Selective targeting of oncogenic KRAS G12D using peptide nucleic acid oligomers attached to cell-penetrating peptides

Mondal, J.; Lam, D.; Gerritsen, M. E.; Brotz, T. M.; Kennedy, J. G.; Rehlaender, B.; Ross, A. J.; Levy, D. E.; Bonagura, C. A.; Lanzilotta, W. N.; McCormick, F.; Rothman, J. H.; Wolfe, A. L.

doi:10.1101/2025.03.28.645837

bioRxiv · 10.1101/2025.03.28.645837

Selective targeting of oncogenic KRAS G12D using peptide nucleic acid oligomers attached to cell-penetrating peptides

Mondal, J.·Lam, D.·Gerritsen, M. E.·Brotz, T. M.·Kennedy, J. G.·Rehlaender, B.·Ross, A. J.·Levy, D. E.·Bonagura, C. A.·Lanzilotta, W. N.·McCormick, F.·Rothman, J. H.·Wolfe, A. L.

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Abstract

KRAS is a proto-oncogene that contains activating mutations in up to 30% of tumors. Many conventional therapies inhibit both cancerous and normal cells, which may cause toxicity. Thus, programmable mutant-selective targeted inhibitors are needed. Peptide nucleic acids (PNAs) incorporate base sequences analogous to DNA, with modified peptide backbones instead of ribose-phosphate backbones, allowing PNAs to hybridize with DNA with high avidity to suppress transcription. Here, we developed KRAS G12D-selective PNA oligomers with novel cell-penetrating flanking regions. Fluorescein-labeled PNA oligomers displayed high uptake rates in cells and nuclei. Exposure to PNA-delivery peptide conjugates resulted in repression of KRAS G12D mRNA and protein expression within 2 hours and lasting up to 48 hours. Varying cell-penetrating peptide (CPP) compositions and lengths of complementary KRAS sequences were tested using dose-response cell viability assays. These experiments identified configurations that were effective at selectively preventing growth of on-target KRAS G12D cells, while relatively sparing off-target KRAS G12C cells. Electrophoretic mobility shift assays demonstrated in vitro binding and selectivity for KRAS G12D DNA sequences. CPP-PNA-G12D-1 was effective against a panel of pancreatic ductal adenocarcinoma cell lines and patient-derived xenografts in vivo. These results show promise for an enhanced PNA-delivery peptide conjugate strategy as both a tool for studying tumors driven by oncogenic point mutations and as a potential therapeutic strategy to selectively target mutant cancer cells.

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Mondal, J., Lam, D., Gerritsen, M. E., Brotz, T. M., Kennedy, J. G., Rehlaender, B., Ross, A. J., Levy, D. E., Bonagura, C. A., Lanzilotta, W. N., McCormick, F., Rothman, J. H., Wolfe, A. L.. 2025-04-02. Selective targeting of oncogenic KRAS G12D using peptide nucleic acid oligomers attached to cell-penetrating peptides. https://doi.org/10.1101/2025.03.28.645837

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