Distinct Mechanisms of Receptor Activation Are Associated with CSF3R Gain-of-Function Mutations

Activating mutations in granulocyte colony-stimulating factor receptor (CSF3R), are found in the extracellular, the membrane proximal, the transmembrane and the cytoplasmic domain of the receptor in various leukemia. To expand the spectrum of CSF3R mutations that can arise to transform Ba/F3 cells initially transfected with wild type CSF3R, we performed a spontaneous cellular transformation assay. We identified several new CSF3R activating mutations. These mutations are located in the extracellular, the membrane proximal, and the transmembrane domains of CSF3R. We further characterized that these mutations transform cells via four distinct mechanisms: (1) cysteine and disulfide bond mediated dimerization, leading to increased STAT3 activation (S581C); (2) polar, non-charged amino acid substitution at the transmembrane helix dimer interface T640, inducing increased STAT3 and ERK activation; (3) increased internalization and STAT3 activation by an E524 substitution; and (4) hydrophobic amino acid substitutions in the membrane proximal O glycosylation sites (T612, T615 and T618), leading to enhanced activation of STAT3 and ERK. Furthermore, we showed that the distinct signaling activation is related to different localization of CSF3R mutants. To this end, we have expanded the region of the CSF3R extracellular and transmembrane domain in which missense mutations exhibit leukemogenic capacity and further elucidated the mechanistic underpinnings that underlie distinct signaling activation and leukemogenic mechanisms. Cumulatively, these findings will facilitate improved evaluation of the biological and clinical relevance of CSF3R mutations identified in patients.

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