Characterising neutrophil subtypes in cancer using scRNA sequencing demonstrates the importance of IL-1β/CXCR2 axis in generation of metastasis specific neutrophils

Neutrophils are a highly heterogeneous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy has not been performed. We utilized single-cell RNA sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and developmental lineages in health and malignancy. Datasets of lung, breast, and colorectal cancer were integrated to establish and validate neutrophil gene signatures. Pseudotime analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand–receptor interactions and signaling pathways between neutrophils and other immune cell populations in primary colorectal cancer and metastatic colorectal cancer were investigated. We define two main neutrophil subtypes in primary tumors: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumor-specific subtype. This signature is conserved in murine and human cancer, across different tumor types. In colorectal cancer metastases, neutrophils are more heterogeneous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL1β/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Functional analysis of neutrophil-tumoroid cocultures and T-cell proliferation assays using orthotopic metastatic mouse models lacking Cxcr2 in neutrophils support our transcriptional analysis. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by the IL1β/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of neutrophil transcriptomic programming could optimize immunotherapeutic interventions into early and late interventions, targeting different neutrophil states

Contribution of the GATA2 transcription factor to the development and progression of myeloid disorders

The GATA2 transcription factor has an essential role in the proliferation and differentiation of hematopoietic cells. GATA2 contributes to myeloid malignancies through various mechanisms, including GATA2 overexpression in acute myeloid leukemia (AML), somatic GATA2 mutations in chronic myeloid leukemia in blast crisis and AML with biallelic CEBPA mutations, and germline mutations of GATA2, associated with four related familial syndromes that predispose to AML, among other myeloid malignancies. We hypothesized that deregulation of either GATA2 expression or GATA2 aberrant function caused by mutations could alter key downstream target genes, contributing to the pathogenesis of these diseases. Our general aim was to identify and functionally characterize novel target genes of the GATA2 transcription factor in AML, and to assess the effect of the GATA2 mutated proteins in the transcription of known target genes of GATA2. The results indicate that MYB is a key transcriptional target of GATA2 in AML, and that leukemic cells depend on MYB to maintain their proliferative phenotype. Moreover, MYB and GATA2 expression is positively correlated in three independent cohorts of patients with AML. On the other hand, human GATA2 contributes to its own transcription, and GATA2 mutations p.Thr354Met, p.Thr355del and p.Arg396Gln are unable to activate the GATA2 promoter, causing a haploinsufficiency that may be responsible for GATA2 deficit in affected individuals. Moreover, the expression of genes with important functions in myeloid development is deregulated in a patient with p.Arg396Gln mutation, including GATA2. Finally, in silico and in vitro studies indicate that p.Arg396Gln is unable to retain the immature phenotype of hematopoietic stem and progenitor cells, and that p.Arg396Gln might contribute to alter the leukocyte profile in affected individuals

Overexpression of SET is a recurrent event associated with poor outcome and contributes to protein phosphatase 2A inhibition in acute myeloid leukemia

BACKGROUND: Protein phosphatase 2A is a novel potential therapeutic target in several types of chronic and acute leukemia, and its inhibition is a common event in acute myeloid leukemia. Upregulation of SET is essential to inhibit protein phosphatase 2A in chronic myeloid leukemia, but its importance in acute myeloid leukemia has not yet been explored. DESIGN AND METHODS: We quantified SET expression by real time reverse transcriptase polymerase chain reaction in 214 acute myeloid leukemia patients at diagnosis. Western blot was performed in acute myeloid leukemia cell lines and in 16 patients' samples. We studied the effect of SET using cell viability assays. Bioinformatics analysis of the SET promoter, chromatin immunoprecipitation, and luciferase assays were performed to evaluate the transcriptional regulation of SET. RESULTS: SET overexpression was found in 60/214 patients, for a prevalence of 28%. Patients with SET overexpression had worse overall survival (P<0.01) and event-free survival (P<0.01). Deregulation of SET was confirmed by western blot in both cell lines and patients' samples. Functional analysis showed that SET promotes proliferation, and restores cell viability after protein phosphatase 2A overexpression. We identified EVI1 overexpression as a mechanism involved in SET deregulation in acute myeloid leukemia cells. CONCLUSIONS: These findings suggest that SET overexpression is a key mechanism in the inhibition of PP2A in acute myeloid leukemia, and that EVI1 overexpression contributes to the deregulation of SET. Furthermore, SET overexpression is associated with a poor outcome in acute myeloid leukemia, and it can be used to identify a subgroup of patients who could benefit from future treatments based on PP2A activators

Overexpression of SET is a recurrent event associated with poor outcome and contributes to protein phosphatase 2A inhibition in acute myeloid leukemia

BACKGROUND: Protein phosphatase 2A is a novel potential therapeutic target in several types of chronic and acute leukemia, and its inhibition is a common event in acute myeloid leukemia. Upregulation of SET is essential to inhibit protein phosphatase 2A in chronic myeloid leukemia, but its importance in acute myeloid leukemia has not yet been explored. DESIGN AND METHODS: We quantified SET expression by real time reverse transcriptase polymerase chain reaction in 214 acute myeloid leukemia patients at diagnosis. Western blot was performed in acute myeloid leukemia cell lines and in 16 patients' samples. We studied the effect of SET using cell viability assays. Bioinformatics analysis of the SET promoter, chromatin immunoprecipitation, and luciferase assays were performed to evaluate the transcriptional regulation of SET. RESULTS: SET overexpression was found in 60/214 patients, for a prevalence of 28%. Patients with SET overexpression had worse overall survival (P<0.01) and event-free survival (P<0.01). Deregulation of SET was confirmed by western blot in both cell lines and patients' samples. Functional analysis showed that SET promotes proliferation, and restores cell viability after protein phosphatase 2A overexpression. We identified EVI1 overexpression as a mechanism involved in SET deregulation in acute myeloid leukemia cells. CONCLUSIONS: These findings suggest that SET overexpression is a key mechanism in the inhibition of PP2A in acute myeloid leukemia, and that EVI1 overexpression contributes to the deregulation of SET. Furthermore, SET overexpression is associated with a poor outcome in acute myeloid leukemia, and it can be used to identify a subgroup of patients who could benefit from future treatments based on PP2A activators

Supplementary Figure S3 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Figure S3. Scoring for the M_enriched signature in NSCLC primary tumour dataset and outgoing signals from cell populations in CRCLM.</p

Supplementary Figure S6 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Figure S6. L-R interactions from the significant signalling pathways that mediate communication between macrophages and other immune-cell subtypes in CRCLM.</p

TABLE 1 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Description of public datasets used in this study</p

Supplementary Figure S8 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Figure S8. CXCR2-lacking neutrophils from healthy mice exhibit no functional differences from wild type in co-culture with KPN organoids</p

Supplementary Tables from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Table S1: Mouse data for ScRNAsequencingTable S2: Primer sequences for qPCRTable S3. Top 5 markers in the integrated mouse dataset neutrophil clustersTable S4: Pseudotime Lineages of the Neutrophil datasetsTable S5: Lineage-specific differentialy expressed genes (Start Vs End) in PYMT dataset.Table S6: Lineage-specific differentialy expressed genes (Start Vs End) in CRC dataset.Table S7: Lineage-specific differentialy expressed genes (Start Vs End) in human NSCLC dataset.Table S8. Top 10 markers in the human CRCLM dataset neutrophil clustersTable S9: Top 10 Differentially expressed genes in neutrophils derived from primary tumors and metastatic tissue.Table S10: Differentially expressed genes in CRCLM neutrophilsTable S11: Differentially expressed genes in CD4 T cells in LM vs PTTable S12: Differentially expressed genes in CD8 T cells in LM vs PTTable S13: Differentially expressed genes in CD4 T cells in LM vs PT used in KEGG analysis(Threshold >3)Table S14: Description of the different neutrophil subtypes identified in CRCLM</p

Supplementary Figure S2 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Figure S2. Gene expression and neutrophil signatures in neutrophils from healthy and tumour tissue.</p