Abstract
Patients with the pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have an approximately 15% risk of developing acute myeloid leukemia (AML; called here CN/AML). Most CN/AML patients co-acquire CSF3R and RUNX1 mutations, which play cooperative roles in the development of AML. To establish an in vitro model of leukemogenesis, we utilized bone marrow lin− cells from transgenic C57BL/6-d715 Csf3r mice expressing a CN patient–mimicking truncated CSF3R mutation. We transduced these cells with vectors encoding RUNX1 wild type (WT) or RUNX1 mutant proteins carrying the R139G or R174L mutations. Cells transduced with these RUNX1 mutants showed diminished in vitro myeloid differentiation and elevated replating capacity, compared with those expressing WT RUNX1. mRNA expression analysis showed that cells transduced with the RUNX1 mutants exhibited hyperactivation of inflammatory signaling and innate immunity pathways, including IL-6, TLR, NF-kappaB, IFN, and TREM1 signaling. These data suggest that the expression of mutated RUNX1 in a CSF3R-mutated background may activate the pro-inflammatory cell state and inhibit myeloid differentiation.
Highlights
Patients with the inborn pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have a very low level or even a complete lack of mature neutrophilic granulocytes in the peripheral blood which is caused by blockade of the terminal differentiation of bone marrow myeloid progenitor cells at the promyelocytes/myelocyte stage [1, 2]
D715 Csf3r lin− cells transduced with mutated RUNX1 had markedly diminished capacities to form myeloid colonies, including CFUG and CFU-GM, as compared with cells transduced with wild type (WT) RUNX1 (Fig. 1d)
FACS was used to count the absolute numbers of myeloid CD11b+ and Gr-1+ cells on day 11 of liquid culture, and the results showed that d715 Csf3r lin− cells transduced with each of the RUNX1 mutants exhibited reduced myeloid differentiation, compared with WT RUNX1–overexpressing samples (Fig. 2b)
Summary
Patients with the inborn pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have a very low level (less than 500 cells/μl blood) or even a complete lack of mature neutrophilic granulocytes in the peripheral blood which is caused by blockade of the terminal differentiation of bone marrow myeloid progenitor cells at the promyelocytes/myelocyte stage [1, 2]. 15% of CN patients develop myelodysplastic syndrome or acute myeloid leukemia (MDS or AML). CSF3R mutations resulting in the production of truncated G-CSFR proteins that lack from one to four phospho-tyrosine residues and exhibit defective receptor. Transgenic d715 Csf3r mice lacking three tyrosines do not develop AML or MDS [3,4,5,6,7,8,9], suggesting that additional genetic alterations in combination with CSF3R mutation are needed for the progression of AML. We recently examined a large cohort of CN/AML patients (31 patients) and found cooperative acquired mutations of CSF3R and RUNX1 (runt-related transcription factor 1) in 55% of CN patients with overt AML or MDS [10]. The detailed mechanism underlying the leukemogenic transformation downstream of CSF3R and RUNX1 mutations remained unknown
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