Background. Severe congenital neutropenia (SCN) is an inherited bone marrow failure syndrome that can transform to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). The most common recurrent mutation that causes SCN involve neutrophil elastase (ELANE). Mutations in ELANE result in the unfolded protein response stress. The treatment of choice for SCN is the chronic administration of high-dose granulocyte-colony stimulating factor (G-CSF), which elevates the neutrophil count, helps resolve pre-existing infections, diminishes the number of new infections, and significantly improves the survival and quality of life. G-CSF treatment also leads to enhanced oxidative stress. Long-term survival with G-CSF is frequently associated with development of MDS/AML. Of note, approximately 70% of SCN patients with MDS/AML acquire nonsense mutations in the region of CSF3R that encodes the cytoplasmic domain. These somatic CSF3R mutations are characterized by a truncation variant found in the cytoplasmic domain of the CSF3R and are associated with a hyper-proliferative/impaired differentiative phenotype that might contribute to the MDS/AML transformation. We hypothesize that the terminal exon of CSF3R constitutes a hotspot vulnerable to mutations resulting from excessive oxidative stress or endoplasmic reticulum (ER) stress.
Results. Murine factor-dependent Ba/F3 cells were used to measure the effect of induced oxidative or ER stress on the mutation rate involving the hypothesized hotspot of the exogenous human CSF3R, the corresponding region in the endogenous Csf3r, and Runx1 (a transcription factor involved in leukemogenesis). Ba/F3 cells transduced with the cDNA for the hypothesized hotspot of CSF3R (partial C-terminal) fused in-frame with m-NeonGreen, a yellow-green fluorescent protein, were subjected to stress-inducing treatment for 30 days (~51 doubling times). The amplicon-based targeted deep-sequencing data for days 15 and 30 samples showed increased mutagenesis of the coding nucleotide sequences for CSF3R, Csf3r, and Runx1. There was no correlation between the stress-inducing chemical treatments and overall level of mutagenesis in Ba/F3 cells. Interestingly, the GC-rich partial CSF3R region was less mutated as compared to the mNeonGreen region, having much lower GC content. However, analysis of our data, including the site-frequency spectra, indicated effects that may be due to clonal selection, specifically at the Csf3r gene.
Conclusion. Our data suggested that oxidative or ER stress induction did not promote genomic instability affecting the 3' exonic end of CSF3R, the endogenous Csf3R, and the endogenous Runx1 in Ba/F3 cells that could account for these targets being mutational hotspots. We conclude that other mechanisms, such as stochastic events, result in mutations of CSF3R drive the evolution of SCN to MDS/AML.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.