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ANIMAL MODELS

A CRISPR/Cas9 engineered MplS504N mouse model recapitulates human myelofibrosis

Leukemia volume 36, pages 2535–2538 (2022)Cite this article

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The genetic etiology of clonal hematopoiesis characterizing >90% of all myeloproliferative neoplasms (MPNs) involve somatic mutations that induce signaling downstream of cytokine receptors and demonstrate significant overlap in clinical phenotype [1]. Among these mutated genes is MPL, encoding for the thrombopoietin receptor which plays a central role in the regulation of megakaryopoiesis, HSC maintenance, and proliferation of progenitors [2,3,4]. The most frequent MPL mutations, MPL S505N and MPL W515K/L/R/S/A, reside in exon 10 encoding for the transmembrane domain and lead to TPO-independent receptor activation [5]. MPL W515K/L/R/S/A occurs at a much higher frequency in MPN, comprising 82% of MPL mutant cases in one study [6]. These somatic mutations are often found in the heterozygous state, but homozygous mutations do occur in primary myelofibrosis (PMF), typically through acquired copy-neutral loss of heterozygosity [7]. Both MPLS505N and MPLW515 mutations are also found as heterozygous autosomal dominant germline mutations with incomplete penetrance in hereditary thrombocytosis [8, 9]. To date, two studies have generated retroviral overexpression murine models to investigate the impact of human MPLW515 mutations on murine hematopoietic cells, both resulting in rapid onset of disease [10, 11]. However, the ability of the MPLS505N mutation has not yet been evaluated for its ability to perturb hematopoiesis, induce an MPN phenotype and recapitulate the pathology found in patients with MPL mutations [6, 8, 12]. Herein, we describe a CRISPR/Cas9 engineered mouse model harboring a germline MplS504N mutation that can aid further investigation into the complex etiology of MPNs and essential thrombocythemia.

A single guide RNA (sgRNA) was designed by the Center for Advanced Genome Engineering (CAGE, St. Jude Children’s Research Hospital, Memphis, TN). The serine to asparagine substitution was accomplished at codon 504 of Mpl, corresponding to the human S505N residue (Supplementary Fig. 1A). Further information on CRISPR/Cas9 engineering of the mouse model, the founder lines (#3, #15), and genotyping strategies are provided in supplemental methods. Animals were bred at the Animal Research Center of St Jude Children’s Research Hospital and studies were conducted under protocols approved by the institutional animal care and use committee. Hematopoiesis was characterized by flow cytometry, histology, and colony formation assays as described in supplemental methods. JAK-STAT activation was determined by western blot as described in supplemental methods.

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Fig. 1: MplS504N causes perturbations in megakaryopoiesis leading to thrombocytosis.
Fig. 2: MplS504N confers TPO independent megakaryocyte production.

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The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

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Acknowledgements

We thank the St. Jude Flow Cytometry and Cell Sorting Core, the Center for Advanced Genome Engineering, the Animal Research Center, the Transgenic/Gene Knockout Shared Resource and the Veterinary Pathology Core Laboratory. This work was supported by the American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children’s Research Hospital.

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Authors and Affiliations

  1. Department of Pediatric Oncology, Erasmus MC-Sophia’s Children’s Hospital, Rotterdam, The Netherlands

    Fabienne R. S. Adriaanse & C. Michel Zwaan

  2. Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands

    Fabienne R. S. Adriaanse, Ronald W. Stam & C. Michel Zwaan

  3. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Jennifer L. Kamens & Tanja A. Gruber

  4. Department of Pathology, St Jude Children’s Research Hospital, Memphis, TN, USA

    Peter Vogel

  5. Center for Advanced Genome Engineering (CAGE), St. Jude Children’s Research Hospital, Memphis, TN, USA

    Sadie M. Sakurada & Shondra M. Pruett-Miller

Authors
  1. Fabienne R. S. Adriaanse
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  2. Jennifer L. Kamens
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  7. C. Michel Zwaan
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Contributions

Contribution: FRSA, JLK, and SMS conducted laboratory experiments and analyzed data. PV supervised histologic tissue processing and provided murine pathology interpretation/expertise. SMP-M designed guide RNAs and supervised CRISPR/Cas9 engineering. RWS, CMZ, and TAG conceived and supervised the study. FRSA wrote the first draft of the manuscript. All authors provided critical feedback and helped shape the research, analysis and final manuscript.

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Correspondence to Tanja A. Gruber.

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Adriaanse, F.R.S., Kamens, J.L., Vogel, P. et al. A CRISPR/Cas9 engineered MplS504N mouse model recapitulates human myelofibrosis. Leukemia 36, 2535–2538 (2022). https://doi.org/10.1038/s41375-022-01684-0

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