Nanobody-Mediated c-MYC Degradation Inhibits Tumor Cell Progression

Abstract

The c-MYC oncogene, a critical driver of malignancies, is frequently associated with poor prognosis because it promotes unchecked cell proliferation and alters gene expression. Effective targeting of c-MYC using conventional therapeutic strategies has been difficult, largely because of its unstructured nature. In the present study, we identified a myc-binding nanobody named as M4 from a synthetic phage-display nanobody library. We conjugated M4 with a cell-penetrating peptide (CPP) to generate a molecule CPM4 and examined the effects and action mechanisms of CPM4 in inhibition of tumor cell growth in vitro and in vivo. CPM4 exhibited efficient nuclear localization, caused c-MYC reduction, and induced apoptosis in MYC-expressing cells. Hydrogen/deuterium exchange mass spectrometry revealed that CPM4 binds to the central PEST sequence (241–263 epitope) of c-MYC with high affinity. Further analysis revealed that CPM4 promotes c-MYC degradation via enhanced phosphorylation at Thr58, disrupts the c-MYC/MAX heterodimer, and downregulates c-MYC-targeted downstream genes. Xenograft studies further validated the therapeutic efficacy of CPM4, showing a significant reduction in tumor growth. These results underscore the therapeutic potential of CPM4 as an effective drug candidate for inhibiting c-MYC-driven tumor growth.

Keywords

c-MYC degradation / cell-penetrating peptide (CPP) / nanobody / targeted cancer therapy

References

[1] F. Bray, M. Laversanne, H. Sung, et al., “Global Cancer Statistics 2022: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries,” CA: A Cancer Journal for Clinicians 74 (2024): 229–263.

[2] C. V. Dang, “MYC on the Path to Cancer,” Cell 149, no. 1 (2012): 22–35.

[3] S. C. Casey, V. Baylot, and D. W. Felsher, “The MYC Oncogene Is a Global Regulator of the Immune Response,” Blood 131, no. 18 (2018): 2007–2015.

[4] J. A. Nilsson and J. L. Cleveland, “Myc Pathways Provoking Cell Suicide and Cancer,” Oncogene 22, no. 56 (2003): 9007–9021.

[5] A. Papadimitropoulou, M. Makri, and G. Zoidis, “MYC the Oncogene From Hell: Novel Opportunities for Cancer Therapy,” European Journal of Medicinal Chemistry 267 (2024): 116194.

[6] C. V. Dang, L. M. Resar, E. Emison, et al., “Function of the c-Myc Oncogenic Transcription Factor,” Experimental Cell Research 253, no. 1 (1999): 63–77.

[7] C. Y. Lin, J. Lovén, P. B. Rahl, et al., “Transcriptional Amplification in Tumor Cells With Elevated c-Myc,” Cell 151, no. 1 (2012): 56–67.

[8] C. Lourenco, D. Resetca, C. Redel, et al., “MYC Protein Interactors in Gene Transcription and Cancer,” Nature Reviews Cancer 21, no. 9 (2021): 579–591.

[9] C. J. Poole and J. van Riggelen, “MYC—master Regulator of the Cancer Epigenome and Transcriptome,” Genes (Basel) 8, no. 6 (2017): 142.

[10] C. Sequera, M. Grattarola, A. Holczbauer, et al., “MYC and MET Cooperatively Drive Hepatocellular Carcinoma With Distinct Molecular Traits and Vulnerabilities,” Cell Death & Disease 13, no. 11 (2022): 994.

[11] L. Nguyen, P. Papenhausen, and H. Shao, “The Role of c-MYC in B-cell Lymphomas: Diagnostic and Molecular Aspects,” Genes (Basel) 8, no. 4 (2017): 116.

[12] J. Xu, Y. Chen, and O. I. Olopade, “MYC and Breast Cancer,” Genes & Cancer 1, no. 6 (2010): 629–640.

[13] L. Tan, D. Peng, and Y. Cheng, “Significant Position of C-myc in Colorectal Cancer: A Promising Therapeutic Target,” Clinical & Translational Oncology 24, no. 12 (2022): 2295–2304.

[14] R. Dhanasekaran, A. S. Hansen, J. Park, et al., “MYC Overexpression Drives Immune Evasion in Hepatocellular Carcinoma That Is Reversible Through Restoration of Proinflammatory Macrophages,” Cancer Research 83, no. 4 (2023): 626–640.

[15] C. V. Dang, A. Le, and P. Gao, “MYC-Induced Cancer Cell Energy Metabolism and Therapeutic Opportunities,” Clinical Cancer Research 15, no. 21 (2009): 6479–6483.

[16] Z. E. Stine, Z. E. Walton, B. J. Altman, A. L. Hsieh, and C. V. Dang, “MYC, Metabolism, and Cancer,” Cancer Discovery 5, no. 10 (2015): 1024–1039.

[17] T. Wahlström and M. Arsenian Henriksson, “Impact of MYC in Regulation of Tumor Cell Metabolism,” Biochimica Et Biophysica Acta 1849, no. 5 (2015): 563–569.

[18] A. Wolfer, B. S. Wittner, D. Irimia, et al., “MYC Regulation of a “Poor-prognosis” Metastatic Cancer Cell state,” Proceedings in the National Academy of Sciences of the United States of America 107, no. 8 (2010): 3698–3703.

[19] K. J. Savage, N. A. Johnson, S. Ben-Neriah, et al., “MYC Gene Rearrangements Are Associated With a Poor Prognosis in Diffuse Large B-cell Lymphoma Patients Treated With R-CHOP Chemotherapy,” Blood 114, no. 17 (2009): 3533–3537.

[20] H. Chen, H. Liu, and G. Qing, “Targeting Oncogenic Myc as a Strategy for Cancer Treatment,” Signal Transduction and Targeted Therapy 3 (2018): 5.

[21] K. I. Chan, S. Zhang, G. Li, et al., “MYC Oncogene: A Druggable Target for Treating Cancers With Natural Products,” Aging and Disease 15, no. 2 (2024): 640–697.

[22] M. J. Duffy and J. Crown, “Drugging “Undruggable” Genes for Cancer Treatment: Are We Making Progress?,” International Journal of Cancer 148, no. 1 (2021): 8–17.

[23] D. Lama, T. Vosselman, C. Sahin, et al., “A Druggable Conformational Switch in the c-MYC Transactivation Domain,” Nature Communications 15, no. 1 (2024): 1865.

[24] L. A. Carabet, P. S. Rennie, and A. Cherkasov, “Therapeutic Inhibition of Myc in Cancer. Structural Bases and Computer-Aided Drug Discovery Approaches,” International Journal of Molecular Sciences 20, no. 1 (2018): 120.

[25] A. D'Avola, K. Kluckova, and A. J. Finch, “Riches JC. Spotlight on New Therapeutic Opportunities for MYC-Driven Cancers,” OncoTargets and Therapy 16 (2023): 371–383.

[26] B. Amati, “Myc Degradation: Dancing With Ubiquitin Ligases,” PNAS 101, no. 24 (2004): 8843–8844.

[27] F. Ji, J. Ren, and C. Vincke, “Nanobodies: From Serendipitous Discovery of Heavy Chain-only Antibodies in Camelids to a Wide Range of Useful Applications,” in Single-Domain Antibodies: Methods and Protocols, ed. G. Hussack and K. A. Henry (2022), 3–17.

[28] S. Muyldermans, “Nanobodies: Natural Single-domain Antibodies,” Annual Review of Biochemistry 82 (2013): 775–797.

[29] Q. Su, W. Shi, X. Huang, et al., “Recent Advances of Nanobody Applications in Diagnosis and Detection,” MedComm – Biomaterials and Applications 2, no. 3 (2023): e54.

[30] P. Merikhian, B. Darvishi, N. Jalili, et al., “Recombinant Nanobody Against MUC1 Tandem Repeats Inhibits Growth, Invasion, Metastasis, and Vascularization of Spontaneous Mouse Mammary Tumors,” Molecular Oncology 16, no. 2 (2022): 485–507.

[31] M. Liu, L. Li, D. Jin, and Y. Liu, “Nanobody—A Versatile Tool for Cancer Diagnosis and Therapeutics,” Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 13, no. 4 (2021): e1697.

[32] Y. Wu, X. Hao, and M. Li, “Application of Nanobody in Cancer Treatment,” Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology 33, no. 7 (2017): 1085–1090.

[33] M. Kijanka, B. Dorresteijn, S. Oliveira, and P. M. P. van Bergen en Henegouwen, “Nanobody-based Cancer Therapy of Solid Tumors,” Nanomedicine (London) 10, no. 1 (2015): 161–174.

[34] J. Xu, K. Xu, S. Jung, et al., “Nanobodies From Camelid Mice and Llamas Neutralize SARS-CoV-2 Variants,” Nature 595, no. 7866 (2021): 278–282.

[35] W. Kang, C. Ding, D. Zheng, et al., “Nanobody Conjugates for Targeted Cancer Therapy and Imaging,” Technology in Cancer Research & Treatment 20 (2021): 15330338211010117.

[36] S. Steeland, R. E. Vandenbroucke, and C. Libert, “Nanobodies as Therapeutics: Big Opportunities for Small Antibodies,” Drug Discovery Today 21, no. 7 (2016): 1076–1113.

[37] D. M. Copolovici, K. Langel, E. Eriste, and Ü. Langel, “Cell-penetrating Peptides: Design, Synthesis, and Applications,” ACS Nano 8, no. 3 (2014): 1972–1994.

[38] S. L. Y. Teo, J. J. Rennick, D. Yuen, H. Al-Wassiti, and A. P. R. Johnston, “Pouton CW. Unravelling Cytosolic Delivery of Cell Penetrating Peptides With a Quantitative Endosomal Escape Assay,” Nature Communications 12, no. 1 (2021): 3721.

[39] Y. Xue, H. Jiang, T. Li, et al., “Inhibition of Tumor Growth Using a Conjugated Nanobody That Specifically Targets c-MYC,” Oncogene 44, no. 35 (2025): 3213–3224.

[40] A. S. Farrell and R. C. Sears, “MYC Degradation,” Cold Spring Harbor Perspectives in Medicine 4, no. 11 (2014): a014365.

[41] J. Jiang, J. Wang, M. Yue, et al., “Direct Phosphorylation and Stabilization of MYC by Aurora B Kinase Promote T-cell Leukemogenesis,” Cancer Cell 37, no. 2 (2020): 200–215.

[42] M. A. Contreras, Y. Serrano-Rivero, A. González-Pose, et al., “Design and Construction of a Synthetic Nanobody Library: Testing Its Potential With a Single Selection Round Strategy,” Molecules (Basel, Switzerland) 28, no. 9 (2023): 3708.

[43] J. Yan, P. Wang, M. Zhu, et al., “Characterization and Applications of Nanobodies Against human Procalcitonin Selected From a Novel Naïve Nanobody Phage Display Library,” Journal of Nanobiotechnology 13 (2015): 33.

[44] I. Zimmermann, P. Egloff, C. A. J. Hutter, et al., “Generation of Synthetic Nanobodies Against Delicate Proteins,” Nature Protocols 15, no. 5 (2020): 1707–1741.

[45] H. Xu, L. Li, B. Deng, et al., “Construction of a T7 Phage Display Nanobody Library for Bio-panning and Identification of Chicken Dendritic Cell-specific Binding Nanobodies,” Scientific Reports 12, no. 1 (2022): 12122.

[46] E. Pardon, T. Laeremans, S. Triest, et al., “A General Protocol for the Generation of Nanobodies for Structural Biology,” Nature Protocols 9, no. 3 (2014): 674–693.

[47] C. G. Acevedo-Rocha, M. T. Reetz, and Y. Nov, “Economical Analysis of Saturation Mutagenesis Experiments,” Scientific Reports 5 (2015): 10654.

[48] S. Tsukiji and T. Nagamune, “Sortase-mediated Ligation: A Gift From Gram-positive Bacteria to Protein Engineering,” Chembiochem 10, no. 5 (2009): 787–798.

[49] M. W. Popp and H. L. Ploegh, “Making and Breaking Peptide Bonds: Protein Engineering Using Sortase,” Angewandte Chemie (International ed in English) 50, no. 22 (2011): 5024–5032.

[50] M. A. Gregory and S. R. Hann, “c-Myc Proteolysis by the Ubiquitin-proteasome Pathway: Stabilization of c-Myc in Burkitt's Lymphoma Cells,” Molecular and Cellular Biology 20, no. 7 (2000): 2423–2435.

[51] E. Garralda, M. E. Beaulieu, V. Moreno, et al., “MYC Targeting by OMO-103 in Solid Tumors: A Phase 1 Trial,” Nature Medicine 30, no. 3 (2024): 762–771.

[52] X. Yin, C. Giap, and J. S. Lazo, “Prochownik EV. Low Molecular Weight Inhibitors of Myc-Max Interaction and Function,” Oncogene 22, no. 40 (2003): 6151–6159.

[53] H. Han, A. D. Jain, M. I. Truica, et al., “Small-molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy,” Cancer Cell 36, no. 5 (2019): 483–497.e15.

[54] J. E. Delmore, G. C. Issa, M. E. Lemieux, et al., “BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc,” Cell 146, no. 6 (2011): 904–917.

[55] M. Bekes, D. R. Langley, and C. M. Crews, “PROTAC Targeted Protein Degraders: The Past Is Prologue,” Nature Reviews Drug Discovery 21, no. 3 (2022): 181–200.

[56] L. Kent, Targeting the N-Myc Oncoprotein Using Nanobody Technology [dissertation] (University of Cambridge, 2018).

[57] M. Welcker, A. Orian, J. Jin, et al., “The Fbw7 Tumor Suppressor Regulates Glycogen Synthase Kinase 3 Phosphorylation-dependent c-Myc Protein Degradation,” PNAS 101, no. 24 (2004): 9085–9090.

[58] D. Clift, W. A. McEwan, L. I. Labzin, et al., “A Method for the Acute and Rapid Degradation of Endogenous Proteins,” Cell 171, no. 7 (2017): 1692–1706.e18.

[59] R. E. Kontermann, “Strategies for Extended Serum Half-life of Protein Therapeutics,” Current Opinion in Biotechnology 22, no. 6 (2011): 868–876.

[60] A. Sahni, Z. Qian, and D. Pei, “Cell-penetrating Peptides Escape the Endosome by Inducing Vesicle Budding and Collapse,” Acs Chemical Biology 15, no. 9 (2020): 2485–2492.

RIGHTS & PERMISSIONS

2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.