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DCC-2618: a new drug against mastocytosis
informed consent had been given. Patients were classified as having indolent SM (ISM; n=3), smoldering SM (SSM; n=1), ASM (n=2), SM-AHN (n=4) and MCL (n=1) according to WHO criteria.8-11 In addition, neoplastic cells were obtained from ten patients suffering from CMML, ten with AML and three with hypereosinophilia. The patients’ characteristics are shown in Table 1 (SM patients) and Online Supplementary Table S1 (other hematologic disorders). Heparinized bone marrow cells were layered over Ficoll to isolate mononuclear cells. The study was approved by the ethics committee of the Medical University of Vienna.
Culture of human cell lines
The following human MCL-like cell lines were employed in this study: HMC-1.1 and HMC-1.2,37 three ROSA sub-clones (ROSAKIT WT, ROSAKIT D816V, ROSAKIT K509I)38 and four MCPV-1 sub- clones (MCPV-1.1, MCPV-1.2, MCPV-1.3, MCPV-1.4).39 In addi- tion, we examined several AML cell lines, the CEL-related cell line EOL-1, the microvascular endothelial cell line HMEC-1, and cultured human umbilical vein endothelial cells (HUVEC). A description of cell lines is provided in the Online Supplement.
Evaluation of growth, survival of neoplastic cells
Drug-exposed cells (cell lines or primary cells) were analyzed for proliferation and survival. The bioassays employed are described in the Online Supplementary Methods.
Western blotting
For evaluation of KIT and BTK signaling, HMC-1.1, HMC-1.2, ROSAKIT WT and ROSAKIT D816V cells were incubated in control medium or in DCC-2618 (0.5-5 μM) for 4 h at 37°C. Western blotting was performed essentially as described elsewhere.26,40 For evaluation of downstream signaling pathways of KIT, HMC- 1.1, HMC-1.2, ROSAKIT WT and ROSAKIT D816V cells were first pre- incubated overnight in Iscove modified Dulbecco medium devoid of fetal calf serum and of stem cell factor. Cells (106) from each line were then treated with DCC-2618 (0.001-10 μM) for 90 min at 37°C. At the end of the treatment, ROSAKIT WT cells were stimulated with stem cell factor-containing supernatants (10%) of Chinese hamster ovary cells transfected with the murine scf (kl) gene (CHO-KL) at room temperature for 10 min. Thereafter, Western blotting was performed essentially as described previously.26,40 Antibodies against phosphorylated (p)Kit, STAT5, pSTAT5, ERK1/2, pERK1/2 were purchased from Cell Signaling (Danvers, MA, USA), antibodies against pBTK were bought from NovusBiologicals (Littleton, CO, USA) and antibodies against total KIT and total BTK were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Measurement of mediator release
Drug-exposed cells (blood basophils obtained from healthy individuals and HMC-1 cells) were analyzed for histamine- and tryptase release as described in the Online Supplementary Methods.
Evaluation of apoptosis in basophils
Drug-exposed blood basophils obtained from healthy donors by dextran sedimentation were analyzed for cell survival by flow cytometry. Technical details are described in the Online Supplementary Methods.
Statistical analysis
To determine the level of significance the Student t-test was applied. Results were considered to be statistically significantly different when P was <0.05.
Results
DCC-2618 and its metabolite DP-5439 inhibit proliferation of neoplastic mast cells
DCC-2618 and its active metabolite, DP-5439 were found to inhibit 3H-thymidine uptake and thus prolifera- tion in a dose-dependent manner in all MC lines tested, with slightly lower IC50 values obtained in HMC-1.1 cells lacking KIT D816V and ROSAKIT WT cells compared to the KIT D816V-positive cell lines HMC-1.2 and ROSAKIT D816V (Figure 1A and Table 2). IC50 values obtained in HMC-1.1 cells with DCC-2618 were also lower than IC50 values obtained with midostaurin.25,26 In addition, DCC-2618 and DP-5439 were found to inhibit proliferation of ROSAKIT K509I cells with lower IC50 values (DCC-2618, IC50: 34±10 nM) compared to ROSAKIT D816V cells (Figure 1A). Unexpectedly, DCC-2618 and its metabolite also produced growth-inhi- bition in the multi-resistant MC lines MCPV-1.1, MCPV- 1.2, MCPV-1.3 and MCPV-1.4 (Figure 1B and Table 2). Finally, we were able to show that DCC-2618 and DP- 5439 induced dose-dependent inhibition of growth of pri- mary neoplastic bone marrow cells obtained from patients suffering from various forms of SM, including ASM and MCL (Figure 1C, Table 1). Interestingly, the effects of DCC-2618 on primary neoplastic BM cells and the related IC50 values obtained in different SM variants were compa- rable (Figure 1C, Table 1).
DCC-2618 inhibits KIT, STAT5, AKT, and ERK activation in neoplastic mast cells
As expected, DCC-2618 was found to suppress phos- phorylation of KIT in ROSAKIT WT and ROSAKIT D816V cells as well as in both HMC-1 sub-clones (Figure 2A). In addition, DCC-2618 was found to decrease the expression of phos- phosphorylated (p)STAT5, pAKT and pERK1/2 in all cell lines tested (Figure 2B). As expression levels of pSTAT5 in
Table 2. Effects of DCC-2618 and DP-5439 on growth of various human cell types.
Cell line / cell type
HMC-1.1
HMC-1.2
ROSA KIT WT
ROSA KIT D816V
ROSA KIT K509I
MCPV-1.1
MCPV-1.2
MCPV-1.3
MCPV-1.4
MOLM-13
MV4-11
KG-1
U937
EOL-1
HMEC-1
HUVEC
DCC-2618, IC50
12.3±3.7 nM
123±36 nM
41±5 nM
168±65 nM
34±10 nM
298±77 nM
1000±932 nM
724±511 nM
670±418 nM
132±95 nM
DP-5439, IC50
13±4 nM
96±23 nM
56±21 nM
188±60 nM
28±13 nM
357±179 nM
913±378 nM
756±435 nM
697±410 nM
73±31 nM
147±88 nM
76±24 nM
2.7±3.7 μM
5.2±4.4 μM
7.8±5.4 μM
2.5±0.3 μM
1.8±1.3 nM
1±0.8 nM
3.7±2.2 μM
2±1.3 μM
haematologica | 2018; 103(5)
707±224 nM
605±222 nM
Cell lines were incubated with various concentrations of DCC-2618 or DP-5439 at 37°C for 48 h.Then, proliferation was determined by measuring uptake of 3H-thymidine and IC50 values were calculated. Values represent the mean±S.D. from three independent experiments. IC50: half maximal inhibitory concentration.
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