Severe congenital neutropenia (SCN) is a rare blood disorder characterized by abnormally low levels of circulating neutrophils. Mutations in multiple genes like neutrophil elastase gene (ELANE) and granulocyte colony stimulating factor receptor (CSF3R) may cause SCN. The treatment of choice for SCN is the administration of granulocyte-colony stimulating factor (G-CSF) which elevates the neutrophil count and hence improves the survival and quality of life. Long term survivorship on G-CSF is however linked to development of MDS (myelodysplastic syndrome)/AML (acute myeloid leukemia). About 70% of MDS/AML patients acquire nonsense mutations affecting the cytoplasmic domain of CSF3R. In this project, we hypothesized that this coding region of CSF3R constitutes a hotspot, vulnerable to mutations resulting from excessive oxidative stress or endoplasmic reticulum (ER) stress. We used the murine Ba/F3 cell line to study the effect of induced oxidative or ER stress on the mutation rate in our hypothesized hotspot of the exogenous human CSF3R, the corresponding region in the endogenous Csf3r, and a leukemia-associated gene Runx1. Ba/F3 cells transduced with the cDNA for partial C-terminal of CSF3R fused in-frame with a Green Fluorescent Protein (GFP) tag was subjected to cellular stress inducing mutagen treatment for a prolonged period of time (30 days). The amplicon based targeted deep sequencing data for days 15 and 30 samples show that although there was increased mutagenesis observed in all genes, there were more mutations in the GFP region as compared to the GC-rich partial CSF3R region. Our findings also indicate that there is no correlation between the stress-inducing chemical treatments and mutagenesis in Ba/F3 cells. Thus, we conclude that there are other mechanisms to acquired mutations of CSF3R that help drive the evolution of SCN to MDS/AML. To test this hypothesis, further experiments using unique barcoding system are in progress to characterize the clonal competition between different mutant CSF3R and ELANE expressing cell lines. This study will shed further light on the selection advantage that is provided to cells because cooperativity between mutations in different genes.

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