparent) = 1.17 ± 0.21 μM) but the native Sp1 did not bind to KIX in the 3–16 μM 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.

Discussion

The ongoing efforts to identify molecules able to directly bind, interfere, and suppress MYC activity have been the scope of intense research in numerous laboratories. Here, we demonstrated strong activation ability of the c-Myc 9aaTAD (comparable to the p53 activator) and very strong binding ability of the c-Myc 9aaTAD to the KIX domain of general coactivator CPB. A previous study (Faiola et al. 2005) clearly demonstrated acetylation of the c-Myc region 1–103 promoting activation function. Together with our observation, the region around 69–103, called here acetyl-TAD, might be a new co-activating domain supporting the 9aaTAD (100–108) function. We observed significant activation function independent of 9aaTAD in constructs with the region 69–103, although 3- and fivefold less, respectively, until the 9aaTAD was added (constructs 1–108 and 69–108 including 9aaTAD versus constructs 1–103 and 69–103 without 9aaTAD). The acetyl-TAD is clearly independent but synergizes with the 9aaTAD domain. Our study was conducted with purified proteins without the acetylation modifications and further in vivo studies are needed to investigate cellular function affected by the c-Myc.

Conclusions

In this study, we tested the human c-Myc 9aaTADs for their ability to activate transcription. We discovered a very strong interaction at the nano-molar scale between the KIX domain of general coactivator CPB and the 9aaTADs of c-Myc and N-Myc proteins. Moreover, we observed the co-activation function of the acetyl-TAD (region 69–103) as a new domain collaborating with the 9aaTAD (region 100–108). For further studies in c-Myc driven tumors, the close-fitting Myc and KIX binary interactions represent new promising druggable targets.

Supplementary Information

Additional file 1. (75KB, pdf)
Additional file 2. (41.5KB, pdf)

Acknowledgements

We thank Alena Hofrova for technical support and Apeptide Shanghai Chutide Biotechnology for excellent quality of supplied peptides.

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

Author 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.

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 Structural 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, Crystallization and Proteomics Core Facilities, CEITEC-Masaryk University.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors have no competing interests to declare.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Andrea Knight, Josef Houser and Martin Piskacek should be considered as joint lead, senior and corresponding authors.

Contributor Information

Andrea Knight, Email: andrea.knight@aru.ac.uk.

Martin Piskacek, Email: piskacek@med.muni.cz.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional file 1. (75KB, pdf)
Additional file 2. (41.5KB, pdf)

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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