DataSheet_1_CRISPR/Cas9-engineering of HMC-1.2 cells renders a human mast cell line with a single D816V-KIT mutation: An improved preclinical model for research on mastocytosis.pdf

The HMC-1.2 human mast cell (huMC) line is 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. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9-engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single mono-allelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells, 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. 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. Despite these similarities in liquid culture, survival of HMC-1.3 cells 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 thus reveals that the additional V560G-KIT oncogenic 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.</p

table_1.PDF

<p>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 (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 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 isoforms 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 DDR 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 the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.</p

table_2.PDF

<p>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 (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 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 isoforms 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 DDR 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 the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.</p

data_sheet_1.PDF

<p>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 (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 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 isoforms 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 DDR 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 the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.</p

Adoptive transfer of mastocytoma P815 murine cells into DBA/2 mice causes serum alterations in DJ-1 and ROS that are reversed by anti-IL-6R antibody treatment.

<p>(A-C) Serum levels of IL-6 (A), ROS (B), and DJ-1 (C) in mice injected with P815 cells and given daily injections of 200 μg/mouse of anti-IL-6R antibody, isotype control antibody or PBS on days 10 through 16 as indicate. (D) Representative histologic images of dorsal skin and spleen samples stained with alcian blue/safranin to detect mast cell in naïve and P185-injected mice untreated or treated with either an isotype control or anti-IL-6R antibody. Below, average number of mast cells per field (5 fields/mouse, n = 4–5) (Axioskop2 plus microscope, total magnification 400X). (E) Representative plots of APC-KIT positive cells (P815 cells lack FcεRI in culture) and APC-KIT and PE-FcεRI double positive cells from blood sorted by FACS in the same experiment. Shown below are the average percentages of mast cells (KIT and FcεRI double positive) in circulation from the various groups of mice. The percentages of KIT positive cells in circulation were as follows: naïve: 0.36±0.07; P815-PBS: 0.91±0.03; P815-Isotype Ab: 0.58±0.03; P815-Anti-IL-6R Ab: 0.21±0.09. (F) Body weights of mice before adoptive transfer of P815 cells and at day 16 after transfer in all the indicated groups. Values represent mean ± SEM of a representative experiment using 4–5 mice per condition. *<i>P</i><0.05 and **<i>P</i><0.01. The experiment was repeated twice with similar results.</p

IL-6 increases extracellular ROS levels in HMC-1 cells.

<p>(A) Changes in intracellular (left) and extracellular ROS (right) levels induced by IL-6 (50 ng/ml) in HMC-1.2 cells compared to LAD2 cells. LAD2 were pre-stimulated with SCF for 48 h (100 ng/ml) prior to IL-6 stimulation. (B) Changes in intracellular (left) and extracellular ROS (right) levels induced by 50 ng/ml IL-6 in P815 cells compared to normal BMMC. ROS was measured using a fluorescence assay. All experiments were repeated at least 3 times and data represents mean±SEM. <i>*P<0</i>.<i>05</i> and <i>**P<0</i>.<i>01</i>.</p

Proposed mechanisms for the alterations in ROS and DJ-1 homeostasis in SM.

<p>ISM patients with lower mast cell burden, tryptase values and IL-6 levels show a high ratio of ROS to DJ-1 (>22) as compared to healthy controls (<4) due to their reduced levels of DJ-1. This is driven by constitutive KIT activity in <i>KIT</i> mutated MCs which induces ROS production and causes oxidation and proteosomal degradation of oxidized DJ-1 (left panel). In ISM patients with higher mast cell burden and with advanced disease, the increasing levels of DJ-1 offset the ROS to DJ-1 ratio toward normal values (<8; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162831#pone.0162831.g001" target="_blank">Fig 1C</a>). This is driven by elevated levels of IL-6 in these patients, which causes DJ-1 transcription and compensates for the enhanced DJ-1 turnover. Reduction of oxidative imbalances may allow for MC expansion.</p

Constitutive KIT activation in cells with gain-of function KIT mutations causes increased ROS levels and decreased DJ-1.

<p>(A) Comparison of the intracellular levels of ROS (left panel) and (B) oxidized DJ-1 (Cys 106) and total DJ-1 in human mast cells carrying normal <i>KIT</i> (LAD2) or mutated <i>KIT</i> (HMC-1.1 and HMC-1.2). DJ-1 expression was silenced in HMC-1.2 using specific sh-RNA sequences to target DJ-1 (DJ-1 KD) or sh-RNA non-target control sequences (sh-RNA Con) (B, right panels). ROS levels in DJ-1 KD and sh-RNA Con cells were measured side by side, but for simplicity, ROS levels in sh-RNA Con are not shown in A since the levels were identical to untreated HMC-1.2 cells (sh-RNA Con: 4.24 μM±0.075 μM). Levels of ROS (C) and DJ-1 (D) in murine MCBS1 mast cells transduced with WT-human <i>KIT</i>, D816V-human <i>KIT</i> or empty vector as indicated. Presence of KIT and phosphorylated KIT in cells with D816V are shown by western blot (D, bottom panels). The values under the blots indicate average fold increases in the band intensities of DJ-1 or oxidized DJ-1 (corrected to their respective β-actin loading control) as compared to non-stimulated cells (n = 3); SDs were less than 10% of the mean</p

Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis

<div><p>Hermansky-Pudlak Syndrome type-1 (HPS-1) is an autosomal recessive disorder caused by mutations in <i>HPS1</i> which result in reduced expression of the HPS-1 protein, defective lysosome-related organelle (LRO) transport and absence of platelet delta granules. Patients with HPS-1 exhibit oculocutaneous albinism, colitis, bleeding and pulmonary fibrosis postulated to result from a dysregulated immune response. The effect of the <i>HPS1</i> mutation on human mast cells (HuMCs) is unknown. Since HuMC granules classify as LROs along with platelet granules and melanosomes, we set out to determine if HPS-1 cutaneous and CD34+ culture-derived HuMCs have distinct granular and cellular characteristics. Cutaneous and cultured CD34+-derived HuMCs from HPS-1 patients were compared with normal cutaneous and control HuMCs, respectively, for any morphological and functional differences. One cytokine-independent HPS-1 culture was expanded, cloned, designated the HP proMastocyte (HPM) cell line and characterized. HPS-1 and idiopathic pulmonary fibrosis (IPF) alveolar interstitium showed numerous HuMCs; HPS-1 dermal mast cells exhibited abnormal granules when compared to healthy controls. HPS-1 HuMCs showed increased CD63, CD203c and reduced mediator release following FcɛRI aggregation when compared with normal HuMCs. HPM cells also had the duplication defect, expressed FcɛRI and intracytoplasmic proteases and exhibited less mediator release following FcɛRI aggregation. HPM cells constitutively released IL-6, which was elevated in patients’ serum, in addition to IL-8, fibronectin-1 (FN-1) and galectin-3 (LGALS3). Transduction with <i>HPS1</i> rescued the abnormal HPM morphology, cytokine and matrix secretion. Microarray analysis of HPS-1 HuMCs and non-transduced HPM cells confirmed upregulation of differentially expressed genes involved in fibrogenesis and degranulation. Cultured HPS-1 HuMCs appear activated as evidenced by surface activation marker expression, a decrease in mediator content and impaired releasibility. The near-normalization of constitutive cytokine and matrix release following rescue by <i>HPS1</i> transduction of HPM cells suggests that HPS-1 HuMCs may contribute to pulmonary fibrosis and constitute a target for therapeutic intervention.</p></div

Characteristics of HPM cells.

<p>A) Representative dot plots of HPM clones show that HPM cells are FcɛRI+/CD117^low/-; B) Histamine content of HPM clones ranges from 0.1–0.3 pg/cell; C) Representative flow cytometry from clone #4 of permeabilized HPM cells in the presence (blue line) or absence (yellow line) of rhSCF and rhIL-6 shows similar expression of tryptase, chymase and carboxypeptidase; D) β-Hex release (upper graph) of HPM clones #3 and #4 at 1 ng/ml antigen is minimum and ranged from 3–5% following crosslinking with antigen. In the presence of 0.1 ng/ml Ag, the addition of increasing concentrations of SCF does not enhance release (lower graph) from either clone. Data are from 2 separate experiments performed in duplicate (A-D); E) Thapsigargin but not FcɛɛRI crosslinking with SA induces calcium influx in HPM clones; F) In contrast to low migration of normal (red column) and HPS-1 (white column) HuMCs to all concentrations of SCF, representative chemotaxis of HPM clone #4 shows HPM migrated cell numbers (black column) are significantly elevated and show increasing migration to increasing concentrations of SCF. Data are representative of 2 separate experiments performed in duplicate (E-F), and G) Representative light microscopy of extracellular matrix formation in HPM clones 3 and 4 (arrow–matrix).</p