Dimerization, internalization, and intracellular trafficking are key processes that regulate surface expression and signaling by cytokine receptors. In the case of the wild type CSF3R, the receptor for granulocyte colony-stimulating factor (G-CSF), ligand binding induces receptor dimerization, internalization, and trafficking to the endosome where the receptor is degraded, ultimately leading to signal termination. Our group previously reported a truncated CSF3R (d716) identified in patients with severe congenital neutropenia transforming to AML that lacks a distal dileucine motif and transduces enhanced growth signals due to defective receptor internalization and prolonged signaling. More recently, membrane-proximal CSF3R mutations that result in constitutive activation of the receptor have been identified in most patients with chronic neutrophilic leukemia (CNL). The T618I mutation is the most common CSF3R mutation in patients with CNL. The mutation at Thr-618 has been shown to inhibit O-glycosylation of the receptor leading to increased receptor dimerization and ligand-independent activation of the receptor that drives the overproduction of neutrophils. The precise mechanisms by which the T618I mutation induces the distinct neutrophilia phenotype observed in CNL remain unknown.
We hypothesized that internalization and trafficking of the constitutively active T618I CSF3R differ from the ligand stimulated wild type CSF3R. To further investigate this, cell lines expressing the wild type and T618I CSF3R were generated. We initially examined surface expression of each receptor form by flow cytometry and detected a significantly reduced steady state expression level of the T618I receptor on the cell surface relative to the wild type CSF3R, despite equivalent mRNA expression levels of the two receptor forms as assessed by real-time PCR. Immunoblot analysis also demonstrated similar total cellular receptor levels in cells expressing the T618I or wild type CSF3R. To further investigate the mechanisms responsible for decreased surface expression of the T618I CSF3R, cells expressing the T618I or the wild type CSF3R were incubated with cycloheximide, an inhibitor of new protein synthesis. Treatment of T618I-expressing cells led to rapid disappearance of the receptor from the cell surface, irrespective of the presence or absence of ligand. Moreover, cells expressing the T618I CSF3R exhibited accelerated ligand-independent degradation that could be inhibited by MG132 but not by chloroquine. Although the addition of ligand accelerated the degradation of the wild type receptor, it did not significantly alter the degradation kinetics of the T618I mutant receptor.
Given the differing pattern of intracellular trafficking observed with the constitutively active T618I receptor compared to the ligand-stimulated wild type receptor, we sought to determine whether the differing trafficking patterns observed with the two different receptor forms might lead to activation of disparate signaling pathways. Whole transcriptome RNA sequencing was performed on unstimulated cells expressing the T618I CSF3R and CSF3-stimulated cells expressing the wild type CSF3R. A distinctly different signature was observed between the T618I-expressing cells and ligand stimulated wild type cells as shown in Figure 1.
Collectively, these data demonstrate that the constitutively active T618I CSF3R exhibits an intracellular trafficking pattern that is distinct from the wild type CSF3R and activates a signaling cascade that is unique from the signaling pathway activated by the wild type receptor in response to ligand stimulation. Although clinical trials of the Jak inhibitor ruxolitinib are currently underway in patients with CNL, our data suggest a potential role for therapies that also target other pathways for patients with CNL.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.