A Novel CSF3R Mutation Uncovers the Importance of Membrane-Proximal N-Glycosylation for Receptor Regulation
Authors: Hughes Seamus B , Spiciarich David , Press Richard D. , Thompson Sarah L , Radich Jerald P. , Bertozzi Carolyn , Oh Stephen T. , Maxson Julia E. 
Published: October 2019

Mutations in CSF3R (aka GCSFR) occur in the majority of patients with Chronic Neutrophilic Leukemia (CNL) and also are rarely found in adult and pediatric Acute Myeloid Leukemia. The most common CSF3R mutation in CNL is T618I (aka T595I), a point mutation in the membrane-proximal extracellular domain that causes ligand independence. Mutations that lead to a premature stop in the cytoplasmic domain are also found in CNL and result in increased expression of CSF3R on the cell surface. Understanding the biology of other novel mutations in CSF3R may lead to insight into both receptor biology and oncogenesis.

The CSF3R N610H mutation was identified in a patient with a myeloproliferative neoplasm, which was most consistent with a JAK2, CALR, MPL mutation negative primary myelofibrosis. This patient had a history of mild leukocytosis for several years with most recent white blood cell counts between 13.3 and 15.3 x 103/uL. A bone marrow biopsy revealed 90% cellularity with mildly increased reticulin fibrosis, increased myeloid to erythroid ratio, no overt dysplasia, and less than 5% blasts. The cells were karotypically normal with a micro deletion of the 3' end of PDGFRB (5q) identified by FISH at 59%. The patient has had minimal symptoms with no anemia or thrombocytopenia and is currently being monitored but not receiving any intervention. The patient's bone marrow was next-generation sequenced using a 42-gene myeloid malignancy targeted mutation hotspot panel which revealed a mutation at N610H in CSF3R at a 50% mutant allele frequency.

Given its proximity to the most common CSF3R mutation found in Chronic Neutrophilic Leukemia (T618I, aka T595I), we were interested in understanding whether the N610H mutation might contribute to disease biology. N610 (also known as N586 in the traditional numbering system that does not include the signal peptide) is part of an N-X-T motif, which is a consensus sequence for N-linked glycosylation. Haniu et al (Biochemistry 1996) demonstrated that N610 is one of 8 sites that are N-glycosylated on CSF3R. We confirm by mass spectrometry (MS) based analysis that N610 is occupied with a bisecting complex N-glycan (in vitro). We further found that the N610H mutation and a more conservative N610Q substitution are highly activating in CSF3R, leading to cytokine-independent growth in the murine Ba/F3 cell line. Furthermore, like the T618I mutation, these mutations render the receptor ligand-independent. N610H and N610Q lead to a robust increase in downstream signaling through the JAK/STAT pathway as demonstrated by an increase in the levels of phospho-STAT3. The loss of N-glycosylation in the membrane-proximal region of CSF3R may therefore increase ligand-independent receptor activation and promote oncogenesis. This study highlights the insight that rare human mutations can provide into the relationship between receptor structure and function.

Disclosures

Radich: Ariad: Consultancy; Novartis: Consultancy, Research Funding; Gilliad: Consultancy; Incyte: Consultancy. Bertozzi:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Catalent Biologics: Membership on an entity's Board of Directors or advisory committees; Verily: Membership on an entity's Board of Directors or advisory committees; Enable Bioscience: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Oh:CTI: Research Funding; Janssen: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.