Contributions
Abstract: EP1066
Type: E-Poster Presentation
Session title: Myeloproliferative neoplasms - Biology & Translational Research
Background
Systemic mastocytosis (SM) is a hematologic stem cell neoplasm characterized by uncontrolled growth and accumulation of neoplastic mast cells (MC) in one or more organs. In a majority of patients with SM, the D816V-mutated variant of KIT is detectable which induces resistance against a number of tyrosine kinase inhibitors (TKI), including imatinib. Midostaurin is a TKI which is known to target KIT D816V. This drug has been approved for the treatment of patients with advanced SM by the FDA and EMA. However, long-lasting hematologic remissions are rarely seen and patients treated with midostaurin may also relapse.
Aims
The aim of our study was to identify novel potential drug targets and associated targeted drugs to overcome mutant-induced drug resistance of neoplastic MC.
Methods
For this purpose, a robot-based high-throughput cell viability screen using 8 different MC lines, including the KIT D816V+ cell lines HMC-1.2 and ROSAKIT D816V was performed.
Results
We identified a number of drugs effectively blocking survival of KIT D816V+ MC lines. Among these, HDAC blockers were the most potent inhibitors. The two most promising HDAC blockers, panobinostat and quisinostat, were used in subsequent validation experiments. Both HDAC inhibitors were found to block proliferation of HMC-1.1 (IC50: 5-25 nM), HMC-1.2 (IC50: 2.5-25 nM), ROSAKIT WT (IC50: 2.5-25 nM), and ROSAKIT D816V cells (IC50: 5-50 nM) as well as the multi-drug resistant cell lines MCPV-1.1, MCPV-1.2, MCPV-1.3 and MCPV-1.4 (IC50: 10-100 nM). Moreover, panobinostat and quisinostat were found to suppress proliferation of primary neoplastic MC obtained from patients with advanced SM (IC50: <10 nM). The two HDAC inhibitors were also found to induce apoptosis in HMC-1 cells. Moreover, combinations of HDAC blockers (panobinostat or quisinostat) and KIT-targeting drugs (midostaurin or avapritinib) produced cooperative growth-inhibitory effects in both HMC-1 subclones as well as ROSAKIT WT and ROSAKIT D816V cells. As assessed by qPCR, all MC-lines tested as well as primary neoplastic MC obtained from patients with various types of SM were found to express HDAC1 and HDAC2 mRNA. Furthermore, all cell lines investigated were found to display HDAC1 and HDAC2 which was evaluated by immunocytochemistry. In Western blot experiments, both HDAC inhibitors were found to promote the expression of acetylated histone H3 and H4 in HMC-1 and ROSA cells. By contrast, no effects of these drugs on phosphorylation of KIT were seen.
Conclusion
HDAC inhibitors are novel potent inhibitors of growth of neoplastic MC. Whether HDAC inhibitors alone or in combination with midostaurin or avapritinb are also effective in vivo in patients with advanced SM remains to be determined in clinical trials.
Keyword(s): Drug resistance, HDAC inhibitor, Mastocytosis, Targeted therapy
Abstract: EP1066
Type: E-Poster Presentation
Session title: Myeloproliferative neoplasms - Biology & Translational Research
Background
Systemic mastocytosis (SM) is a hematologic stem cell neoplasm characterized by uncontrolled growth and accumulation of neoplastic mast cells (MC) in one or more organs. In a majority of patients with SM, the D816V-mutated variant of KIT is detectable which induces resistance against a number of tyrosine kinase inhibitors (TKI), including imatinib. Midostaurin is a TKI which is known to target KIT D816V. This drug has been approved for the treatment of patients with advanced SM by the FDA and EMA. However, long-lasting hematologic remissions are rarely seen and patients treated with midostaurin may also relapse.
Aims
The aim of our study was to identify novel potential drug targets and associated targeted drugs to overcome mutant-induced drug resistance of neoplastic MC.
Methods
For this purpose, a robot-based high-throughput cell viability screen using 8 different MC lines, including the KIT D816V+ cell lines HMC-1.2 and ROSAKIT D816V was performed.
Results
We identified a number of drugs effectively blocking survival of KIT D816V+ MC lines. Among these, HDAC blockers were the most potent inhibitors. The two most promising HDAC blockers, panobinostat and quisinostat, were used in subsequent validation experiments. Both HDAC inhibitors were found to block proliferation of HMC-1.1 (IC50: 5-25 nM), HMC-1.2 (IC50: 2.5-25 nM), ROSAKIT WT (IC50: 2.5-25 nM), and ROSAKIT D816V cells (IC50: 5-50 nM) as well as the multi-drug resistant cell lines MCPV-1.1, MCPV-1.2, MCPV-1.3 and MCPV-1.4 (IC50: 10-100 nM). Moreover, panobinostat and quisinostat were found to suppress proliferation of primary neoplastic MC obtained from patients with advanced SM (IC50: <10 nM). The two HDAC inhibitors were also found to induce apoptosis in HMC-1 cells. Moreover, combinations of HDAC blockers (panobinostat or quisinostat) and KIT-targeting drugs (midostaurin or avapritinib) produced cooperative growth-inhibitory effects in both HMC-1 subclones as well as ROSAKIT WT and ROSAKIT D816V cells. As assessed by qPCR, all MC-lines tested as well as primary neoplastic MC obtained from patients with various types of SM were found to express HDAC1 and HDAC2 mRNA. Furthermore, all cell lines investigated were found to display HDAC1 and HDAC2 which was evaluated by immunocytochemistry. In Western blot experiments, both HDAC inhibitors were found to promote the expression of acetylated histone H3 and H4 in HMC-1 and ROSA cells. By contrast, no effects of these drugs on phosphorylation of KIT were seen.
Conclusion
HDAC inhibitors are novel potent inhibitors of growth of neoplastic MC. Whether HDAC inhibitors alone or in combination with midostaurin or avapritinb are also effective in vivo in patients with advanced SM remains to be determined in clinical trials.
Keyword(s): Drug resistance, HDAC inhibitor, Mastocytosis, Targeted therapy