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HMC-1.1 cells were rather low and difficult to quantify by Western blotting, we also performed intracellular flow cytometry-staining experiments using an antibody against pSTAT5. In these experiments, DCC-2618 was found to counteract pSTAT5 expression in HMC-1.1 and HMC-1.2 cells in a dose-dependent manner (Online Supplementary Figure S1). DCC-2618 did not inhibit phosphorylation of BTK, another important target of tyrosine kinase inhibitors expressed by neoplastic MC (Figure 2C).
DCC-2618 induces apoptosis in neoplastic mast cells
To explore the mechanism of drug action, we analyzed the effects of DCC-2618 on the survival of neoplastic MC. As assessed by light microscopy, DCC-2618 induced apoptosis in HMC-1.1, HMC-1.2, ROSAKIT WT, ROSAKIT D816V and ROSA KIT K509I cells in a dose-dependent manner (Figure 3A). The effects of DCC-2618 on survival were more pronounced in KIT D816V-negative MC lines than in KIT D816V-positive MC lines (Figure 3A). DCC-2618 was also found to produce apoptosis in the multi-resis- tant MCPV-1 cell lines (Figure 3B). The apoptosis-induc- ing effect of DCC-2618 on MC was confirmed by com- bined annexin V/propidium-iodide staining (Figure 3A,B
A
and Online Supplementary Figure S2A,B). The metabolite DP-5439 was found to be equally effective in producing apoptosis in MC lines compared to DCC-2618 (Figure 3A,B and Online Supplementary Figure S2A,B). Together, these data show that DCC-2618 is a novel potent anti- neoplastic compound inducing apoptosis and growth arrest in neoplastic MC.
DCC-2618 produces synergistic growth-inhibitory effects with midostaurin and cladribine (2CdA) in neoplastic mast cells
In advanced SM, drug combinations may be required to suppress malignant cell growth. We found that DCC-2618 and midostaurin produce clear cooperative (synergistic) growth-inhibitory effects in HMC-1.1 cells (Online Supplementary Figure S3A,C). In HMC-1.2 cells, the drug combination also produced cooperative antineoplastic effects, but these effects were additive rather than syner- gistic as defined by Calcusyn software (Online Supplementary Figure S3A,C). In addition, we found that DCC-2618 and 2CdA induce clear synergistic growth- inhibitory effects on HMC-1.1 and HMC-1.2 cells (Online Supplementary Figure S3B,D).
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Figures 1. DCC-2618 and its active metabolite DP-5439 inhibit proliferation of neoplastic mast cells. HMC-1, ROSA (A), MCPV-1 (B) and primary neoplastic mast cells (C) obtained from patients with different variants of systemic mastocytosis (ISM, SSM, ASM, MCL) were incubated in control medium (0 nM) or medium con- taining various concentrations of DCC-2618 or DP-5439, as indicated, at 37°C for 48 h. Thereafter, 3H-thymidine uptake was measured. Results in (A) and (B) are expressed as percent of control and represent the mean±S.D. from three independent experiments. Results in (C) are expressed as percent of control and represent mean±S.D from triplicates. Asterisk (*): P<0.05 compared to control medium.
haematologica | 2018; 103(5)