The CSF3R T618I Mutation Found In Chronic Neutrophilic Leukemia Removes An O-Linked Glycosylation Site and Increases Receptor Dimerization

▪ We have recently identified mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) in ∼60% of chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) patients (Maxson et al, NEJM 2013). These mutations fall into two categories: membrane proximal point mutatio...

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Published in:	Blood 2013-11, Vol.122 (21), p.270-270
Main Authors:	Maxson, Julia E., Gotlib, Jason, Pollyea, Daniel A., Fleischman, Angela G., Agarwal, Anupriya, Luty, Samuel B., Abel, Melissa, Eide, Christopher A., Bottomly, Daniel, Wilmot, Beth, McWeeney, Shannon K., Tognon, Cristina E., Collins, Robert H., Pond, J. Blake, Goueli, Basem, Oh, Stephen T., Deininger, Michael W., Chang, Bill H., Loriaux, Marc M., Druker, Brian J., Tyner, Jeffrey W.
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Language:	eng
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Summary:	▪ We have recently identified mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) in ∼60% of chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) patients (Maxson et al, NEJM 2013). These mutations fall into two categories: membrane proximal point mutations (the most common of which is T618I) and truncation mutations. Drug and siRNA screening of primary patient samples revealed that the two classes of CSF3R mutations exhibit differential sensitivity to inhibition of SRC or JAK kinases. CSF3R truncation mutations conferred sensitivity to SRC family kinase inhibition, while CSF3R membrane proximal mutations (T618I) conferred sensitivity to JAK kinase inhibition. A patient with the T618I membrane proximal mutation responded to treatment with the FDA approved JAK inhibitor, ruxolitinib. CSF3R truncation mutations have also been observed in a subset of severe congenital neutropenia patients who are at high risk for development of acute myeloid leukemia. Prior studies in this context have shown that truncation mutations result in loss of endocytic and degradation motifs, leading to increased expression of the receptor. The differences in signaling and drug sensitivity of these mutation classes suggest that membrane proximal mutations may activate CSF3R signaling through a distinct, as-yet unknown mechanism. Furthermore, a subset of CNL patients harbor both membrane proximal and truncation mutations on the same allele, though the consequences of these compound mutations are not yet known. CSF3R expression level and banding pattern were assessed by immunoblot of lysates from 293T17 cells transfected with wild type, membrane proximal mutant, or truncation mutant CSF3R. O-linked glycosylation was removed from the receptor by treatment with O-glycosidase and neuraminidase. Ligand independence of the CSF3R mutants was analyzed in murine interleukin-3 (IL3)-dependent Ba/F3 cells. CSF3R dimerization was assessed by co-transfecting CSF3R-Flag and CSF3R-V5 tagged constructs and then immunoprecipitating CSF3R-Flag and detecting co-immunoprecipitation of the CSF3R-V5 by immunoblot. Transforming potential of the CSF3R compound mutations relative to the corresponding point or truncation mutations was assessed by analyzing IL3-independent growth of Ba/F3 cells or mouse bone marrow colony formation. To better understand the functional and biochemical differences between membrane proximal and truncation mutant CSF3R, we examined trans
ISSN:	0006-4971 1528-0020