YTX treatment induced apoptosis in B16F10 cells.

<p>B16F10 cells were either left untreated or were treated for indicated times with 10, 30, 50 and 100nM YTX. YTX solvent controls were performed and no effects were observed. Cells were then analyzed for Annexin-V and PI staining by flow cytometry and the percentage of live cells (Annexin V-FITC -/PI -), early apoptotic (Annexin V-FITC +/PI -) and late apoptotic or necrotic cells (Annexin V-FITC +/PI +) was determined. Mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.</p

YTX treatment induced apoptosis and necrosis in the RBL-2H3 cell line.

<p>RBL-2H3 cells were either left untreated or were treated for indicated times with 10, 30, 50 and 100nM YTX. YTX solvent controls were performed and no effects were observed. Cells were then analysed for Annexin V-FITC and PI staining by flow cytometry and the percentage of live cells (Annexin V-FITC -/ PI -), early apoptotic (Annexin V-FITC +/PI -) and late apoptotic or necrotic cells (Annexin V-FITC +/PI +) was determined. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.</p

Effect of YTX on MEC1 and B16F10 cell line viability.

<p>The MEC1 and B16F10 cell lines were incubated with 10, 30, 50 and 100nM YTX for the indicated times and cell viability was assessed by the MTT assay at the indicated time points. Corresponding controls with YTX solvent were performed and cell viability was arbitrarily set to 100%. Of note, solvent did not significantly affect cell viability as compared to non-treated cells even at the highest concentration of vehicle. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.</p

YTX treatment decreases tumour development in the B16F10 melanoma mouse model.

<p>(A) Mice were injected subcutaneous B16F10 melanoma cells and tumours were allowed to develop until they reached a volume of 50 mm³ achieved between days 5 and 10. Mice were then either left untreated or were treated with the indicated concentrations of YTX or vehicle, which was injected subcutaneous right next to the tumour. (B) Representative glucose uptake after FX Pro Kodak image analysis of untreated, vehicle and YTX-treated mice at the day of sacrifice. (C) Corresponding resected tumour weight of animals after sacrifice at day 12 post-1st treatment. (D) Corresponding weight of untreated, vehicle- and YTX-treated mice. (E-G) Results of the haematological analysis performed before mice sacrifice. Data are expressed as mean ± SEM. Significant differences between untreated and YTX: (*) p≤0.05 and (***) p≤0.001.</p

Effect of YTX on β-hexosaminidase release induced by IgE/Ag in RBL-2H3 and BMMCs.

<p>IgE-sensitized RBL-2H3 cells (A) or BMMCs (B) were incubated with 10 and 30nM YTX or vehicle (methanol) for 30 or 60min. Then YTX was removed (+) from the medium or not (-) and cells were stimulated for 45min with DNP-HSA antigen at the indicated concentrations. The percentage release of β-hexosaminidase was determined and compared to vehicle treated cells. No effect was observed after vehicle incubation. Data (percentage release) are the mean ± SEM of three experiments. Significant differences between DNP-HSA- (hatched) and DNP-HSA+YTX-treated cells: (*) p≤0.05.</p

Effect of YTX on RBL-2H3 and BMMCs cell viability.

<p>Cells were incubated with 10, 30, 50 and 100nM YTX for the indicated times and cell viability was assessed by the MTT assay. Corresponding controls with YTX solvent (methanol) were performed and cell viability was arbitrarily set to 100%. Of note, solvent did not significantly affect cell viability as compared to non-treated cells even at the highest concentration of vehicle. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.</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