Abstract 532: Identification of unpaired cysteine-mediated gain and loss of function CSF3R extracellular mutations
Authors: Zhang Haijiao , Means Sophie , Schultz Anna R. , Watanabe-Smith Kevin , Medeiros Bruno C. , Kükenshöner Tim , Hantschel Oliver , Bottomly Daniel , Wilmot Beth , McWeeney Shannon K. , Tyner Jeffrey W. 
Published: July 2017

Abstract
Mutations in the CSF3R extracellular domain have been reported so far only in neutropenia patients. These extracellular mutations are loss of function mutations, which interrupt ligand binding. Here we screened CSF3R extracellular domain variants in a variety of hematological malignances and described for the first time the identification of an activating CSF3R extracellular mutation W341C in a leukemia patient. Functional assays revealed that this mutation conferred Ba/F3 cell cytokine-independent growth and induced constitutive JAK-STAT activation.
We further characterized that cysteine substitutions at other amino acid positions (342, 356 and 477) and other amino acid substitutes of W341 (A/G/K/R/S) did not transform cells, indicating that both the amino acid position and cysteine substitution are essential for the transforming capacity. In agreement, increased dimer formation of W341C was observed in the co-immunoprecipitation assay and non-reducing condition immunoblot, which could be abrogated in an immunoblot run under reducing conditions, highly suggesting that dimerization is mediated by the formation of intermolecular disulfide bonds. Surprisingly, W356C demonstrated loss of function properties, however, showed increased dimer formation similar to W341C, indicating that only the increased dimerization is not sufficient for transforming capacity. Computational modeling based on IL6ST showed opposite directions of W341 and W356, which may orientate the cytoplasmic domain towards or away from one another.
Interestingly, a CSF3R cytoplasmic truncation mutation at amino acid W791 was found to be on the same allele as W341C in this patient. The W341C/W791X compound mutation transformed Ba/F3 with faster kinetics comparing to the W341C single mutation. Furthermore, the compound mutation, but not W341C alone demonstrated delayed receptor degradation, indicating enhanced oncogenic potential. Notably, the primary patient sample and the Ba/F3 cells transformed by W341C or the compound mutation were all sensitive to JAK inhibitors. The patient harboring these CSF3R mutations displayed myelodysplastic morphology with BCOR mutation at disease diagnosis, and showed good response to Azacytidine treatment. However her white blood count (mature neutrophils in particular) increased after 15-18 months’ treatment, which was concomitant with the acquisition and expansion of CSF3R W341C and W791X and disease progression. We further investigated the oncogenic potential of disrupting original cysteine pairs in the CSF3R extracellular domain. Increased dimers are observed in these mutations, whereas two functional consequences were observed: loss of function and constitutive activity. This, therefore, represents the first characterization of unpaired cysteines that mediate both loss and gain of function phenotypes. This paradigm may apply to other cytokine receptors.
Citation Format: Haijiao Zhang, Sophie Means, Anna R. Schultz, Kevin Watanabe-Smith, Bruno C. Medeiros, Tim Kükenshöner, Oliver Hantschel, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Jeffrey W. Tyner. Identification of unpaired cysteine-mediated gain and loss of function CSF3R extracellular mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 532. doi:10.1158/1538-7445.AM2017-532