An increasing number of variants of unknown significance (VUS) are being identified in leukemia patients with the application of deep sequencing and these include CSF3R cytoplasmic mutations. Previous studies have demonstrated oncogenic potential of certain CSF3R truncation mutations prior to internalization motifs. However, the oncogenic potential of truncating the more distal region of CSF3R cytoplasmic domain as well as cytoplasmic missense mutations remains uncharacterized. Here we identified that CSF3R distal cytoplasmic truncation mutations (Q793–Q823) also harbored leukemogenic potential. Mechanistically, these distal cytoplasmic truncation mutations demonstrated markedly decreased receptor degradation, probably due to loss of the de-phosphorylation domain (residues N818–F836). Furthermore, all truncations prior to Q823 demonstrated increased expression of the higher molecular weight CSF3R band, which is shown to be essential for the receptor surface expression and the oncogenic potential. We further demonstrated that sufficient STAT5 activation is essential for oncogenic potential. In addition, CSF3R K704A demonstrated transforming capacity due to interruption of receptor ubiquitination and degradation. In summary, we have expanded the region of the CSF3R cytoplasmic domain in which truncation or missense mutations exhibit leukemogenic capacity, which will be useful for evaluating the relevance of CSF3R mutations in patients and helpful in defining targeted therapy strategies.

Granulocyte colony-stimulating factor (G-CSF or CSF3) and its receptor CSF3R regulate granulopoiesis, neutrophil function, and hematopoietic stem cell mobilization. Recent studies have uncovered an oncogenic role of mutations in the gene in many hematologic malignancies. To find additional mutations that give rise to cell transformation, we performed a cellular transformation assay in which murine interleukin 3 (IL-3)-dependent Ba/F3 cells were transduced with WT CSF3R plasmid and screened for spontaneous growth in the absence of IL-3. Any outgrowth clones were sequenced to identify mutations with transformation capacity. We identified several novel mutations and determined that they transform cells via four distinct mechanisms: 1) cysteine- and disulfide bond-mediated dimerization (S581C); 2) polar, noncharged amino acid substitution at the transmembrane helix dimer interface at residue Thr-640; 3) increased internalization by a Glu-524 substitution that mimics a low G-CSF dose; and 4) hydrophobic amino acid substitutions in the membrane-proximal residues Thr-612, Thr-615, and Thr-618. Furthermore, the change in signaling activation was related to an altered CSF3R localization. We also found that CSF3R-induced STAT3 and ERK activations require CSF3R internalization, whereas STAT5 activation occurred at the cell surface. Cumulatively, we have expanded the regions of the CSF3R extracellular and transmembrane domains in which missense mutations exhibit leukemogenic capacity and have further elucidated the mechanistic underpinnings that underlie altered CSF3R expression, dimerization, and signaling activation.

Exclusive of membrane-proximal mutations seen commonly in chronic neutrophilic leukemia (e.g., T618I), functionally defective mutations in the extracellular domain of the G-CSF receptor (CSF3R) have been reported only in severe congenital and idiopathic neutropenia patients. Here, we describe the first activating mutation in the fibronectin-like type III domain of the extracellular region of CSF3R (W341C) in a leukemia patient. This mutation transformed cells via cysteine-mediated intermolecular disulfide bonds, leading to receptor dimerization. Interestingly, a CSF3R cytoplasmic truncation mutation (W791X) found on the same allele as the extracellular mutation and the expansion of the compound mutation was associated with increased leukocytosis and disease progression of the patient. Notably, the primary patient sample and cells transformed by W341C and W341C/W791X exhibited sensitivity to JAK inhibitors. We further showed that disruption of original cysteine pairs in the CSF3R extracellular domain resulted in either gain- or loss-of-function changes, part of which was attributable to cysteine-mediated dimer formation. This, therefore, represents the first characterization of unpaired cysteines that mediate both gain- and loss-of-function phenotypes. Overall, our results show the structural and functional importance of conserved extracellular cysteine pairs in CSF3R and suggest the necessity for broader screening of CSF3R extracellular domain in leukemia patients.

Purpose: Chronic neutrophilic leukemia (CNL), chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), and unclassified myeloproliferative neoplasms (MPN-U) are a group of heterogeneous disorders belonging to rare entities of myeloproliferative or myelodysplastic/myeloproliferative (MDS/MPN) syndromes. Due to lack of specific molecular markers and the limited understanding of pathogenesis, the treatment of these diseases remains empirical, resulting in poor outcomes. Recently, recurrent mutations in ASXL1, TET2, SRSF2 and cell signaling genes have been identified in these diseases. In this study we aim to analyze the co-occurrence patterns of these gene mutations, as well as the association of different mutations with disease subtypes and treatment outcomes. Methods and results: We performed whole exome sequence and RNA-Seq on primary patient samples. Consistent with previous studies, high frequencies of ASXL1, SRSF2, TET2, SETBP1 and signaling pathway mutations were observed; whereas mutations of MPL, CEBPa, IDH1/2, and TP53 were rare. Further variant allelic frequency analysis demonstrated that mutations of the chromatin modifiers, epigenetic markers and splicing factors are mostly present in the major clones indicating early acquisition of these mutations. In addition, ASXL1/2, splicing factor and signaling pathway mutations co-occur in around 45% of all patients with these mutations, indicating that they drive these diseases in a cooperative manner. RNA-seq analysis demonstrated two major gene expression clusters with high levels of either RAS or JAK-STAT signaling alterations respectively, indicating a potential need for distinct therapeutic targeting of specific subgroups of these diseases. In addition, we observed that 17% of patients have more than one signaling pathway mutation, possibly in distinct subclones, providing rationale for drug combination treatment. Furthermore, we observed that mutations related to RAS signaling pathway are prevalent in CMML; CSF3R and JAK2 mutations are enriched in CNL; whereas, RAS and JAK-STAT pathway double mutations are more frequent in aCML. Similar incidence of ASXL1, splicing factor and epidemics mutations are observed across all these diseases. Conclusions: CNL/aCML/MPN-U/CMML is a group of heterogeneous diseases associated with chromatin modifier, epigenetic, splicing factor and signaling pathway mutations in concomitant manner. We propose future studies of rational drug combinations with agents, targeting epigenetic and splicing factors, together with the appropriate signaling pathway inhibitors. Our study represents a large-scale comprehensive genomic analysis of these diseases, which reveals novel insight into patterns of mutation co-occurrence which could translate into new treatment paradigms for these difficult to treat hematologic malignancies. Citation Format: Haijiao Zhang, Beth Wilmot, Daniel Bottomly, Libbey White, Erik Segerdell, Shannon K. McWeeney, Vishesh Khanna, Angela Rofelty, Sophie Means, Brian Junio, Samantha Savage, Emily Stevens, Kim-Hien Dao, Julia E. Maxson, Jeffrey W. Tyner. Detailed genomic characterization of CNL/aCML/MPN-U/CMML reveals disease subgroups that may benefit from rationally-designed combination therapies [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 2452. doi:10.1158/1538-7445.AM2017-2452

<p>The file contains a table demonstrating CSF3R targeted deep sequencing result</p>