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.

Characterization of human Fc alpha receptor transgenic mice: comparison of CD89 expression and antibody-dependent tumor killing between mouse strains.

Since mice do not express a homologue of the human Fc alpha receptor (FcαRI or CD89), a transgenic mouse model was generated in four different backgrounds (C57BL/6, BALB/c, SCID and NXG) expressing the FcαRI under the endogenous human promoter. In this study, we describe previously unknown characteristics of this model, such as the integration site of the FCAR gene, the CD89 expression pattern in healthy male and female mice and in tumor-bearing mice, expression of myeloid activation markers and FcγRs and IgA/CD89-mediated tumor killing capacity. In all mouse strains, CD89 expression is highest in neutrophils, intermediate on other myeloid cells such as eosinophils and DC subsets and inducible on, among others, monocytes, macrophages and Kupffer cells. CD89 expression levels are highest in BALB/c and SCID, lower in C57BL/6 and lowest in NXG mice. Additionally, CD89 expression on myeloid cells is increased in tumor-bearing mice across all mouse strains. Using Targeted Locus Amplification, we determined that the hCD89 transgene has integrated in chromosome 4. Furthermore, we established that wildtype and hCD89 transgenic mice have a similar composition and phenotype of immune cells. Finally, IgA-mediated killing of tumor cells is most potent with neutrophils from BALB/c and C57BL/6 and less with neutrophils from SCID and NXG mice. However, when effector cells from whole blood are used, SCID and BALB/c are most efficient, since these strains have a much higher number of neutrophils. Overall, hCD89 transgenic mice provide a very powerful model to test the efficacy of IgA immunotherapy against infectious diseases and cancer.

Enhancing neutrophil cytotoxicity of a panel of clinical EGFR antibodies by Fc engineering to IgA3.0.

The epidermal growth factor receptor (EGFR) plays an essential role in cellular signaling pathways that regulate cell growth, proliferation and survival, and is often found dysregulated in cancer. Several monoclonal IgG antibodies have been clinically tested over the years which exert their function via blocking the ligand binding domain (thereby inhibiting downstream signaling) and induction of Fc-related effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). However, these IgG antibodies do not optimally recruit neutrophils, by far the most abundant white blood cell population in humans. Therefore, we reformatted six therapeutic EGFR antibodies (cetuximab, panitumumab, nimotuzumab, necitumumab, zalutumumab, and matuzumab) into the IgA3.0 format, which is an IgA2 isotype that has been adapted for clinical application. Reformatting these antibodies preserved Fab-mediated functions such as EGFR binding, growth inhibition and ligand blockade. Additionally, whole leukocyte ADCC was significantly increased when using this panel of IgA3.0 antibodies compared to their respective IgG counterparts, with no major differences between IgA3.0 antibodies. In vivo, IgA3.0 matuzumab outperformed the other antibodies, resulting in the strongest suppression of tumor outgrowth in a long intraperitoneal model. We show that neutrophils are important for the suppression of tumor outgrowth. IgA3.0 matuzumab exhibited reduced receptor internalization compared to the other antibodies, possibly accounting for its superior in vivo Fc-mediated tumor cell killing efficacy. In conclusion, reformatting EGFR antibodies into an IgA3.0 format increased Fc-mediated killing while retaining Fab-mediated functions and could therefore be a good alternative for the currently available antibody therapies.

The Role of Cytokines in Neutrophil Development, Tissue Homing, Function and Plasticity in Health and Disease.

Neutrophils are crucial innate immune cells and comprise 50-70% of the white blood cell population under homeostatic conditions. Upon infection and in cancer, blood neutrophil numbers significantly increase because of the secretion of various chemo- and cytokines by, e.g., leukocytes, pericytes, fibroblasts and endothelial cells present in the inflamed tissue or in the tumor microenvironment (TME). The function of neutrophils in cancer has recently gained considerable attention, as they can exert both pro- and anti-tumorigenic functions, dependent on the cytokine milieu present in the TME. Here, we review the effect of cytokines on neutrophil development, tissue homing, function and plasticity in cancer and autoimmune diseases as well as under physiological conditions in the bone marrow, bloodstream and various organs like the spleen, kidney, liver, lung and lymph nodes. In addition, we address several promising therapeutic options, such as cytokine therapy, immunocytokines and immunotherapy, which aim to exploit the anti-tumorigenic potential of neutrophils in cancer treatment or block excessive neutrophil-mediated inflammation in autoimmune diseases.

Effective, Long-Term, Neutrophil Depletion Using a Murinized Anti-Ly-6G 1A8 Antibody.

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro- and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting antibodies or genetic "knockout" models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition, we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment.

PML-controlled responses in severe congenital neutropenia with ELANE-misfolding mutations.

Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.

RUNX1 Mutations in the Leukemic Progression of Severe Congenital Neutropenia.

Somatic RUNX1 mutations are found in approximately 10% of patients with de novo acute myeloid leukemia (AML), but are more common in secondary forms of myelodysplastic syndrome (MDS) or AML. Particularly, this applies to MDS/AML developing from certain types of leukemia-prone inherited bone marrow failure syndromes. How these RUNX1 mutations contribute to the pathobiology of secondary MDS/AML is still unknown. This mini-review focusses on the role of RUNX1 mutations as the most common secondary leukemogenic hit in MDS/AML evolving from severe congenital neutropenia (SCN).

Secondary CNL after SAA reveals insights in leukemic transformation of bone marrow failure syndromes.

Acquired aplastic anemia and severe congenital neutropenia (SCN) are bone marrow (BM) failure syndromes of different origin, however, they share a common risk for secondary leukemic transformation. Here, we present a patient with severe aplastic anemia (SAA) evolving to secondary chronic neutrophilic leukemia (CNL; SAA-CNL). We show that SAA-CNL shares multiple somatic driver mutations in CSF3R, RUNX1, and EZH2/SUZ12 with cases of SCN that transformed to myelodysplastic syndrome or acute myeloid leukemia (AML). This molecular connection between SAA-CNL and SCN progressing to AML (SCN-AML) prompted us to perform a comparative transcriptome analysis on nonleukemic CD34high hematopoietic stem and progenitor cells, which showed transcriptional profiles that resemble indicative of interferon-driven proinflammatory responses. These findings provide further insights in the mechanisms underlying leukemic transformation in BM failure syndromes.

Malignant Transformation Involving CXXC4 Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.

Rps14, Csnk1a1 and miRNA145/miRNA146a deficiency cooperate in the clinical phenotype and activation of the innate immune system in the 5q- syndrome.

RPS14, CSNK1A1, and miR-145 are universally co-deleted in the 5q- syndrome, but mouse models of each gene deficiency recapitulate only a subset of the composite clinical features. We analyzed the combinatorial effect of haploinsufficiency for Rps14, Csnk1a1, and miRNA-145, using mice with genetically engineered, conditional heterozygous inactivation of Rps14 and Csnk1a1 and stable knockdown of miR-145/miR-146a. Combined Rps14/Csnk1a1/miR-145/146a deficiency recapitulated the cardinal features of the 5q- syndrome, including (1) more severe anemia with faster kinetics than Rps14 haploinsufficiency alone and (2) pathognomonic megakaryocyte morphology. Macrophages, regulatory cells of erythropoiesis and the innate immune response, were significantly increased in Rps14/Csnk1a1/miR-145/146a deficient mice as well as in 5q- syndrome patient bone marrows and showed activation of the innate immune response, reflected by increased expression of S100A8, and decreased phagocytic function. We demonstrate that Rps14/Csnk1a1/miR-145 and miR-146a deficient macrophages alter the microenvironment and induce S100A8 expression in the mesenchymal stem cell niche. The increased S100A8 expression in the mesenchymal niche was confirmed in 5q- syndrome patients. These data indicate that intrinsic defects of the 5q- syndrome hematopoietic stem cell directly alter the surrounding microenvironment, which in turn affects hematopoiesis as an extrinsic mechanism.

ASPP2 controls epithelial plasticity and inhibits metastasis through ß-catenin-dependent regulation of ZEB1.

Epithelial to mesenchymal transition (EMT), and the reverse mesenchymal to epithelial transition (MET), are known examples of epithelial plasticity that are important in kidney development and cancer metastasis. Here we identify ASPP2, a haploinsufficient tumour suppressor, p53 activator and PAR3 binding partner, as a molecular switch of MET and EMT. ASPP2 contributes to MET in mouse kidney in vivo. Mechanistically, ASPP2 induces MET through its PAR3-binding amino-terminus, independently of p53 binding. ASPP2 prevents ß-catenin from transactivating ZEB1, directly by forming an ASPP2-ß-catenin-E-cadherin ternary complex and indirectly by inhibiting ß-catenin's N-terminal phosphorylation to stabilize the ß-catenin-E-cadherin complex. ASPP2 limits the pro-invasive property of oncogenic RAS and inhibits tumour metastasis in vivo. Reduced ASPP2 expression results in EMT, and is associated with poor survival in hepatocellular carcinoma and breast cancer patients. Hence, ASPP2 is a key regulator of epithelial plasticity that connects cell polarity to the suppression of WNT signalling, EMT and tumour metastasis.