G-CSF Receptor Mutations Found in Patients with Severe Congenital Neutropenia Confer a Strong Competitive Advantage at the Hematopoeitic Stem Cell Level That Is Dependent on Increased Systemic Levels of G-CSF.
Authors: Grenda David S. , Liu Fulu , Richards Michael , Link Daniel C. 
Published: April 2020

Patients with severe congenital neutropenia (SCN) have a markedly increased risk of developing myelodysplasia (MDS) or acute myeloid leukemia (AML). Though the genetic basis for this increased susceptibility is unknown, gain-of-function mutations of the G-CSF receptor (G-CSFR) have been found in the great majority of patients with SCN who develop MDS/AML. These mutations are somatic and produce a truncated G-CSFR that, though remaining ligand dependent, transmits a hyperproliferative signal. We and others have shown that targeted transgenic mice expressing a representative G-CSFR mutation (termed d715) have markedly exaggerated neutrophil responses to G-CSF treatment. Based on these observations, it has been suggested that these gain-of-function G-CSFR mutations contribute to leukemogenesis. However, direct evidence supporting this hypothesis is scant. Moreover, it is unclear how hematopoietic cells expressing the mutant G-CSFR gain clonal dominance. Finally, it is not clear why these G-CSFR mutations are uniquely associated with SCN and rarely seen in de novo AML.

To address these questions, we generated a series of bone marrow chimeras reconstituted with both wild type and d715 G-CSFR hematopoietic cells, thus reproducing, in part, the mixed bone marrow populations found in patients with SCN. Equal numbers of wild type or d715 G-CSFR bone marrow cells were transplanted into irradiated syngeneic hosts and donor chimerism periodically assessed by flow cytometry. At 5 weeks post-transplantation the contribution of d715 cells to the myeloid (percentage of d715 cells ± SD: 45.7 ± 12.0%, n=9), B-lymphocyte (63.5 ± 5.8%), and T-lymphocyte (46.6 ± 6.4%) lineages was near the expected level of 50%. Surprisingly, this level of chimerism was stable over the 6-month observation period, showing that the d715 G-CSFR does not confer a competitive advantage under basal conditions. In patients with SCN, systemic levels of G-CSF are elevated either due to increased endogenous production or exogenous G-CSF treatment. To simulate this condition, a cohort of chimeric mice was treated with G-CSF (10μg/kg/day) for 21 days. At the end of the treatment period, the contribution of d715 cells to the myeloid lineage in the blood increased to 97.6 ± 1.2% (n=5). Surprisingly, a marked increase in d715 donor chimerism in the B-lymphocyte lineage in the bone marrow also was observed (89.1 ± 5.7%). Remarkably, this shift in donor chimerism extended to the hematopoietic stem cell (HSC) compartment as defined by Kit+ lineage− Sca+ (KLS) cells; the contribution of d715 to the KLS cell population in G-CSF treated mice was 97.8 ± 0.8% versus 53.3 ± 11.5% in untreated mice. Transplantation of bone marrow cells from these mice into secondary recipients showed that this brief (21 day) exposure to G-CSF was sufficient to significantly expand the d715 HSC.

Collectively, these data show that expression of the d715 G-CSFR results in a strong competitive advantage at the HSC level, but only in the presence of an increased concentration of G-CSF. Furthermore, they provide an explanation for the association of these mutations with SCN since SCN is one of a small number of conditions in which systemic levels of G-CSF are chronically elevated. Finally, the effect of G-CSF signals on HSC function provides further evidence for the contributions of these mutations to leukemogenesis since it is the HSC compartment in which leukemia is thought to arise.