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

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.

Targeting AraC-Resistant Acute Myeloid Leukemia by Dual Inhibition of CDK9 and Bcl-2: A Systematic Review and Meta-Analysis

“…Cells in the human blood are all derived from pluripotent HSCs in the bone marrow. Pluripotent hematopoietic SCs differentiate into myeloid progenitors, and after a series of differentiation maturation processes, myeloid progenitors eventually become erythrocytes, granulocytes, monocytes, and platelets, while lymphoid progenitors differentiate into lymphocytes and plasma cells [ 32 – 35 ]. Each blood cell has important and unique physiological functions, and blood cell abnormalities can lead to the occurrence of disease.…”

Are Induced Pluripotent Stem Cells a Step towards Modeling Pediatric Leukemias?

“…Moreover, RNA-seq performed RUNX1-JAK2-expressing hematopoietic cells showed the constitutive activation of JAK-STAT signaling and the upregulation of MYC targets, confirming the role and interaction between these pathways in the onset of the leukemic process. However, increased clonogenicity or enhanced hematopoietic differentiation upon RUNX1-JAK2 expression were not observed, hypothesizing that hematopoietic progenitors derived from iPSC were not susceptible to oncogenic transformation [37].…”

“…Fortschegger et al [37] established an iPSCs model focusing on the RUNX1-JAK2 fusion gene studying its consequences on hematopoietic development though the insertion of the RUNX1-JAK2 fusion into one endogenous RUNX1 allele employing in trans paired nicking genome editing. They observed a decrease in the hematopoietic progenitor generation that, nonetheless, did not result in the lack of myeloid lineage cell differentiation.…”

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi

“… 50 , 51 Indeed, fostering otherwise inefficient SSB-dependent HDR such as by in trans paired nicking (ITPN) approaches allows for seamless chromosomal editing, including at multi-copy or essential genomic tracts. 45 , 49 Moreover, ITPN could also be suitable for allele-specific editing, 52 , 53 one-step biallelic editing, 48 , 54 or knocking-in whole transgenes at safe harbor loci. 45 , 49 …”