Colony-stimulating Factor 3 Receptor Research Articles

Enhanced MAPK signaling induced by CSF3R mutants confers dependence to DUSP1 for leukemic transformation

AbstractElevated MAPK and the JAK-STAT signaling play pivotal roles in the pathogenesis of chronic neutrophilic leukemia and atypical chronic myeloid leukemia. Although inhibitors targeting these pathways effectively suppress the diseases, they fall short in providing enduring remission, largely attributed to the cytostatic nature of these drugs. Even combinations of these drugs are ineffective in achieving sustained remission. Enhanced MAPK signaling besides promoting proliferation and survival triggers a proapoptotic response. Consequently, malignancies reliant on elevated MAPK signaling use MAPK feedback regulators to intricately modulate the signaling output, prioritizing proliferation and survival while dampening the apoptotic stimuli. Herein, we demonstrate that enhanced MAPK signaling in granulocyte colony-stimulating factor 3 receptor (CSF3R)–driven leukemia upregulates the expression of dual specificity phosphatase 1 (DUSP1) to suppress the apoptotic stimuli crucial for leukemogenesis. Consequently, genetic deletion of Dusp1 in mice conferred synthetic lethality to CSF3R-induced leukemia. Mechanistically, DUSP1 depletion in leukemic context causes activation of JNK1/2 that results in induced expression of BIM and P53 while suppressing the expression of BCL2 that selectively triggers apoptotic response in leukemic cells. Pharmacological inhibition of DUSP1 by BCI (a DUSP1 inhibitor) alone lacked antileukemic activity due to ERK1/2 rebound caused by off-target inhibition of DUSP6. Consequently, a combination of BCI with a MEK inhibitor successfully cured CSF3R-induced leukemia in a preclinical mouse model. Our findings underscore the pivotal role of DUSP1 in leukemic transformation driven by enhanced MAPK signaling and advocate for the development of a selective DUSP1 inhibitor for curative treatment outcomes.

CSF3R T618I Collaborates With RUNX1-RUNX1T1 to Expand Hematopoietic Progenitors and Sensitizes to GLI Inhibition.

Activating colony-stimulating factor-3 receptor gene (CSF3R) mutations are recurrent in acute myeloid leukemia (AML) with t(8;21) translocation. However, the nature of oncogenic collaboration between alterations of CSF3R and the t(8;21) associated RUNX1-RUNX1T1 fusion remains unclear. In CD34+ hematopoietic stem and progenitor cells from healthy donors, double oncogene expression led to a clonal advantage, increased self-renewal potential, and blast-like morphology and distinct immunophenotype. Gene expression profiling revealed hedgehog signaling as a potential mechanism, with upregulation of GLI2 constituting a putative pharmacological target. Both primary hematopoietic cells and the t(8;21) positive AML cell line SKNO-1 showed increased sensitivity to the GLI inhibitor GANT61 when expressing CSF3R T618I. Our findings suggest that during leukemogenesis, the RUNX1-RUNXT1 fusion and CSF3R mutation act in a synergistic manner to alter hedgehog signaling, which can be exploited therapeutically.

Integrated transcriptome, proteome and single-cell sequencing uncover the prognostic and immunological features of colony-stimulating factor 3 receptor in pan-cancer.

Colony-stimulating factor 3 receptor (CSF3R) has been demonstrated to be associated with various hematological tumors, especially chronic neutrophilic leukemia; however, the detailed roles of CSF3R in other cancers remain to be explored. In the present study, we systematically analyzed the expression profiles of CSF3R in pan-cancer by comprehensive bioinformatics databases, such as Tumor Immune Estimation Resource, version 2 (TIMER2.0), Gene Expression Profiling Interactive Analysis, version 2 (GEPIA2.0), etc. GEPIA2.0 was also used to analyze the relationship between CSF3R expression and patients' survival prognosis. We found that the high expression of CSF3R was associated with a poor prognosis in the brain tumor patients, such as brain lower grade glioma and glioblastoma multiforme. In addition, we further investigated the genetic mutation and DNA methylation level of CSF3R in multiple cancers. Immune infiltration analysis showed that CSF3R expression was positively correlated with a variety of tumor-infiltrating immune cells in most cancers. Single cell sequencing indicated that CSF3R levels were correlated with several cancer-associated pathways, such as DNA damage, cell invasion, and stemness. Taken together, the role of CSF3R in multiple cancers might reveal its potential as a novel prognostic biomarker and therapeutic target for cancer patients.

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.

Chronic neutrophilic leukemia/chronic eosinophilic leukemia

Chronic neutrophilic leukemia (CNL) is a clonal disorder that is characterized by increasing mature neutrophils. Colony stimulating factor 3 receptor (CSF3R) T618I mutation was frequently identified in patients with CNL and is defined as a molecular marker of the disease. Ruxolitinib, a JAK2 inhibitor, provided a promising therapeutic effect in a phase II study. In particular, ruxolitinib was more efficient for patients with CSF3R mutation. Allogeneic stem cell transplantation (Allo-SCT) may be a curative treatment for CNL. On the other hand, further studies are needed to define the optimal method of transplantation, source of donor, conditioning therapy, and timing of transplantation. Chronic eosinophilic leukemia (CEL) is a clonal disorder that is characterized by increasing eosinophils. In the World Health Organization Classification 5th edition, diagnostic criteria for CEL are renewed. Because the new criteria will be more specific for CEL than criteria in the older edition, "not otherwise specified (NOS) " is removed from the name of the disease. Anti-CD52 antibody, alemtuzumab, or anti-IL-5 antibody, mepolizumab, are promising drugs to control symptoms that are associated with hypereosinophilic syndrome. Allo-SCT is anticipated as a curative treatment for CEL, but the evidence of Allo-SCT for CEL is still limited. Further study is required to define the treatment strategy.

Identification and exploration of novel M2 macrophage-related biomarkers in the development of acute myocardial infarction.

Acute myocardial infarction (AMI), one of the most severe and fatal cardiovascular diseases, is a major cause of morbidity and mortality worldwide. Macrophages play a critical role in ventricular remodeling after AMI. The regulatory mechanisms of the AMI progression remain unclear. This study aimed to identify hub regulators of macrophage-related modules and provide translational experiments with potential therapeutic targets. The GSE59867 dataset was downloaded from the Gene Expression Omnibus (GEO) database for bioinformatics analysis. The expression patterns of 22 types of immune cells were determined using CIBERSORT. GEO2R was used to identify differentially expressed genes (DEGs) through the limma package. Then, DEGs were clustered into different modules, and relationships between modules and macrophage types were analyzed using weighted gene correlation network analysis (WGCNA). Further functional enrichment analysis was performed using significantly associated modules. The module most significantly associated with M2 macrophages (Mϕ2) was chosen for subsequent analysis. Co-expressed DEGs of AMI were identified in the GSE123342 and GSE97320 datasets and module candidate hub genes. Additionally, hub gene identification was performed in GSE62646 dataset and clinical samples. A total of 8,760 DEGs were identified and clustered into ten modules using WGCNA analysis. The blue and turquoise modules were significantly related to Mϕ2, and 482 hub genes were discerned from two hub modules that conformed to module membership values > 0.8 and gene significance values > 0.25. Subsequent analysis using a Venn diagram assessed 631 DEGs in GSE123342, 1457 DEGs in GSE97320, and module candidate hub genes for their relationship with Mϕ2 in the progression of AMI. Finally, four hub genes (CSF2RB, colony stimulating factor 2 receptor subunit beta; SIGLEC9, sialic acid-binding immunoglobulin-like lectin 9; LRRC25, leucine-rich repeat containing 25; and CSF3R, colony-stimulating factor-3 receptor) were validated to be differentially expressed and to have high diagnostic value in both GSE62646 and clinical samples. Using comprehensive bioinformatics analysis, we identified four novel genes that may play crucial roles in the pathophysiological mechanism of AMI. This study provides novel insights into the impact of macrophages on the progression of AMI and directions for Mϕ2-targeted molecular therapies for AMI.

Truncated CSF3 receptors induce pro-inflammatory responses in severe congenital neutropenia.

Severe congenital neutropenia (SCN) patients are prone to develop myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML). Leukaemic progression of SCN is associated with the early acquisition of CSF3R mutations in haematopoietic progenitor cells (HPCs), which truncate the colony-stimulating factor 3 receptor (CSF3R). These mutant clones may arise years before MDS/AML becomes overt. Introduction and activation of CSF3R truncation mutants in normal HPCs causes a clonally dominant myeloproliferative state in mice treated with CSF3. Paradoxically, in SCN patients receiving CSF3 therapy, clonal dominance of CSF3R mutant clones usually occurs only after the acquisition of additional mutations shortly before frank MDS or AML is diagnosed. To seek an explanation for this discrepancy, we introduced a patient-derived CSF3R-truncating mutation in ELANE-SCN and HAX1-SCN derived and control induced pluripotent stem cells and compared the CSF3 responses of HPCs generated from these lines. In contrast to CSF3R-mutant control HPCs, CSF3R-mutant HPCs from SCN patients do not show increased proliferation but display elevated levels of inflammatory signalling. Thus, activation of the truncated CSF3R in SCN-HPCs does not evoke clonal outgrowth but causes a sustained pro-inflammatory state, which has ramifications for how these CSF3R mutants contribute to the leukaemic transformation of SCN.

Alternatively spliced CSF3R isoforms in SRSF2 P95H mutated myeloid neoplasms.

Alternatively spliced colony stimulating factor 3 receptor (CSF3R) isoforms Class III and Class IV are observed in myelodysplastic syndromes (MDS), but their roles in disease remain unclear. We report that the MDS-associated splicing factor SRSF2 affects the expression of Class III and Class IV isoforms and perturbs granulopoiesis. Add-back of the Class IV isoform in Csf3r-null mouse progenitor cells increased granulocyte progenitors with impaired neutrophil differentiation, while add-back of the Class III produced dysmorphic neutrophils in fewer numbers. These CSF3R isoforms were elevated in patients with myeloid neoplasms harboring SRSF2 mutations. Using in vitro splicing assays, we confirmed increased Class III and Class IV transcripts when SRSF2 P95 mutations were co-expressed with the CSF3R minigene in K562 cells. Since SRSF2 regulates splicing partly by recognizing exonic splicing enhancer (ESE) sequences on pre-mRNA, deletion of either ESE motifs within CSF3R exon 17 decreased Class IV transcript levels without affecting Class III. CD34+ cells expressing SRSF2 P95H showed impaired neutrophil differentiation in response to G-CSF and was accompanied by increased levels of Class IV. Our findings suggest that SRSF2 P95H promotes Class IV splicing by binding to key ESE sequences in CSF3R exon 17, and that SRSF2, when mutated, contributes to dysgranulopoiesis.

The presence of Auer rods in neutrophils and monocytes besides blasts in a case of acute myeloid leukemia with CSF3R mutation

An 81-year-old male with a history of diabetes, hypertension, hypercholesterolemia, previous DVT not on anticoagulation, and arthritis presented to the emergency department with a chief complaint of generalized weakness. Complete blood counts revealed white blood cells (WBC) 165.3 × 109/L, hemoglobin (Hb) 4.4 g/dl, and platelet (PLT) < 10 × 109/L, with 62% blasts. Review of peripheral blood showed blasts of variable sizes with a moderate amount of basophilic cytoplasm (Figure 1A–D; peripheral blood; Leishman stain; 100× objective), and pale-salmon-pink-colored cytoplasmic granules in a subset (Figure 1A, see arrows). Auer rods are easily identified in blasts (Figure 1A,B, see asterisk), occasionally seen in monocytes (Figure 1C, see asterisk) and neutrophils (Figure 1d, see asterisk). Bone marrow was examined to establish diagnosis, showing marked hypercellularity with 72% blasts on aspirate smears with morphologic features similar to those in peripheral blood (Figure 1E [bone marrow biopsy; hematoxylin and eosin, 40× objective] and F [bone marrow aspirate; Leishman stain; 50× objective]). Granulocytic dysplasia with pelgeroid morphology and abnormal nuclear chromatin clumping was seen in both peripheral blood and bone marrow (> 10% and < 50%). Concurrent flow cytometry showed increased myeloblasts with expression of CD34, CD13, CD 117, CD33, HLA-DR, CD19 (dim; minor subset), CD11b (dim; subset), CD14 (dim; minor subset), CD15 (dim; minor subset), myeloperoxidase (dim; subset), and terminal deoxynucleotidyl transferase (dim; minor subset). Conventional cytogenetics showed normal male karyotype. Concurrent FISH studies using acute myeloid leukemia (AML) and myelodysplastic syndrome panels of probes are negative for all loci studied, including 8q22 and 21q22 probes for RUNX1-RUNX1T1 rearrangement. Next generation sequencing showed mutation on the CSF3R gene (p.T640N, variant allele frequency 48.4%). An integrated diagnosis of AML with maturation was established (French–American–British Classification Subtype M2). Auer rods are a morphologic feature in leukemic blasts defining myeloid differentiation. The presence of Auer rods in neutrophils and monocytes are rarely reported, most commonly in association with acute promyelocytic leukemia (APL) with PML-RARA and AML with RUNX1-RUNX1T1, occasionally seen in other subtypes of AML and myelodysplastic syndrome (MDS) in the literature as well [1]. We presented a case of AML with Auer rods in non-blast cells as well as morphologic and immunophenotypic features resembling AML with RUNX1-RUNX1T1: blasts with basophilic cytoplasm, salmon-pink (pale) cytoplasmic granules and perinuclear Hof besides Auer rods as well as granulocytic dysplasia in the background. However, no recurrent cytogenetic and molecular genetic abnormalities as defined by World Health Organization (WHO) classification are detected, while a CSF3R mutation seen. Although mutations in the colony stimulating factor 3 receptor (CSF3R) occurs rarely in AML, comprising only about 1% of adults with AML, it is suggested that CSF3R can be a potential therapeutic target in AML [2]. In summary, we presented for the first time a case of AML with a few rare morphologic features including the presence of Auer rods in non-blast cells coinciding with a potential therapeutic target of CSF3R mutation. Whether the association between the morphologic features and the CSF3R mutation can be established warrants additional studies. In addition, a potential diagnostic pitfall to be taken into consideration in this case is that no reverse transcription-polymerase chain reaction (RT-PCR) was done to further exclude RUNX1-RUNX1T1 rearrangement, although the likelihood of having undetected rearrangement by karyotyping and FISH studies is extremely low. What is the new aspect of your work? Here, we for the first time presented a case of AML with morphologic features resembling AML with RUNX1-RUNX1T1, including Auer rods in non-blast cells, coinciding with CSF3R mutation, which is a rare mutation in AML while representing a potential novel therapeutic target. What is the central finding of your work? A rare case of CSF3R-mutated AML with no recurrent cytogenetic abnormality, showing unusual morphologic features resembling AML with RUNX1-RUNX1T1, including Auer rods in non-blast cells. What is (or could be) the specific clinical relevance of your work? The rare morphological features present in this AML could potentially serve as a trigger to identify cases of AML with CSF3R mutation for targeted therapy. None. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. The authors received no specific funding for this work. Not applicable. Not applicable. Not applicable. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Multiomics Analysis Identifies SOCS1 as Restraining T Cell Activation and Preventing Graft-Versus-Host Disease.

Graft‐versus‐host disease (GVHD) is a major life‐threatening complication of allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Inflammatory signaling pathways promote T‐cell activation and are involved in the pathogenesis of GVHD. Suppressor of cytokine signaling 1 (SOCS1) is a critical negative regulator for several inflammatory cytokines. However, its regulatory role in T‐cell activation and GVHD has not been elucidated. Multiomics analysis of the transcriptome and chromatin structure of granulocyte‐colony‐stimulating‐factor (G‐CSF)‐administered hyporesponsive T cells from healthy donors reveal that G‐CSF upregulates SOCS1 by reorganizing the chromatin structure around the SOCS1 locus. Parallel in vitro and in vivo analyses demonstrate that SOCS1 is critical for restraining T cell activation. Loss of Socs1 in T cells exacerbates GVHD pathogenesis and diminishes the protective role of G‐CSF in GVHD mouse models. Further analysis shows that SOCS1 inhibits T cell activation not only by inhibiting the colony‐stimulating‐factor 3 receptor (CSF3R)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway, but also by restraining activation of the inflammasome signaling pathway. Moreover, high expression of SOCS1 in T cells from patients correlates with low acute GVHD occurrence after HSCT. Overall, these findings identify that SOCS1 is critical for inhibiting T cell activation and represents a potential target for the attenuation of GVHD.

The prognostic factors in acute myeloid leukaemia with double-mutated CCAAT/enhancer-binding protein alpha (CEBPAdm).

The prognostic factors to stratify acute myeloid leukaemia (AML) with double-mutated CCAAT/enhancer-binding protein alpha (CEBPAdm) into different risk groups remains to be determined. In this retrospective study, we evaluated 171 consecutive patients with newly diagnosed AML with CEBPAdm by a Cox proportional hazards regression model. In univariate analyses, colony stimulating factor 3 receptor (CSF3R) and Wilms tumour 1 (WT1) mutations were associated with poor relapse-free survival (RFS). The induction regimens including homoharringtonine (omacetaxine mepesuccinate) or intermediate-dose cytarabine was associated with favourable RFS and overall survival (OS). The induction regimen including both homoharringtonine and intermediate-dose cytarabine was associated with the most favourable RFS (3-year RFS 84.7%) and OS (3-year OS 92.8%) compared to the conventional cytarabine and daunorubicin regimen (3-year RFS 27.7%, hazard ratio [HR] 0.126, 95% confidence interval [CI] 0.051-0.313, Wald p<0.001; and 3-year OS 56.4%, HR 0.179, 95% CI 0.055-0.586, Wald p=0.005). In multivariate analyses, the induction regimen including intermediate-dose cytarabine (HR 0.364, 95% CI 0.205-0.646, Wald p<0.001) and CSF3R mutations (HR 2.667, 95% CI 1.276-5.572, Wald p=0.009) were independently associated with RFS. Taken together, we found that induction regimen and CSF3R mutations were independent prognostic factors for AML with CEBPAdm.

Chronic neutrophilic leukemia: 2022 update on diagnosis, genomic landscape, prognosis, and management

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 2013 seminal discovery of oncogenic driver mutations in colony-stimulating factor 3 receptor (CSF3R) in the majority of patients with CNL not only established its molecular pathogenesis but provided a diagnostic biomarker and rationale for pharmacological targeting. In 2016, the World Health Organization (WHO) recognized 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. There is currently no standard of care for management of CNL, due in large part to the rarity of disease and dearth of formal clinical trials. Most commonly used therapeutic agents include conventional oral chemotherapy (e.g., hydroxyurea), interferon, and Janus kinase (JAK) inhibitors, while hematopoietic stem cell transplant remains the only potentially curative modality. Increasingly comprehensive genetic profiling in CNL, including new data on clonal evolution, has disclosed a complex genomic landscape with additional mutations and combinations thereof driving disease progression and drug resistance. Although accurate prognostic stratification and therapeutic decision-making remain challenging in CNL, emerging data on molecular biomarkers and the addition of newer agents, such as JAK inhibitors, to the therapeutic arsenal, are paving the way toward greater standardization and improvement of patient care.

CEACAM-1 Induced CSF3-receptor Downregulation in Bone Marrow Associated With Refractory Neutropenia in Advanced Cirrhosis.

Background and AimsCirrhosis patients exhibit cytopenia, and, at times refractory neutropenia to granulocyte colony-stimulating factor (G-CSF), which acts through the CSF3-receptor (CSF3R), and changes in CSF3R can affect the response. We conducted this study to assess the CSF3R status and its relevance in cirrhotic patients.MethodsCirrhotic patients (n=127) and controls (n=26) with clinically indicated bone marrow (BM) examination were studied. BM assessment was done by qRT-PCR and immunohistochemistry (IHC) for CSF3R. Circulating G-CSF, CSF3R, and carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) were measured. BM hematopoietic precursor cells and their alterations were examined by flow cytometry. The findings were validated in liver cirrhosis patients who received G-CSF for severe neutropenia.ResultsThe mean age was 48.6±13.4 years, and 80.3% were men. Circulatory CSF3R reduction was noted with the advancement of cirrhosis, and confirmed by qRT-PCR and IHC in BM. CSF3R decline was related to decreased hematopoietic stem cells (HSCs) and downregulation of CSF3R in the remaining HSCs. Cocultures confirmed that CEACAM1 led to CSF3R downregulation in BM cells by possible lysosomal degradation. Baseline low peripheral blood-(PB)-CSF3R also predisposed development of infections on follow-up. Decreased CSF3R was also associated with nonresponse to exogenous G-CSF treatment of neutropenia.ConclusionsAdvanced liver cirrhosis was associated with low CSF3R and high CEACAM1 levels in the BM and circulation, making patients prone to infection and inadequate response to exogenous G-CSF.

Differential Implications of CSF3R Mutations in t(8;21) and CEBPA Double Mutated Acute Myeloid Leukemia

BackgroundFew data are available exploring mutations of the colony-stimulating factor 3 receptor (CSF3R) in acute myeloid leukemia (AML) in an all-round and systematic manner. The purpose of this study was to analyze the CSF3R mutations (CSF3Rmut) in AML with recurrent genetic abnormalities for potential synergistic pathomechanism. Patients and MethodsWe retrospectively screened 1102 adult de novo AML patients with available next-generation sequencing (NGS) information on 132 genes related to hematologic disorders. The χ2, Mann-Whitney U tests were used to analyze their associations with clinicopathologic characteristics, and a propensity score matching (PSM) followed by Kaplan-Meier method was applied to measure their prognostic effects. ResultsOverall, CSF3Rmut were detected in 40 (3.6%) of 1102 patients with adult de novo AML. CSF3Rmut were predominantly enriched in AML with the CEBPA double mutations (CEBPAdm) (16/122, 13.1%), t(8;21) (12/186, 6.5%) and mutated RUNX1 (3/50, 6.0%), respectively. The CSF3Rmut loci and types differed according to AML subtypes, with frameshift-indels and premature stop confined in the t(8;21) AML [10/12 (83.3%)], and missense recurrently aggregated in the CEBPAdm AML [16/16 (100%)]. Cases with CSF3Rmut had a lower WBC count versus those with CSF3R wild-type (CSF3Rwt) in the t(8;21) AML cohort, with a borderline significance [median 5.45 (range 0.94-20.30) × 109/L) vs. 8.80 (range 0.96-155.00) × 109/L, P = .046]. CSF3Rmut were non-significantly associated with higher WBC counts [median 33.6 (range 6.8-287.6) × 109/L vs. 18.1 (range 1.7-196.0) × 109/L, P = .156] and significantly with lower immunophenotypic CD15 positivity [0/8 (0%) vs. 44/80 (55%), P = .009] as compared to CSF3Rwt in the CEBPAdm AML cohort. After propensity score matching followed by Kaplan-Meier analysis, CSF3Rmut cases had comparable disease-free survival (DFS) and overall survival (OS) to those with CSF3Rwt (P = .607 and P = .842, respectively) in the t(8;21) AML cohort. By contrast, CSF3Rmut showed an inclination towards inferior DFS compared to CSF3Rwt in the CEBPAdm AML cohort [median DFS 19.8 (95%CI 3.1-36.5) months vs. not reached (NR), P = .086]. No significant difference was found for OS between CSF3Rmut and CSF3Rwt cases (P = .943). ConclusionWe concluded that CSF3Rmut were frequently enriched in patients with t(8;21) and CEBPAdm subtypes among AML, but showed divergent clinicopathologic features, mutation loci and types and differing prognostic aspects.

The clinical characteristics and prognosis of Chinese acute myeloid leukemia patients with CSF3R mutations.

The colony-stimulating factor 3 receptor (CSF3R) controls the proliferation of myeloid progenitors and differentiation into neutrophils. However, the clinical features and prognostic significance of CSF3R mutations in primary acute myeloid leukemia (AML) patients are still unclear. 158 newly diagnosed AML patients were retrospectively evaluated in our study. Amplicon-based next-generation sequencing (NGS) and multiplex-nested reverse-transcription polymerase chain reaction (RT-PCR) were used to investigate the 34 genes and 43 fusion genes associated with leukemia. In addition, clinical features, mutation incidence, and survival outcomes were compared between patients with CSF3R mutation and patients with wild-type CSF3R. In our study, CSF3R mutations were found in 7.6% (12/158) cases. The membrane-proximal amino acid substitution T618I (58.3%) was the most frequent mutation. CSF3R mutations were associated with higher WBC counts (P=.035). CEBPA mutation, TET2 mutation, and RUNX1-RUNX1T1 translocation were the most common co-mutations of CSF3R. The CSF3R gene was mutually exclusive with signal transduction genes (P=.029), while positively associated with TET2 mutations (P=.014). CSF3R mutations had no effect on CR1 (P=.935), R (P=.625) and OS (P=.1172). Patients with CSF3R mutations had a worse DFS (P=.0352) than those with wild-type CSF3R. Multivariate survival analysis showed that CSF3R mutation was an independent risk factor for DFS of primary AML patients (HR=2.048, 95%CI: 1.006-4.170, P=.048). AML patients with CSF3R mutations had unique clinical features and gene co-mutation spectrum. CSF3R mutation was an independent risk factor for DFS and could be a potential prognostic marker and therapeutic target for Chinese primary AML patients.

Hereditary Chronic Neutrophilic Leukemia in a Four Generation Family without Transformation to Acute Leukemia

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood leukocytosis consisting primarily of segmented neutrophils and band forms, hypercellular bone marrow with granulocytosis, hepatosplenomegaly, and the presence of activating colony-stimulating factor 3 receptor (CSF3R) mutations. Blast transformation occurs frequently in patients with acquired CNL, with a median overall survival of 21 months from diagnosis. Typically, CSF3R mutations in CNL are thought to be somatic; however, we and others have reported rare cases of germline activating CSF3R mutations producing a familial CNL. Here we report the clinical course of a patient with CNL along with definitive evidence of inherited germline transmission of the CSF3R T618I mutation. Spanning four generations, with affected family members of ages 1.6 - 51 years, this is the largest reported pedigree of a family with familial CNL (Figure 1A).The proband is a 49-year-old female referred to our center with a history of lifelong leukocytosis and leukocyte count of 115.1 x 10 9/L with 75% granulocytes and 11% bands, platelet count of 341 x 10 9/L, and hemoglobin of 12.5 g/dL with hematocrit of 38%. The family history was also remarkable for leukocytosis. Prior therapies for the proband included imatinib, splenectomy, and hydroxyurea. Additional testing by our center revealed a T618I CSFR3 mutation, and the absence of mutations in ASXL1 and SETBP1 or a BCR-ABL translocation. Treatment with ruxolitinib resulted in improvement of her leukocyte count to 43.0 x 10 9/L with 73% granulocytes, and reduction in her alkaline phosphatase from 732 IU/L to 296 IU/L. There has been no evidence of gain of any known deleterious somatic mutations that frequently co-occur with somatic T618I CSF3R mutations in CNL in the patient to date.Germline analysis of genomic DNA extracted from cultured mesenchymal stromal cells from the proband and Sanger sequencing demonstrated a heterozygous T618I mutation. Mutational analysis of the proband's family members confirmed a heterozygous CSF3R T618I mutation in all living affected family members, while all unaffected family members tested were homozygous wild type. There has been no evidence of leukemic transformation in any affected family members to date. Mutational analysis was not feasible on the proband's deceased mother and brother with a putative CNL diagnosis due to lack of DNA samples; however, there was no evidence of transformation to acute leukemia in either of the two deceased family members.Because CSF3R can produce anti-apoptotic signaling, we hypothesized that autoactivating T618I mutations could prolong neutrophil survival. Polymorphonuclear cells (PMNs) isolated from the proband and from normal donors were cultured in vitro and apoptosis assessed at 24-hour intervals. Neutrophils expressing the CSF3R T618I had prolonged survival with a >40% decrease in apoptosis after 48 hours in culture (Figure 1B). RNA-seq followed by pathway analysis demonstrated significant decreases in activation of canonical apoptotic pathways in PMNs, including both the extrinsic and mitochondrial dependent pathways. Immunoblotting for candidate anti- and pro-apoptotic proteins revealed increased expression of the anti-apoptotic BCL2 family member MCL1 in T618I-expressing PMNs. Notably, inhibition of MCL1 using S63845 reversed the anti-apoptotic effect induced by ligand-activation of the CSF3R receptor in PMNs (P < 0.001, Figure 1C).In conclusion, we demonstrate hereditary CNL within a large family tree with no observed transformation to acute leukemia in any affected individuals up to age 51, suggesting a potentially more indolent course. Nonetheless, our observations highlight the need for germline testing of patients with CNL to better understand the natural history of CNL. Moreover, our data provide further insight into the pathobiology of CNL and potential novel targets for therapy. [Display omitted] DisclosuresVoorhees: Bristol-Myers Squibb Company.: Other: Data Safety & Monitoring; AbbVie Inc, Bristol-Myers Squibb Company; Consulting Agreement: GlaxoSmithKline, Novartis, Oncopeptides: Other: Advisory Committee.

Efficacy of Low-Dose rhGM-CSF Treatment in a Patient With Severe Congenital Neutropenia Due to CSF3R Deficiency: Case Report of a Novel Biallelic CSF3R Mutation and Literature Review.

This study reports the clinical manifestations, genetics, and efficacy of treatment with the efficacy of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-GSF) of a 2-year-old female patient with severe congenital neutropenia (SCN) type 7 (SCN7) caused by novel biallelic mutations in the colony-stimulating factor 3 receptor (CSF3R) gene. Genetic diagnosis of the patient was performed by whole-exome and Sanger sequencing. Expression of the CSF3R gene in the peripheral neutrophils of the patient was detected by real-time PCR and Western blotting. The patient presented with recurrent suppurative tonsillitis and decreased absolute neutrophil count <0.5 × 109/L. Novel heterozygous mutations were found to be inherited from each parent (maternal c.690delC [p.met231Cysfs*32] and paternal c.64+5G>A). The patient's neutrophils had lower CSF3R mRNA and protein levels than those of the parents. Low-dose rhGM-CSF (3 μg/kg/day once a week) prevented recurrent infection in the patient. These results demonstrate that the clinical manifestations of SCN7 with biallelic CSF3R mutations and downregulated CSF3R can be effectively treated with rhGM-CSF.

Colony-stimulating factor 3 receptor (CSF3R) M696T mutation does not impact on clinical outcomes of a Ph+ acute lymphoblastic leukemia patient

Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in a variety of myeloid disorders. Although CSF3R point mutations (eg, T618I) are emerging as key players in chronic neutrophilic leukemia/atypical chronic myelogenous leukemia , the significance of rarer CSF3R mutations is unknown. Here, we report a 32-year-old female who was diagnosed as Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) with the CSF3R M696T mutation and was undergone unrelated donor hematopoietic stem cell transplantation. The patient achieved complete remission with chemotherapy in combination with tyrosine kinase inhibitor (TKI) and long-term survival by unrelated donor transplantation. Meanwhile, we performed a series of experiments using murine interleukin 3 (IL-3)-dependent Ba/F3 cell line to evaluate the transforming capacity of the CSF3R M696T mutation. We confirmed the presence of a CSF3R M696T germline mutation in this patient which was inherited from her mother. The in vitro experiment results showed that the CSF3R M696T mutation contributes marginally to the tumor transformation of Ba/F3 cells, indicating that CSF3R M696T mutation was neutral in tumor transformation ability. We concluded that TKI is effective in patients with the CSF3R M696T mutation in Ph+ ALL and donors with CSF3R M696T mutation might still be selected as the candidate for transplantation.

Current Management of Chronic Neutrophilic Leukemia.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by oncogenic driver mutations in colony-stimulating factor 3 receptor (CSF3R). Due in large part to the rarity of the disease and dearth of clinical trials, there is currently no standard of care for CNL. Available therapies range from conventional oral chemotherapy to targeted JAK inhibitors to hematopoietic stem cell transplant (HSCT), the latter representing the only potentially curative modality. For this reason, coupled with CNL's typically aggressive clinical course, allogeneic HSCT remains the primary recommended therapy for eligible patients. For ineligible patients, a number of nontransplant therapies have been evaluated in limited trials. These agents may additionally be considered "bridging" therapies pre-transplant in order to control myeloproliferation and alleviate symptoms. Historically, the most commonly utilized first-line agent has been hydroxyurea, though most patients ultimately require second (or subsequent)-line therapy; still hydroxyurea remains the conventional frontline option. Dasatinib has demonstrated efficacy in vitro in cases of CSF3R terminal membrane truncation mutations and may cautiously be considered upfront in such instances, though no substantive studies have validated its efficacy in vivo. Numerous other chemotherapy agents, practically re-appropriated from the pharmaceutical arsenal of MPN, have been utilized in CNL and are typically reserved for second/subsequent-line settings; these include interferon-alpha (IFN-a), hypomethylating agents, thalidomide, cladribine, and imatinib, among others. Most recently, ruxolitinib, a JAK1/2 inhibitor targeting JAK-STAT signaling downstream from CSF3R, has emerged as a potentially promising new candidate for the treatment of CNL. Increasingly robust data support the clinical efficacy, with associated variable reductions in allele burden, and tolerability of ruxolitinib in patients with CNL, particularly those carrying the CSF3RT618I mutation. Similar to conventional nontransplant strategies, however, no disease-modifying or survival benefits have been demonstrated. While responses to JAK-STAT inhibition in CNL have not been uniform, data are sufficient to recommend consideration of ruxolitinib in the therapeutic repertory of CNL. There remains a major unmet need for prospective trials with investigational therapies in CNL.

Leukemia Cell Lines: In Vitro Models for the Study of Chronic Neutrophilic Leukemia.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm that is genetically characterized by the absence of both the Philadelphia chromosome and BCR-ABL1 fusion gene and the high prevalence of mutations in the colony-stimulating factor 3 receptor (CSF3R). Additional disease-modifying mutations have been recognized in CNL samples, portraying a distinct mutational landscape. Despite the growing knowledge base on genomic aberrations, further progress could be gained from the availability of representative models of CNL. To address this gap, we screened a large panel of available leukemia cell lines, followed by a detailed mutational investigation with focus on the CNL-associated candidate driver genes. The sister cell lines CNLBC-1 and MOLM-20 were derived from a patient with CNL and carry CNL-typical molecular hallmarks, namely mutations in several genes, such as CSF3R, ASXL1, EZH2, NRAS, and SETBP1. The use of these validated and comprehensively characterized models will benefit the understanding of the pathobiology of CNL and help inform therapeutic strategies.