KIT D816V mast cells derived from induced pluripotent stem cells recapitulate systemic mastocytosis transcriptional profile
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ABSTRACT:
PROVIDER: PRJNA928728 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23254561_1.fastq.gz | Fastqsanger.gz | |||
| SRR23254561_2.fastq.gz | Fastqsanger.gz | |||
| SRR23254562_1.fastq.gz | Fastqsanger.gz | |||
| SRR23254562_2.fastq.gz | Fastqsanger.gz | |||
| SRR23254563_1.fastq.gz | Fastqsanger.gz |
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Project description:Mast cells (MC) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MC occur in low abundance which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells of the body and established a robust protocol for human iPS cell differentiation towards MC. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MC that recapitulate SM disease features: increased number of MC, abnormal maturation kinetics and activated phenotype, CD2 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MC are a reliable and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.
2023-04-05 | GSE223883 | GEO
Project description:Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated CDC25c depletion and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DNA damage response in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.
2018-03-30 | GSE111060 | GEO
Project description:The HMC-1.1 and 1.2 human mast cell (huMC) lines are often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. Both cell lines express mutated and constitutively active KIT, an essential growth factor receptor for huMC survival and function, with one KIT mutation in HMC-1.1 cells (V560G-KIT) and two in HMC-1.2 cells (D816V-KIT and V560G-KIT). D816V-KIT is commonly associated with systemic mastocytosis, while V560G-KIT appears more frequently in other human neoplasms. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9 engineering to reverse the V560G-KIT mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single monoallelic D816V-KIT mutation. Transcriptome analyses of differentially expressed genes in HMC1.3 compared to HMC-1.2 predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable, albeit increased compared to HMC-1.1 cells. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells but increased compared to HMC-1.1 cells. Despite these similarities, survival of HMC-1.3 cells in liquid culture was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study reveals that the additional V560G-KIT variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.
2023-03-09 | GSE216446 | GEO
Project description:Global phosphoproteomics can reveal signaling cascades in unprecedented detail. The SCF/KIT axis represents a vital hematopoietic receptor tyrosine kinase (RTK) system with gain-of-function mutations prompting the development of mastocytosis and leukemia. Here, we employed a label-free phosphoproteomics profiling strategy to provide the first comprehensive portrayal of SCF-triggered phosphosignaling. Employing untransformed skin-derived mast cells expressing vast amounts of KIT, we uncover around 10,500 class-I-phosphosites following ligand-induced KIT dimerization. Apart from STAT5, the MEK/ERK cascade (including upstream and downstream events) stood out as highly stimulatable by SCF compared to less impacted PI3K/Akt>p38>JNK, a pattern validated by time-resolved immunoblotting. Direct comparison between MEK/ERKÕs and PI3KÕs support of basic programs (anti-apoptosis, cell cycle progression) revealed equipotency with substantial redundancy between the modules. In functional outputs, ERK showed dominance over PI3K regarding cytokine stimulation (TNF-a, IL-8, LIF, OSM) and was the only contributor in the induction of immediate early genes (Fos, JunB, Egr1). Our proteome-wide screen also found Å1,000 phosphosites currently undescribed in databases, pertaining to multiple protein classes. Among candidates, capicua (and components of its network) experienced massive phosphorylation by SCF. Perturbation with capicua by RNA interference unveiled its function as a potent KIT repressor, whose deregulation may contribute to the development of mastocytosis. We demonstrate the utility of an unbiased approach to uncover single protein entities and entire networks activated by RTKs, that can be surveyed experimentally. This rich resource enhances our understanding of RTK-elicited signaling networks and can serve as a benchmark to identify pathological processes, e.g. under circumstances of KIT deregulation.
2022-11-15 | PXD028252 | Pride
Project description:We investigated the metabolic regulation of mast cell (MC) functions to control them. MCs are sentinels of the immune system with functions involved not only in host defense but also, if not properly controlled, in MC disorders such as systemic mastocytosis (SM). We identified N-acetyl-D-glucosamine (GlcNAc or NAG) as both a circulating biomarker for SM severity and an oncometabolite that promotes uncontrolled degranulation and proliferation of MCs expressing the Kit kinase domain mutant allele, D816V, that is both an oncogene and characterizes patients with SM. Given the known role of GlcNAc-related protein glycosylation in modifying factors involved in epigenetic regulation of gene expression and the effects of GlcNAc on neoplastic MCs effector functions, we asked whether the increased susceptibility of KIT D816V MCs to GlcNAc was due to chromatin state changes.
2023-04-20 | GSE149106 | GEO
Project description:Transcriptional profiling of pooled adult kidneys from transgenic zebrafish expressing human KIT-D816V mutant gene versus wild-type zebrafish was performed. The aim of this experiment was to determine the expression and cellular changes caused by expression of KIT-D816V oncogene in the hematopoietic organ in zebrafish. This transgenic zebrafish represents a model of systemic mastocytosis with many features of this disease present in adults
2014-02-03 | GSE54614 | GEO
Project description:Transcriptional profiling of 28 hpf zebrafish transgenic expressing human KIT-D816V mutant gene versus wild-type embryos was performed. The aim of this experiment was to determine the expression changes caused by expression of KIT-D816V oncogene. This transgenic zebrafish represents a model of systemic mastocytosis with many features of this disease present in adults, but no very clear phenotypes in embryos. Thus, we attempted to determine if there are certain expression changes, which can be used as molecular features of active KIT expressed in these embryos.
2014-02-03 | GSE54613 | GEO
Project description:Transcriptional profiling of 28 hpf zebrafish transgenic expressing human KIT-D816V mutant gene versus wild-type embryos was performed. The aim of this experiment was to determine the expression changes caused by expression of KIT-D816V oncogene. This transgenic zebrafish represents a model of systemic mastocytosis with many features of this disease present in adults, but no very clear phenotypes in embryos. Thus, we attempted to determine if there are certain expression changes, which can be used as molecular features of active KIT expressed in these embryos. Two-condition experiment, Tg(actb2:KIT-D816V) versus wild-type(AB), 4 replicate experiments with a dye-swap each time
2014-02-03 | E-GEOD-54613 | biostudies-arrayexpress
Project description:Transcriptional profiling of pooled adult kidneys from transgenic zebrafish expressing human KIT-D816V mutant gene versus wild-type zebrafish was performed. The aim of this experiment was to determine the expression and cellular changes caused by expression of KIT-D816V oncogene in the hematopoietic organ in zebrafish. This transgenic zebrafish represents a model of systemic mastocytosis with many features of this disease present in adults Two-condition experiment, Tg(actb2:KIT-D816V) versus wild-type(AB), one replicate of each prepared from 3 adult kidneys of each genotype and a dye-swap hybridization of these RNA samples.
2014-02-03 | E-GEOD-54614 | biostudies-arrayexpress
Project description:Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT-containing EVs have not been thoroughly investigated. Here, we isolated KIT-EV subpopulations released by neoplastic huMCs using an immunocapture approach that selectively isolates EVs containing KIT in its proper topology. Proteomic profiling was performed on KIT-containing EVs compared to KIT-depleted EV populations, and on KIT-EVs that are microvesicle- and exosome-like.
2023-03-10 | PXD033889 | Pride