Mutational screens highlight glycosylation as a modulator of colony-stimulating factor 3 receptor (CSF3R) activity.

The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia. However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in chronic neutrophilic leukemia and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues-the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at WT threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain. Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.

T618I CSF3R mutations in chronic neutrophilic leukemia induce oncogenic signals through aberrant trafficking and constitutive phosphorylation of the O-glycosylated receptor form.

Activating mutations in the membrane-proximal region of the colony-stimulating factor 3 receptor (CSF3R) are a hallmark of chronic neutrophilic leukemia (CNL) with the T618I mutation being most common. The mechanisms underlying constitutive activation of the T618I CSF3R and its signal propagation are poorly understood. Ligand-independent activation of the T618I CSF3R has previously been attributed to loss of receptor O-glycosylation and increased receptor dimerization. Here, we show that the T618I CSF3R is indeed glycosylated but undergoes enhanced spontaneous internalization and degradation that results in a marked decrease in its surface expression. Inhibition of the proteasome dramatically increases expression of the O-glycosylated T618I CSF3R. We also demonstrate that the O-glycosylated wild-type CSF3R is tyrosine phosphorylated in response to ligand but constitutively phosphorylated in cells expressing T618I CSF3R. Constitutive tyrosine phosphorylation of the O-glycosylated T618I receptor form correlated with activation of JAK2 and both the mutant receptor and JAK2 were found to be constitutively ubiquitinated. These observations provide novel insights into the mechanisms of oncogenic signaling by T618I CSF3R mutations in CNL.

Chronic neutrophilic leukemia: 2020 update on diagnosis, molecular genetics, prognosis, and management.

DISEASE OVERVIEW: Chronic neutrophilic leukemia (CNL) is a rare, often aggressive myeloproliferative neoplasm (MPN) defined by persistent mature neutrophilic leukocytosis, bone marrow granulocyte hyperplasia, and frequent hepatosplenomegaly. The seminal discovery of oncogenic driver mutations in colony-stimulating factor 3 receptor (CSF3R) in the majority of patients with CNL in 2013 anchored a new scientific framework, deepening our understanding of its molecular pathogenesis, providing a diagnostic biomarker, and rationalizing the use of pharmacological targeting. DIAGNOSTIC CRITERIA: In 2016, the World Health Organization (WHO) included the presence of activating CSF3R mutations as a central diagnostic feature of CNL. Other criteria include leukocytosis of ≥25 × 109 /L comprising >80% neutrophils with <10% circulating precursors and rare blasts, and absence of dysplasia or monocytosis, while not fulfilling criteria for other MPN. DISEASE UPDATES: Increasingly comprehensive genetic profiling of CNL has disclosed a complex genomic landscape and additional prognostically relevant mutational combinations. Though prognostic determination and therapeutic decision-making remain challenging, emerging data on prognostic markers and the use of newer therapeutic agents, such as JAK inhibitors, are helping to define state-of-the-art management in CNL.

Genomics of chronic neutrophilic leukemia.

Chronic neutrophilic leukemia (CNL) is a distinct myeloproliferative neoplasm with a high prevalence (>80%) of mutations in the colony-stimulating factor 3 receptor (CSF3R). These mutations activate the receptor, leading to the proliferation of neutrophils that are a hallmark of CNL. Recently, the World Health Organization guidelines have been updated to include CSF3R mutations as part of the diagnostic criteria for CNL. Because of the high prevalence of CSF3R mutations in CNL, it is tempting to think of this disease as being solely driven by this genetic lesion. However, recent additional genomic characterization demonstrates that CNL has much in common with other chronic myeloid malignancies at the genetic level, such as the clinically related diagnosis atypical chronic myeloid leukemia. These commonalities include mutations in SETBP1, spliceosome proteins (SRSF2, U2AF1), and epigenetic modifiers (TET2, ASXL1). Some of these same mutations also have been characterized as frequent events in clonal hematopoiesis of indeterminate potential, suggesting a more complex disease evolution than was previously understood and raising the possibility that an age-related clonal process of preleukemic cells could precede the development of CNL. The order of acquisition of CSF3R mutations relative to mutations in SETBP1, epigenetic modifiers, or the spliceosome has been determined only in isolated case reports; thus, further work is needed to understand the impact of mutation chronology on the clonal evolution and progression of CNL. Understanding the complete landscape and chronology of genomic events in CNL will help in the development of improved therapeutic strategies for this patient population.

Granulocyte colony-stimulating factor receptor signaling in severe congenital neutropenia, chronic neutrophilic leukemia, and related malignancies.

Granulocyte colony-stimulating factor is a hematopoietic cytokine that stimulates neutrophil production and hematopoietic stem cell mobilization by initiating the dimerization of homodimeric granulocyte colony-stimulating factor receptor. Different mutations of CSF3R have been linked to a unique spectrum of myeloid disorders and related malignancies. Myeloid disorders caused by the CSF3R mutations include severe congenital neutropenia, chronic neutrophilic leukemia, and atypical chronic myeloid leukemia. In this review, we provide an analysis of granulocyte colony-stimulating factor receptor, various mutations, and their roles in the severe congenital neutropenia, chronic neutrophilic leukemia, and malignant transformation, as well as the clinical implications and some perspective on approaches that could expand our knowledge with respect to the normal signaling mechanisms and those associated with mutations in the receptor.

Chronic neutrophilic leukaemia and plasma cell-related neutrophilic leukaemoid reactions.

Many cases reported as 'chronic neutrophilic leukaemia' have had an associated plasma cell neoplasm. Recent evidence suggests that the great majority of such cases represent a neutrophilic leukaemoid reaction to the underlying multiple myeloma or monoclonal gammopathy of undetermined significance. We have analysed all accessible reported cases to clarify the likely diagnosis and to ascertain whether toxic granulation, Döhle bodies and an increased neutrophil alkaline phosphatase score were useful in making a distinction between chronic neutrophilic leukaemia and a neutrophilic leukaemoid reaction. We established that all these changes occur in both conditions. Toxic granulation and Döhle bodies are more consistently present in leukaemoid reactions but also occur quite frequently in chronic neutrophilic leukaemia. The neutrophil alkaline phosphatase score is increased in both conditions and is of no value in making a distinction.

Jak-2 positive myeloproliferative neoplasms.

Originally described by Dameshek in 1951, myeloproliferative disorders are today classified as myeloproliferative Neoplasms (MPNs) in WHO's Classification of Tumors of Hematopoietic and Lymphoid Tissues. The term includes a range of conditions, [ie, BCR-ABL-positive chronic myelogenous leukemia (CML), chronic neutrophilic leukemia (CNL), polycythemia vera (PV), primary myelofibrosis (PMF), essential thromobocythemia (ET), chronic eosinophilic leukemia not otherwise specified (CEL-NOS), mastocytosis, and unclassifiable myeloproliferative neoplasm]. In the specific case of CML, a better understanding of the pathogenesis and pathophysiology of the disease has led to a targeted therapy. The presence of chromosome Philadelphia, t(9;22)(q34;11) results in the oncogene BCR-ABL, which characterizes the disease; this molecular rearrangement gives rise to a tyrosine-kinase, which in turn triggers the proliferation of the myeloid line through the activation of the signaling pathways downstream. Tyrosine-kinase inhibitors (TKIs) have altered the therapy and monitoring of CML patients and improved both their prognosis and quality of life. In 2005, various groups of investigators described a new point mutation of the gene JAK2 associated to MPNs. Although the presence of this mutation has led to a modification in the diagnostic criteria of these conditions, the impact of the use of JAK2 inhibitors on the prognosis and course of the disease continues to be controversial.

Ligand independence of the T618I mutation in the colony-stimulating factor 3 receptor (CSF3R) protein results from loss of O-linked glycosylation and increased receptor dimerization.

Mutations in the CSF3 granulocyte colony-stimulating factor receptor CSF3R have recently been found in a large percentage of patients with chronic neutrophilic leukemia and, more rarely, in other types of leukemia. These CSF3R mutations fall into two distinct categories: membrane-proximal mutations and truncation mutations. Although both classes of mutation have exhibited the capacity for cellular transformation, several aspects of this transformation, including the kinetics, the requirement for ligand, and the dysregulation of downstream signaling pathways, have all been shown to be discrepant between the mutation types, suggesting distinct mechanisms of activation. CSF3R truncation mutations induce overexpression and ligand hypersensitivity of the receptor, likely because of the removal of motifs necessary for endocytosis and degradation. In contrast, little is known about the mechanism of activation of membrane-proximal mutations, which are much more commonly observed in chronic neutrophilic leukemia. In contrast with CSF3R truncation mutations, membrane-proximal mutations do not exhibit overexpression and are capable of signaling in the absence of ligand. We show that the Thr-615 and Thr-618 sites of membrane-proximal mutations are part of an O-linked glycosylation cluster. Mutation at these sites prevents O-glycosylation of CSF3R and increases receptor dimerization. This increased dimerization explains the ligand-independent activation of CSF3R membrane-proximal mutations. Cytokine receptor activation through loss of O-glycosylation represents a novel avenue of aberrant signaling. Finally, the combination of the CSF3R membrane proximal and truncation mutations, as has been reported in some patients, leads to enhanced cellular transformation when compared with either mutation alone, underscoring their distinct mechanisms of action.

CSF3R is mutated in chronic neutrophilic leukemia and atypical CML.

Colony-stimulating factor 3 receptor (CSF3R) is mutated in 59% of CNL or atypical CML cases.

Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia.

Human neutrophils were found to express members of the inhibitor of apoptosis (IAP) family, namely cellular IAP1 (cIAP1), cIAP2, and X-linked IAP. Among these members, cIAP2 expression was selectively up-regulated by stimulation with granulocyte colony-stimulating factor (G-CSF), but not with granulocyte-macrophage CSF. The increased expression of cIAP2 mRNA was detected as early as 30 minutes after in vitro stimulation with G-CSF, and the elevated level of cIAP2 protein was detected at 1 hour. The elevated level of cIAP2 protein was also detected in peripheral blood neutrophils obtained from healthy donors receiving G-CSF administration. G-CSF-induced up-regulation of cIAP2 mRNA and protein, phosphorylation of signal transducer and activator of transcription 3 (STAT3), and the antiapoptotic effects were inhibited by pretreatment of cells with AG490, a specific inhibitor of Janus kinase 2 (JAK2). Mature neutrophils from a patient with chronic neutrophilic leukemia exhibited remarkable overexpression of cIAP2 mRNA and prolongation of survival, whereas cIAP2 mRNA expression and survival in mature neutrophils from patients with chronic myelogenous leukemia were essentially similar to those in normal neutrophils. These findings suggest that cIAP2 expression is up-regulated by G-CSF through activation of the JAK2-STAT3 pathway, and increased expression of cIAP2 protein may contribute to G-CSF-mediated antiapoptosis. In addition, overexpression of cIAP2 may be partly responsible for sustained neutrophilia at least in some cases of chronic neutrophilic leukemia.

Expression of mu-BCR-ADL transcripts in chronic neutrophilic leukemia.

The classification of chronic neutrophilic leukemia (CNL) is controversial. Our purpose was to correlate clinical, pathologic, and molecular analyses in 2 cases of CNL. In both cases, the patients were referred because of a substantially increased peripheral WBC count noted during routine examination. Bone marrow biopsies and aspirate smears revealed hypercellularity with myeloid/erythroid ratios of 4:1 and 11:1, respectively. The bone marrow aspirate results were as follows: case 1: blasts, 2%; promyelocytes, 2%; myelocytes, 6%; metamyelocytes, 16%; band neutrophils, 13%; segmented neutrophils, 34%; and case 2: blasts, 1%; promyelocytes, 2%; myelocytes, 15%; metamyelocytes, 20%; band neutrophils, 24%; neutrophils, 19%. Reverse transcriptase in situ polymerase chain reaction studies demonstrated expression of mu-BCR-ABL transcripts in 13% and 25% of the bone marrow cells, respectively. In both cases, the positive signal was noted mainly in the early granulocytic precursors and was present in occasional mature neutrophils. To our knowledge, this is thefirst in situ demonstration of mu-BCR-ABL expression in CNL Ourfindings reinforce the usefulness of this messenger RNA as a molecular marker of CNL.

Granulocyte-colony stimulating factor concentrations in a patient with plasma cell dyscrasia and clinical features of chronic neutrophilic leukaemia.

In order to study the pathogenesis of plasma cell dyscrasias with associated clinical features of chronic neutrophilic leukaemia, the concentration of granulocyte-colony stimulating factor (G-CSF) was measured in a patient, a 73 year old man, who underwent steroid pulse therapy. High G-CSF concentrations and leucocyte counts prior to treatment declined rapidly on administration of dexamethazone, but rose subsequently. G-CSF was not detected in primary cultures of bone marrow cells, but large amounts of interleukin-6 were found in the culture supernatant. These observations suggest that the neutrophilia observed in the patient represented a reactive response to G-CSF secreted from abnormal plasma cells or stromal cells rather than the existence of a genuine myeloproliferative disorder.

Chronic neutrophilic leukemia. A study of four cases.

Chronic neutrophilic leukemia (CNL) is a very rare entity, which has to be included among the chronic myeloid leukemias. Once an underlying cause of neutrophilia is excluded, the diagnosis of CNL is based on exclusion of chronic granulocytic and other types of chronic myeloid leukemias. The classification proposed by Sheperd et al. has proven to be helpful, but it must be completed by cytogenetic analysis and the search for bcr rearrangement by molecular biology methods, in order to confirm the absence of Philadelphia chromosome and of bcr-abl hybrid gene. We report here four cases of CNL, with confirmed absence of bcr rearrangement in two cases. Two patients died, 12 and 8 years after diagnosis, the second one following transformation into myelofibrosis with myeloid metaplasia. The other two died of acute myelogenous leukemia, the first one, 25 years after diagnosis of CNL, following a 3-year phase of acceleration. The last patient presented combined features of CNL and refractory anemia with excess of blasts, and was characterized by both progressive leukocytosis and severe thrombocytopenia; acute transformation into acute myelogenous leukemia occurred 6 months after diagnosis and death 1 month later. Among the 30 cases reported so far, plus the four presented here, combined myelodysplastic features were observed in five cases and transformation into acute myelogenous leukemia in six. Chronic neutrophilic leukemias should be reported regularity, in view of the uncertain and low frequency of this hematological disease.

Chronic neutrophilic leukemia and multiple myeloma. An association with lambda light chain expression.

Two patients with plasma cell dyscrasia and IgG lambda paraproteinemia in association with the rare disorder, chronic neutrophilic leukemia (CNL), are described. Cytogenetic studies excluded Philadelphia + chronic myeloid leukemia and molecular analysis of the breakpoint cluster region (bcr) revealed no evidence of clonal gene rearrangement. Nine similar cases of coexistent CNL and paraproteinemia have been identified in the literature and attention is drawn to the disproportionate excess of lambda light chain restriction in this subset of patients. Evidence supporting a clonal origin of CNL is considered and the nature of the relationship between the chronic myeloid expansion and the development of multiple myeloma is discussed.

The discrepancy between chemotaxis and leukotriene B4 production in a patient with chronic neutrophilic leukemia.

Chronic neutrophilic leukemia (CNL) is a rare type of leukemia. We diagnosed a 81-year-old woman as CNL because she showed that sustained leukocytosis dominated by mature neutrophils, hepatosplenomegaly, high neutrophilic alkaline phosphatase (NAP) score, absence of the Ph1 chromosome and no evidence of leukemoid reaction. During the clinical course, she did not manifest hemorrhagic tendency or infection. We also examined neutrophilic function including chemotaxis, chemiluminescence, nitroblue tetrazolium (NBT) dye reduction, which all indicated normal neutrophil function. Using a reversed phase-high pressure liquid chromatography (HPLC), we detected the production of leukotriene B4 (LTB4) in neutrophils. We found that the LTB4 production was decreased in neutrophils whereas they showed normal chemotaxis. This discrepancy has never, to our knowledge, been reported before in case of CNL.