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Mutations in the histone methyltransferase Ezh2 drive context-dependent leukemia in Xenopus tropicalis

Leukemia volume 37, pages 2404–2413 (2023)Cite this article

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Abstract

CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.

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Fig. 1: The notch1/ezh2 crispant cohort shows acute penetrant disease with short latency.
Fig. 2: The notch1/ezh2 crispant cohort develops acute leukemia of myeloid, but not T-cell origin.
Fig. 3: Biallelic inactivation of ezh2 drives leukemogenesis independent from notch1 editing status.
Fig. 4: AML cells show engraftment upon transplantation in an irradiated frog.
Fig. 5: notch1/ezh2/dnm2 crispants show a dual leukemic phenotype with lymphoid and myeloid features.
Fig. 6: Differential engraftment at particular body sites upon transplantation of a leukemic liver of a sick notch1/ezh2/dnm2 animal in a rag2−/− host.

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Data availability

All data supporting the findings of this study are available within the article and its supplementary information and from the corresponding author upon reasonable request.

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Acknowledgements

DT was funded by a Ph.D. fellowship from the Research Foundation—Flanders (FWO-Vlaanderen, 3F021818). Research in the authors’ laboratory is supported by the Research Foundation—Flanders (FWO-Vlaanderen) (grants 3G0A6922 and 3G0D8716) and by the Concerted Research Actions from Ghent University (01G01115). Further support was obtained by the Hercules Foundation, Flanders (grant AUGE/11/14), the Desmoid Tumor Research Foundation, the Desmoid Tumor Foundation of Canada and SOS Desmoïde. We would like to acknowledge Amanda Gonçalves and Benjamin Pavie (VIB Bioimaging Core) for generating the QuPath script for positive cell detection. Furthermore, we are thankful to Tim Deceuninck for the good animal care. Finally, we would like to thank Joeri Tulkens for critical proofreading of the manuscript.

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Authors and Affiliations

  1. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Dieter Tulkens, Marthe Boelens, Thomas Naert, Marjolein Carron, Suzan Demuynck, Sylviane Dewaele, Gert Van Isterdael & Kris Vleminckx

  2. Cancer Research Institute Ghent (CRIG), Ghent, Belgium

    Dieter Tulkens, Marthe Boelens, Thomas Naert, Suzan Demuynck, David Creytens, Tim Pieters, Steven Goossens, Pieter Van Vlierberghe & Kris Vleminckx

  3. Center for Medical Genetics, Ghent University, Ghent, Belgium

    Marjolein Carron, Tim Pieters & Pieter Van Vlierberghe

  4. Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

    Marjolein Carron, Tim Pieters, Pieter Van Vlierberghe & Kris Vleminckx

  5. VIB Center for Inflammation Research, Ghent, Belgium

    Sylviane Dewaele

  6. VIB Flow Core, VIB Center for Inflammation Research, Ghent, Belgium

    Gert Van Isterdael

  7. Department of Pathology, Ghent University and Ghent University Hospital, Ghent, Belgium

    David Creytens

  8. Department of Diagnostic Sciences, Ghent University, Ghent, Belgium

    David Creytens & Steven Goossens

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  1. Dieter Tulkens
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Contributions

DT, TP, SG, PVV, and KV designed the study. DT and MB performed all genome engineering experiments. DT, SDem, TN, and MB performed general phenotyping experiments of the leukemia models. DT, MB, and SDew performed RNA expression analysis. WT executed the irradiation procedure. DT and GVI performed flow cytometry experiments. DT, MB, and SDem performed all stainings. TN performed binominal statistics. MC did the BATCH-GE analysis of amplicon deep sequencing data. DC provided histopathological feedback on blinded organ slides. DT and KV wrote the manuscript.

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Correspondence to Kris Vleminckx.

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Tulkens, D., Boelens, M., Naert, T. et al. Mutations in the histone methyltransferase Ezh2 drive context-dependent leukemia in Xenopus tropicalis. Leukemia 37, 2404–2413 (2023). https://doi.org/10.1038/s41375-023-02052-2

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