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Novel read through agent: ZKN-0013 demonstrates efficacy in APCmin model of familial adenomatous polyposis

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Abstract

Familial adenomatous polyposis (FAP) is a precancerous, colorectal disease characterized by hundreds to thousands of adenomatous polyps caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). Approximately 30% of these mutations are premature termination codons (PTC), resulting in the production of a truncated, dysfunctional APC protein. Consequently, the β-catenin degradation complex fails to form in the cytoplasm, leading to elevated nuclear levels of β-catenin and unregulated β-catenin/wnt-pathway signaling. We present in vitro and in vivo data demonstrating that the novel macrolide, ZKN-0013, promotes read through of premature stop codons, leading to functional restoration of full-length APC protein. Human colorectal carcinoma SW403 and SW1417 cells harboring PTC mutations in the APC gene showed reduced levels of nuclear β-catenin and c-myc upon treatment with ZKN-0013, indicating that the macrolide-mediated read through of premature stop codons produced bioactive APC protein and inhibited the β-catenin/wnt-pathway. In a mouse model of adenomatous polyposis coli, treatment of APCmin mice with ZKN-0013 caused a significant decrease in intestinal polyps, adenomas, and associated anemia, resulting in increased survival. Immunohistochemistry revealed decreased nuclear β-catenin staining in the epithelial cells of the polyps in ZKN-0013-treated APCmin mice, confirming the impact on the β-catenin/wnt-pathway. These results indicate that ZKN-0013 may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene.

Key messages

• ZKN-0013 inhibited the growth of human colon carcinoma cells with APC nonsense mutations.

• ZKN-0013 promoted read through of premature stop codons in the APC gene.

• In APCmin mice, ZKN-0013 treatment reduced intestinal polyps and their progression to adenomas.

• ZKN-0013 treatment in APCmin mice resulted in reduced anemia and increased survival.

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Data availability

The data used in this study for analysis and presented in the results are available upon reasonable request.

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Funding

The authors declare that no external funds, grants, or other support were received during the preparation of this manuscript. All studies were funded by Eloxx Pharmaceuticals.

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Authors and Affiliations

  1. Eloxx Pharmaceuticals, Watertown, MA, 02472, USA

    Martin R. Graf, Shruti Apte, Esteban Terzo, Simran Padhye, Shuhao Shi, Megan K. Cox, Roger B. Clark, Vijay Modur & Vasudeo Badarinarayana

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  1. Martin R. Graf
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Contributions

All authors contributed to the study’s design and experimental procedures. S.S. and R.C. designed and synthesized ZKN-0013; S.A. and S.P. conducted the cell proliferation and western blot experiments and analysis; E.T. provided oversight of the in vitro studies; M.C., V.M., and V.B. critically reviewed the manuscript; V.B. designed and managed the APCmin studies; V.B., V.M., and M.G. analyzed in vivo data and histopathology; M.G. wrote the manuscript. All authors approved the final manuscript.

Corresponding author

Correspondence to Vasudeo Badarinarayana.

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Ethics approvals

Pharmacokinetic studies were performed at WuXi AppTec Laboratory Testing Division (Cranbury, NJ), which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and compliant with the Office of Laboratory Animal Welfare (OLAW) Assurance to Conduct Public Health Service (PHS)-funded studies. Efficacy studies were conducted at the Crown Biosciences, Loughborough, UK, in compliance with the UK Animals Scientific Procedures Act 1986 (ASPA) and in line with Directive 2010/63/EU of the European Parliament and the Council of September 22, 2010, on the protection of animals used for scientific purposes.

Competing interests

All authors are employees and shareholders of Eloxx Pharmaceuticals.

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Graf, M.R., Apte, S., Terzo, E. et al. Novel read through agent: ZKN-0013 demonstrates efficacy in APCmin model of familial adenomatous polyposis. J Mol Med 101, 375–385 (2023). https://doi.org/10.1007/s00109-023-02291-x

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