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

Klaus Fortschegger,Anna-Maria Husa,Karin Nebral,Sabine Strehl,Dagmar Schinnerl

doi:10.3390/ijms22147576

Abstract

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 RUNX1-JAK2 fusion into one endogenous RUNX1 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 RUNX1 regulatory elements at physiological levels. Functional analysis reveals that RUNX1-JAK2 knock-in cell lines yield fewer hematopoietic progenitors, due to RUNX1 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.

Highlights

B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most frequent pediatric malignancy and a clinically and genetically heterogeneous disease [1,2,3,4,5]
Since in vitro differentiation of human induced pluripotent stem cells (hiPSCs) toward B-lymphoid cells remains a challenging task, we aimed to investigate a JAK2 fusion, which occurs in leukemia of the myeloid and lymphoid lineages, and whose expression is driven by an N-terminal partner at the onset of hematopoietic development
We addressed the question of whether the expression of RUNX1-JAK2 leads to constitutive activation of the JAK-signal transducers and activators of transcription (STATs) pathway in hiPSC-derived progenitors as it does in Ba/F3 cells (Supplementary Figure S8B,C)

Summary

Introduction

B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most frequent pediatric malignancy and a clinically and genetically heterogeneous disease [1,2,3,4,5]. A genetically diverse B-ALL subgroup comprises cases with rearrangements affecting genes involved in cytokine-receptor or kinase signaling, such as ABL1, ABL2, PDGFRB, CSF1R, JAK2, EPOR, and CRLF2 [6,7]. These alterations elicit similar gene expression signatures and often confer failure to standard multidrug treatment [1]. At least some of these fusion proteins may constitute a dual-hit oncogenic mutation. Constitutive kinase-activation induces proliferative and/or antiapoptotic signaling pathways. Interference with the function of the other fusion partner—for example, a developmental transcription factor—blocks differentiation, as is the case for EBF1-PDGFRB and PAX5-JAK2 [8,9,10]

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