Expression of RUNX1-JAK2 in Human Induced Pluripotent Stem Cell-Derived Hematopoietic Cells Activates the JAK-STAT and MYC Pathways

• Main Authors: Klaus Fortschegger, Anna-Maria Husa, Dagmar Schinnerl, Karin Nebral, Sabine Strehl
• Format: Article
• Language: English
• Published: MDPI AG 2021-07-01
• Series: International Journal of Molecular Sciences
• Subjects: leukemia; oncogenic fusion; CRISPR/Cas9; hematopoiesis; hiPSC; JAK-STAT signaling
• Online Access: https://www.mdpi.com/1422-0067/22/14/7576
• ISSN: 1661-6596; 1422-0067

A heterogeneous genetic subtype of B-cell precursor acute lymphoblastic leukemia is driven by constitutive kinase-activation, including patients with JAK2 fusions. In our study, we model the impact of a novel JAK2 fusion protein on hematopoietic development in human induced pluripotent stem cells (hiPSCs). We insert the <i>RUNX1-JAK2</i> fusion into one endogenous <i>RUNX1</i> allele through employing in trans paired nicking genome editing. Tagging of the fusion with a degron facilitates protein depletion using the heterobifunctional compound dTAG-13. Throughout in vitro hematopoietic differentiation, the expression of RUNX1-JAK2 is driven by endogenous <i>RUNX1</i> regulatory elements at physiological levels. Functional analysis reveals that <i>RUNX1-JAK2</i> knock-in cell lines yield fewer hematopoietic progenitors, due to <i>RUNX1</i> haploinsufficiency. Nevertheless, these progenitors further differentiate toward myeloid lineages to a similar extent as wild-type cells. The expression of the RUNX1-JAK2 fusion protein only elicits subtle effects on myeloid differentiation, and is unable to transform early hematopoietic progenitors. However, phosphoprotein and transcriptome analyses reveal that RUNX1-JAK2 constitutively activates JAK-STAT signaling in differentiating hiPSCs and at the same time upregulates MYC targets—confirming the interaction between these pathways. This proof-of-principle study indicates that conditional expression of oncogenic fusion proteins in combination with hematopoietic differentiation of hiPSCs may be applicable to leukemia-relevant disease modeling.