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Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells with D816V-KIT

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE111060 | GEO | 2018/03/30

REPOSITORIES: GEO

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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.

Project description:Background: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. Sphingosine-1-phosphate (S1P) is a lipid mediator that regulates immune cell trafficking by signaling via five G protein-coupled receptors (S1PRs). We investigated the role of S1P in the RTM of aortic intimal MCs. Methods: Intravenous injection of lipopolysaccharide (LPS) was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. Results: In wild-type C57BL/6 mice, LPS induced intimal cell expression of S1pr1, S1pr3, and sphingosine kinase 1 (Sphk1, a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked LPS-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked LPS-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima and blunted LPS-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and LPS-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1 +/+ and -/- bone marrow. Stimulation with LPS increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. Conclusions: Functional and expression studies support a novel role for S1P signaling in the regulation of LPS-induced RTM as well as the homeostatic maintenance of aortic intimal MCs. Our data provides insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

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Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells with D816V-KIT

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