CITEViz: interactively classify cell populations in CITE-Seq via a flow cytometry-like gating workflow using R-Shiny.

BACKGROUND: The rapid advancement of new genomic sequencing technology has enabled the development of multi-omic single-cell sequencing assays. These assays profile multiple modalities in the same cell and can often yield new insights not revealed with a single modality. For example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) simultaneously profiles the RNA transcriptome and the surface protein expression. The surface protein markers in CITE-Seq can be used to identify cell populations similar to the iterative filtration process in flow cytometry, also called "gating", and is an essential step for downstream analyses and data interpretation. While several packages allow users to interactively gate cells, they often do not process multi-omic sequencing datasets and may require writing redundant code to specify gate boundaries. To streamline the gating process, we developed CITEViz which allows users to interactively gate cells in Seurat-processed CITE-Seq data. CITEViz can also visualize basic quality control (QC) metrics allowing for a rapid and holistic evaluation of CITE-Seq data. RESULTS: We applied CITEViz to a peripheral blood mononuclear cell CITE-Seq dataset and gated for several major blood cell populations (CD14 monocytes, CD4 T cells, CD8 T cells, NK cells, B cells, and platelets) using canonical surface protein markers. The visualization features of CITEViz were used to investigate cellular heterogeneity in CD14 and CD16-expressing monocytes and to detect differential numbers of detected antibodies per patient donor. These results highlight the utility of CITEViz to enable the robust classification of single cell populations. CONCLUSIONS: CITEViz is an R-Shiny app that standardizes the gating workflow in CITE-Seq data for efficient classification of cell populations. Its secondary function is to generate basic feature plots and QC figures specific to multi-omic data. The user interface and internal workflow of CITEViz uniquely work together to produce an organized workflow and sensible data structures for easy data retrieval. This package leverages the strengths of biologists and computational scientists to assess and analyze multi-omic single-cell datasets. In conclusion, CITEViz streamlines the flow cytometry gating workflow in CITE-Seq data to help facilitate novel hypothesis generation.

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.

Mutated SETBP1 activates transcription of Myc programs to accelerate CSF3R-driven myeloproliferative neoplasms.

Colony stimulating factor 3 receptor (CSF3R) mutations lead to JAK pathway activation and are the molecular hallmark of chronic neutrophilic leukemia (CNL). Approximately half of patients with CNL also have mutations in SET binding protein 1 (SETBP1). In this study, we developed models of SETBP1-mutated leukemia to understand the role that SETBP1 plays in CNL. SETBP1 mutations promote self-renewal of CSF3R-mutated hematopoietic progenitors in vitro and prevent cells from undergoing terminal differentiation. In vivo, SETBP1 mutations accelerate leukemia progression, leading to the rapid development of hepatosplenomegaly and granulocytosis. Through transcriptomic and epigenomic profiling, we found that SETBP1 enhances progenitor-associated programs, most strongly upregulating Myc and Myc target genes. This upregulation of Myc can be reversed by LSD1 inhibitors. In summary, we found that SETBP1 mutations promote aggressive hematopoietic cell expansion when expressed with mutated CSF3R through the upregulation of Myc-associated gene expression programs.

Combined inhibition of JAK/STAT pathway and lysine-specific demethylase 1 as a therapeutic strategy in CSF3R/CEBPA mutant acute myeloid leukemia.

Acute myeloid leukemia (AML) is a deadly hematologic malignancy with poor prognosis, particularly in the elderly. Even among individuals with favorable-risk disease, approximately half will relapse with conventional therapy. In this clinical circumstance, the determinants of relapse are unclear, and there are no therapeutic interventions that can prevent recurrent disease. Mutations in the transcription factor CEBPA are associated with favorable risk in AML. However, mutations in the growth factor receptor CSF3R are commonly co-occurrent in CEBPA mutant AML and are associated with an increased risk of relapse. To develop therapeutic strategies for this disease subset, we performed medium-throughput drug screening on CEBPA/CSF3R mutant leukemia cells and identified sensitivity to inhibitors of lysine-specific demethylase 1 (LSD1). Treatment of CSF3R/CEBPA mutant leukemia cells with LSD1 inhibitors reactivates differentiation-associated enhancers driving immunophenotypic and morphologic differentiation. LSD1 inhibition is ineffective as monotherapy but demonstrates synergy with inhibitors of JAK/STAT signaling, doubling median survival in vivo. These results demonstrate that combined inhibition of JAK/STAT signaling and LSD1 is a promising therapeutic strategy for CEBPA/CSF3R mutant AML.

Genomic landscape of neutrophilic leukemias of ambiguous diagnosis.

Chronic neutrophilic leukemia (CNL), atypical chronic myeloid leukemia (aCML), and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are a group of rare and heterogeneous myeloid disorders. There is strong morphologic resemblance among these distinct diagnostic entities as well as a lack of specific molecular markers and limited understanding of disease pathogenesis, which has made diagnosis challenging in certain cases. The treatment has remained empirical, resulting in dismal outcomes. We, therefore, performed whole-exome and RNA sequencing of these rare hematologic malignancies and present the most complete survey of the genomic landscape of these diseases to date. We observed a diversity of combinatorial mutational patterns that generally do not cluster within any one diagnosis. Gene expression analysis reveals enrichment, but not cosegregation, of clinical and genetic disease features with transcriptional clusters. In conclusion, these groups of diseases represent a continuum of related diseases rather than discrete diagnostic entities.

Gain-of-function mutations in granulocyte colony-stimulating factor receptor (CSF3R) reveal distinct mechanisms of CSF3R activation.

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 CSF3R gene in many hematologic malignancies. To find additional CSF3R 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 CSF3R 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.

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.

Neogenin Facilitates the Induction of Hepcidin Expression by Hemojuvelin in the Liver.

Hemojuvelin (HJV) regulates iron homeostasis by direct interaction with bone morphogenetic protein (BMP) ligands to induce hepcidin expression through the BMP signaling pathway in the liver. Crystallography studies indicate that HJV can simultaneously bind to both BMP2 and the ubiquitously expressed cell surface receptor neogenin. However, the role of the neogenin-HJV interaction in the function of HJV is unknown. Here we identify a mutation in HJV that specifically lowers its interaction with neogenin. Expression of this mutant Hjv in the liver of Hjv(-/-) mice dramatically attenuated its induction of BMP signaling and hepcidin mRNA, suggesting that interaction with neogenin is critical for the iron regulatory function of HJV. Further studies revealed that neogenin co-immunoprecipitated with ALK3, an essential type-I BMP receptor for hepatic hepcidin expression. Neogenin has also been shown to facilitate the cleavage of HJV by furin in transfected cells. Surprisingly, although cleavage of HJV by furin has been implicated in the regulation of HJV function in cell culture models and furin-cleaved soluble Hjv is detectable in the serum of mice, mutating the furin cleavage site did not alter the stimulation of hepcidin expression by Hjv in mice. In vivo studies validated the important role of HJV-BMP interaction for Hjv stimulation of BMP signaling and hepcidin expression. Together these data support a model in which neogenin acts as a scaffold to facilitate assembly of the HJV·BMP·BMP receptor complex to induce hepcidin expression.

Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML.

BACKGROUND: The molecular causes of many hematologic cancers remain unclear. Among these cancers are chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL1-negative) chronic myeloid leukemia (CML), both of which are diagnosed on the basis of neoplastic expansion of granulocytic cells and exclusion of genetic drivers that are known to occur in other myeloproliferative neoplasms and myeloproliferative-myelodysplastic overlap neoplasms. METHODS: To identify potential genetic drivers in these disorders, we used an integrated approach of deep sequencing coupled with the screening of primary leukemia cells obtained from patients with CNL or atypical CML against panels of tyrosine kinase-specific small interfering RNAs or small-molecule kinase inhibitors. We validated candidate oncogenes using in vitro transformation assays, and drug sensitivities were validated with the use of assays of primary-cell colonies. RESULTS: We identified activating mutations in the gene encoding the receptor for colony-stimulating factor 3 (CSF3R) in 16 of 27 patients (59%) with CNL or atypical CML. These mutations segregate within two distinct regions of CSF3R and lead to preferential downstream kinase signaling through SRC family-TNK2 or JAK kinases and differential sensitivity to kinase inhibitors. A patient with CNL carrying a JAK-activating CSF3R mutation had marked clinical improvement after the administration of the JAK1/2 inhibitor ruxolitinib. CONCLUSIONS: Mutations in CSF3R are common in patients with CNL or atypical CML and represent a potentially useful criterion for diagnosing these neoplasms. (Funded by the Leukemia and Lymphoma Society and others.).

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.

The new genetics of chronic neutrophilic leukemia and atypical CML: implications for diagnosis and treatment.

Although activation of tyrosine kinase pathways is a shared theme among myeloproliferative neoplasms, the pathogenetic basis of chronic neutrophilic leukemia (CNL) has remained elusive. Recently, we identified high-frequency oncogenic mutations in the granulocyte-colony stimulating factor receptor (CSF3R) in CNL and in some patients with atypical chronic myeloid leukemia. Inhibition of Janus kinase 2 or SRC kinase signaling downstream of mutated CSF3R is feasible and should be explored therapeutically. Herein, we discuss the potential impact of these findings for the classification and treatment of these disorders.

The CSF3R T618I mutation causes a lethal neutrophilic neoplasia in mice that is responsive to therapeutic JAK inhibition.

We have recently identified targetable mutations in CSF3R (GCSFR) in 60% of chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL-negative) chronic myeloid leukemia (aCML) patients. Here we demonstrate that the most prevalent, activating mutation, CSF3R T618I, is sufficient to drive a lethal myeloproliferative disorder in a murine bone marrow transplantation model. Mice transplanted with CSF3R T618I-expressing hematopoietic cells developed a myeloproliferative disorder characterized by overproduction of granulocytes and granulocytic infiltration of the spleen and liver, which was uniformly fatal. Treatment with the JAK1/2 inhibitor ruxolitinib lowered the white blood count and reduced spleen weight. This demonstrates that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder resembling aCML and CNL that is sensitive to pharmacologic JAK inhibition. This murine model is an excellent tool for the further study of neutrophilic myeloproliferative neoplasms and implicates the clinical use of JAK inhibitors for this disease.

Cancer- and endotoxin-induced cachexia require intact glucocorticoid signaling in skeletal muscle.

Cachexia is a wasting condition defined by skeletal muscle atrophy in the setting of systemic inflammation. To explore the site at which inflammatory mediators act to produce atrophy in vivo, we utilized mice with a conditional deletion of the inflammatory adaptor protein myeloid differentiation factor 88 (MyD88). Although whole-body MyD88-knockout (wbMyD88KO) mice resist skeletal muscle atrophy in response to LPS, muscle-specific deletion of MyD88 is not protective. Furthermore, selective reexpression of MyD88 in the muscle of wbMyD88KO mice via electroporation fails to restore atrophy gene induction by LPS. To evaluate the role of glucocorticoids as the inflammation-induced mediator of atrophy in vivo, we generated mice with targeted deletion of the glucocorticoid receptor in muscle (mGRKO mice). Muscle-specific deletion of the glucocorticoid receptor affords a 71% protection against LPS-induced atrophy compared to control animals. Furthermore, mGRKO mice exhibit 77% less skeletal muscle atrophy than control animals in response to tumor growth. These data demonstrate that glucocorticoids are a major determinant of inflammation-induced atrophy in vivo and play a critical role in the pathogenesis of endotoxemic and cancer cachexia.

Asxl1 deletion disrupts MYC and RNA polymerase II function in granulocyte progenitors.

Mutations in the gene Additional Sex-Combs Like 1 (ASXL1) are recurrent in myeloid malignancies as well as the pre-malignant condition clonal hematopoiesis, where they are universally associated with poor prognosis. However, the role of ASXL1 in myeloid lineage maturation is incompletely described. To define the role of ASXL1 in myelopoiesis, we employed single cell RNA sequencing and a murine model of hematopoietic-specific Asxl1 deletion. In granulocyte progenitors, Asxl1 deletion leads to hyperactivation of MYC and a quantitative decrease in neutrophil production. This loss of granulocyte production was not accompanied by significant changes in the landscape of covalent histone modifications. However, Asxl1 deletion results in a decrease in RNAPII promoter-proximal pausing in granulocyte progenitors, indicative of a global increase in productive transcription. These results suggest that ASXL1 inhibits productive transcription in granulocyte progenitors, identifying a new role for this epigenetic regulator in myeloid development.

Disruption of the MYC Superenhancer Complex by Dual Targeting of FLT3 and LSD1 in Acute Myeloid Leukemia.

Mutations in Fms-like tyrosine kinase 3 (FLT3) are common drivers in acute myeloid leukemia (AML) yet FLT3 inhibitors only provide modest clinical benefit. Prior work has shown that inhibitors of lysine-specific demethylase 1 (LSD1) enhance kinase inhibitor activity in AML. Here we show that combined LSD1 and FLT3 inhibition induces synergistic cell death in FLT3-mutant AML. Multi-omic profiling revealed that the drug combination disrupts STAT5, LSD1, and GFI1 binding at the MYC blood superenhancer, suppressing superenhancer accessibility as well as MYC expression and activity. The drug combination simultaneously results in the accumulation of repressive H3K9me1 methylation, an LSD1 substrate, at MYC target genes. We validated these findings in 72 primary AML samples with the nearly every sample demonstrating synergistic responses to the drug combination. Collectively, these studies reveal how epigenetic therapies augment the activity of kinase inhibitors in FLT3-ITD (internal tandem duplication) AML. IMPLICATIONS: This work establishes the synergistic efficacy of combined FLT3 and LSD1 inhibition in FLT3-ITD AML by disrupting STAT5 and GFI1 binding at the MYC blood-specific superenhancer complex.

Expression of myeloid differentiation factor 88 in neurons is not requisite for the induction of sickness behavior by interleukin-1ß.

BACKGROUND: Animals respond to inflammation by suppressing normal high-energy activities, including feeding and locomotion, in favor of diverting resources to the immune response. The cytokine interleukin-1 beta (IL-1ß) inhibits normal feeding and locomotor activity (LMA) via its actions in the central nervous system (CNS). Behavioral changes in response to IL-1ß are mediated by myeloid differentiation factor 88 (MyD88) in non-hematopoietic cells. It is unknown whether IL-1ß acts directly on neurons or requires transduction by non-neuronal cells. METHODS: The Nestin-cre mouse was crossed with MyD88lox mice to delete MyD88 from neurons and glia in the CNS (MyD88ΔCNS). These mice were compared to total body MyD88KO and wild type (WT) mice. Mice had cannulae stereotactically placed in the lateral ventricle and telemetry transponders implanted into the peritoneum. Mice were treated with either intracerebroventricular (i.c.v.) IL-1ß (10 ng) or vehicle. Food intake, body weight and LMA were continuously monitored for 24 h after treatment. I.c.v. tumor necrosis factor (TNF), a MyD88-independent cytokine, was used to control for normal immune development. Peripheral inflammation was modeled using intraperitoneal lipopolysaccharide (LPS). Groups were compared using two-way ANOVA with Bonferroni post-test. Efficacy of recombination was evaluated using tdTomato reporter mice crossed with the Nestin-cre mouse. MyD88 deletion was confirmed by Western blot. RESULTS: I.c.v. IL-1ß treatment caused a significant reduction in feeding, body weight and LMA in WT mice. MyD88KO mice were protected from these changes in response to i.c.v. IL-1ß despite having intact behavioral responses to TNF. Cre-mediated recombination was observed in neurons and astrocytes, but not microglia or endothelial cells. In contrast to MyD88KO mice, the behavioral responses of MyD88ΔCNS mice to i.c.v. IL-1ß or intraperitoneal (i.p.) LPS were indistinguishable from those of WT mice. CONCLUSION: Sickness behavior is mediated by MyD88 and is dependent on the activity of cytokines within the brain. Our results demonstrate that MyD88 is not required in neurons or astrocytes to induce this behavioral response to IL-1ß or LPS. This suggests that a non-Nestin expressing cell population responds to IL-1ß in the CNS and transduces the signal to neurons controlling feeding and activity.