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A NOVEL EXON SPECIFIC ISOFORM EXPRESSION REPORTER SYSTEM (EXSISERS) APPLICABLE TO REAL-TIME, LIVE-CELL, AND HIGH-THROUGHPUT QUANTIFICATION OF P53 PROTEIN ISOFORMS

Deniz Tümen 1, Jeffery-Jiunn Dong Truong 2, Barbara Volz 1, Elisabeth Aschenbrenner 1, Jan Peter Teubner 1, Katja Neumeyer 1, Manuela Gunckel 1, Kirstin Pollinger 1, Patricia Mester-Pavel 3, Marlen Michalski 1, Veruschka Albert 1, Claudia Kunst 1, Karsten Gülow 1, Martina Müller-Schilling 1

Affiliations

1 University Hospital Regensburg, Regensburg, Germany

2 Helmholtz Center Munich, Munich, Germany

3 Universitätsklinikum Regensburg, Regensburg, Germany

Summary

AI Generated

A novel intein-luciferase reporter system (EXSISERS) enables real-time quantification of p53 isoforms and identified drug candidates that selectively kill colorectal cancer cells by upregulating pro-apoptotic p53 isoforms.

EXSISERS was integrated into TP53 coding exons 2, 4, and 7 of HCT116 cells using CRISPR/Cas9 to non-invasively distinguish FLp53, Δ40p53, Δ133p53, and Δ160p53 in real-time, overcoming limitations of immunoblot, immunoprecipitation, and mass spectrometry methods.
Screening 4,863 anti-cancer compounds identified drug candidates that upregulate pro-apoptotic p53 isoform members and induce 60-90% cell death in colon cancer cell lines at nanomolar concentrations.
Drug treatment of primary colonic organoids predominantly induced cell death in malignant cells but not in healthy colonic organoids.
Higher Δ40p53:FLp53 ratios are linked to increased apoptotic and decreased tumor recurrence rates, while Δ133p53 and Δ160p53 are negative regulators with tumor-progressive abilities.
This work provides a universally applicable method to expand colorectal cancer treatment options by targeting p53 isoforms, which have been considered undruggable.

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This summary was generated by an AI large language model based on the content transcript. It is for informational purposes only and should not be considered a substitute for clinical judgment. Always rely on your professional expertise and the full clinical context when making clinical decisions.

Abstract

Introduction

p53 is frequently mutated in cancer which leads to attenuated DNA-binding or complete loss of p53. Further assuming, that p53 primarily acts as a transcription factor, it has therefore long been considered as undruggable. The presence of multiple protein isoforms and their respective cellular ratios often determine therapeutic success and therefore provide novel targets for drug treatment of p53. Those mainly include FLp53, Δ40p53, Δ133p53, and Δ160p53. FLp53 is responsible for the proliferative and metabolic regulation. Δ40p53, an N-terminally truncated isoform lacking TAD I (1 – 40 aa), is transcriptionally inactive. Together with FLp53, Δ40p53 can address other tumor-suppressive genes. Higher Δ40p53:FLp53 ratios are linked to increased apoptotic and decreased tumor recurrence rates. The Δ133p53 and Δ160p53 isoforms are known for their tumor-progressive abilities and are considered as negative regulators of tumor suppressive p53. We introduced an exon‑resident and intein-luciferase-based reporter system (Truong, Tümen et al., 2021, Nat. Cell Biol.) that first successfully quantifies FLp53, Δ40p53, Δ133p53, and Δ160p53 non-invasively and in real-time. Its access to high-throughput screenings allowed us to monitor the differential p53 isoform expression for 4,863 anti-cancer compounds and identified two potential drug candidates to provide new p53-tailored therapeutic options for the effective treatment of colorectal cancer (CRC).

Aims & Methods

We introduced three exon-specific intein luciferase reporter systems (EXSISERS) into tumor suppressor p53 (TP53) coding exon 2, 4, and 7 of HCT116 cells. Knock-in of the reporter systems was conducted with CRISPR/Cas9. We demonstrated spatial separation and integrity of both, p53 and the intein-luciferase reporters by Western blot and immunofluorescence. The luminescent read-out of EXSISERS provides the quantity of cellular p53 isoforms in real-time. We screened the p53 isoform expression via EXSISERS for over 4,863 anti-cancer compounds (MCE, HY-L025) and measured the luminescent signal alterations with a Multiplate Reader during the drug treatment.

Results

With the implementation of EXSISERS, we demonstrated the advantageous application over conventional protein detection methods. Immunoblot and immunoprecipitation, for instance, failed to quantify the isoform diversity of p53. Moreover, absolute p53-peptide quantification via mass spectrometry showed low FLp53-peptide sensitivity, which exclusively allowed distinction between tumor suppressive vs. oncogenic p53. EXSISERS, however, was able to non-invasively distinguish FLp53, Δ40p53, Δ133p53, and Δ160p53. The screen of 4,863 small molecules led to the identification of specific drug candidates upregulating pro-apoptotic p53 isoform members. We validated the efficacy of these drugs on different colon cancer cell lines. The drugs were applicable in nanomolar concentrations and resulted in efficient induction of cell death (60-90%). Drug treatment of primary colonic organoids has shown to predominantly induce cell death in malignant cells, but not in healthy colonic organoids.

Conclusion

In summary, our study demonstrated the integration of EXSISERS into TP53 and allowed us to rapidly quantify essential p53 isoforms to provide new approaches to treat CRC via p53. By screening various anti-cancer compounds, we identified potential drug candidates efficiently upregulating tumor suppressive p53 and consequently inducing cell death in CRC. Here, we underline the particular importance of our new universally applicable method to expand the CRC treatment options by drugging p53.

References

Truong, D.-J.J.; Phlairaharn, T.; Eßwein, B.; Gruber, C.; Tümen, D.; Baligács, E.; Armbrust, N.; Vaccaro, F.L.; Lederer, E.-M.; Beck, E.M.; et al. Non-Invasive and High-Throughput Interrogation of Exon-Specific Isoform Expression. Nat. Cell Biol. 2021, doi:10.1038/s41556-021-00678-x .

Disclosure

Nothing to disclose

Conference

UEG Week Copenhagen 2023

Topics

Colorectal

Submission format

Abstract

Session

New molecules and tools in GI cancer (Posters)

Citation

United European Gastroenterology Journal 2023; 11 (Supplement 8)

Published

2023

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