Abstract 2760: CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation
Authors: Maxson Julia , Luty Samuel , MacManiman Jason , Savage Samantha , Abel Melissa , Bahamadi Swaleh , Druker Brian , Tyner Jeffrey 
Published: May 2015

Abstract
We have recently discovered that a large percentage (59%) of patients with chronic neutrophilic leukemia (CNL) or atypical chronic myeloid leukemia (aCML) have mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) (Maxson et al, NEJM 2013). These mutations are either point mutations in the extracellular domain (T618I, T615A) or truncations of the cytoplasmic domain. The most frequent mutation is T618I (aka T595I). The two classes of CSF3R mutations operate through distinct mechanisms that result in differential sensitivity to inhibition of tyrosine kinases downstream of CSF3R. While the mechanism of action of CSF3R truncation mutations has been well characterized-they have increased cell surface expression due to loss of negative regulatory motifs-the way in which the CSF3R point mutations activate the receptor is unclear.
Consistent with a previous report, the CSF3R T618I mutation is ligand independent in both mouse bone marrow colony assays and the Ba/F3 pro-B cell line. WT CSF3R consists of two molecular weight species suggestive of differential posttranslational modification. Analysis of CSF3R point mutations T618I and T615A revealed an alteration of banding pattern of these mutations. Since extracellular threonine residues are capable of being O-glycosylated, we employed endoglycosidase digestion to reveal that the larger CSF3R species is O-glycosylated. Furthermore the T618I and T615A mutations reduce O-glycosylation of the receptor. This analysis was confirmed by direct labeling of O-glycosylation using azide-modified galactosamine, which labeled WT CSF3R, but had minimal labeling of CSF3R T618I. Receptor dimerization studies showed increased dimerization of the CSF3R T618I mutant relative to WT.
Sequencing of CSF3R from an atypical CML patient samples revealed an additional mutation not previously observed in this disease, T640N. The T640N mutation (aka T617N) is a ligand-independent mutation originally found in a family with hereditary chronic neutrophilia (Plo et al, JEM 2009). Plo et al found that this mutation resides within the transmembrane domain and predicted by molecular modeling that it increased receptor dimerization through interaction of the transmembrane helices. In mouse bone marrow colony assays we confirmed the ligand-independence of the mutation and also found that it had similar potency as T618I in colony and cytokine-independent growth assays. Furthermore, like T618I, the T640N mutation conferred sensitivity to a JAK kinase inhibitor in colony assays. Surprisingly, the T640N mutation had an alteration of banding pattern very similar to T618I, suggesting that it may also have altered glycosylation status, even though the mutation resides within the transmembrane domain. Our studies demonstrate that O-glycosylation is critical for regulation of CSF3R dimerization and that the T618I and T640N mutations can activate the receptor to promote oncogenesis.
Citation Format: Julia Maxson, Samuel Luty, Jason MacManiman, Samantha Savage, Melissa Abel, Swaleh Bahamadi, Brian Druker, Jeffrey Tyner. CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2014-2760