Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Fosselteder, J.
Functional characterization of CALR mutations in human HSPCs using CRISPR/Cas9 genome engineering.
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medizinische Universität Graz; 2023. pp. 113
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- Autor*innen der Med Uni Graz:
- Foßelteder Johannes
- Betreuer*innen:
- Kargl Julia
- Reinisch Andreas
- Wölfler Albert
- Altmetrics:
- Abstract:
- Myeloproliferative neoplasms are a group of clonal hematopoietic stem cell disorders, characterized by overproduction of various mature myeloid lineage-derived cell types. Calreticulin mutations are important oncogenic drivers of Philadelphia chromosome negative myeloproliferative neoplasms including essential thrombocythemia and primary myelofibrosis, where patients face a high thrombotic risk caused by excessive platelet counts and the risk for transformation into secondary acute myeloid leukemia. Mutant calreticulin, while causing and fueling these diseases, presents a promising target with high therapeutic potential. Current knowledge of mutant calreticulin’s mechanism-of-action is derived from genetically engineered mouse models or immortalized cancer cell lines. While these models proofed very valuable for gaining new insights in the past, ectopic over-expression of calreticulin and cross-species differences in molecular mechanisms in these models impede clinical translation of the findings. To overcome these limitations, we developed the first human gene-engineered model of calreticulin mutant myeloproliferative neoplasms. We utilized a sophisticated approach combining the CRISPR/Cas9 technology and adeno-associated viral vector transfer to site specifically introduce the two most common calreticulin mutations in primary human hematopoietic stem and progenitor cells. This approach allowed the establishment of a reproducible and trackable myeloproliferative phenotype in vitro and in xenografted mice. The novel model described here, represents important clinical disease hallmarks like thrombopoietin-independent megakaryopoiesis, bone marrow fibrosis with linked splenomegaly and excessive growth of megakaryocyte progenitors. The introduction of calreticulin mutations in healthy hematopoietic stem and progenitor cells allowed to reveal early transcriptional rewiring upon mutation acquisition and the induction of an endoplasmic reticulum stress response. Novel insights gained through our model enabled the identification of mutation-specific vulnerabilities that can be exploited by proteasome and chaperone inhibition to selectively eradicate mutant cells. The humanized primary cell-based model described here, improves current calreticulin mutant models and provides a platform for molecular investigations as well as for testing of novel therapeutic compounds.