Contributions
Abstract: EP903
Type: E-Poster Presentation
Session title: Myelodysplastic syndromes - Biology & Translational Research
Background
Myelodysplastic syndrome (MDS) is the clonal myeloid neoplasm which is characterized by blood cytopenia and an increased risk of transformation to acute myelogenous leukemia (AML). About 30 to 40% of MDS patients develop AML and these patients are chemoresistance. Moreover, iron overload (IOL) often begins to develop in patients with MDS through repeated blood transfusion. Because the outcome of MDS is poor, alternative therapeutic strategies are required to improve the survival of MDS patients.
Aims
The inhibition of the DNA damage response pathway including Poly (ADP-ribose) polymerase 1 (PARP-1) in the treatment of cancer has recently approved in clinically. Wee1 is also an oncogenic nuclear kinase, which regulate the cell cycle as a crucial G2/M checkpoint. Because overexpression of Wee1 can be observed in various cancer types, Wee1 inhibitor could suppress MDS and AML cells in combination with PARP-1 inhibitor.
Methods
In this study, we investigated whether Wee1 was involved in MDS progress. We also investigated the efficacy of Wee1 inhibitor, MK-1775 and PARP1 inhibitor, talazoparib by using MDS and AML cell line, SKM-1, MDS-L, U937, THP-1, MV4;11 and osteoblastic cell line, MC3T3-E1.
Results
We first investigated the relationship between Wee1 expression and MDS patients by microarray gene expression data from the online Gene Expression Omnibus (GEO). Wee1 expression was increased in AML transformed cells and AML cells compared to refractory anemia with excess blasts cells (GSE14468). In contrast, PARP-1 expression was not changed. We next evaluated the effect of MK-1775 or talazoparib on proliferation of MDS and AML cell lines. 72 h treatment of MDS and AML cells were inhibited by MK-1775 or talazoparib in a dose dependent manner. Cellular cytotoxicity and caspase 3/7 activity was also increased. We next investigated the relationship between MK-1775 and talazoparib. We found that cotreatment with MK-1775 and talazoparib showed superior effect than single drug treatment. We found that cellular cytotoxicity, caspase 3/7 activity, intracellular reactive oxygen species (ROS) and beta-galactosidase staining positive cells were also increased after MK-1775 and talazoparib treatment. Cotreatment with MK-1775 and talazoparib reduced colony formation. Cell cycle analysis showed significant cell population in sub G1 and induced apoptosis. The study of gene expression profiling of cells revealed that the expression of anti-apoptotic genes such as BCL11A and BCL2 were reduced. We also found γ-H2AX was increased and BCL2 was reduced after MK-1775 and talazoparib treatment by immunoblot analysis. The change of mitochondrial transmembrane potential is one of the early intracellular events to occur following induction of apoptosis. Mitochondrial membrane potential was changed after MK-1775 and talazoparib treatment. Wee1 shRNA transfected cells reduced cellular proliferation. Cell cycle analysis showed G2/M arrest. We found that sensitivity of talazoparib was increased and induced apoptosis in Wee1 shRNA transfected cells. We next examined IOL by using osteoblastic cell line, MC3T3-E1. The cellular proliferation was reduced by ferric ammonium citrate (FAC) in a dose dependent manner. Intracellular ROS was increased. Talazoparib treatment protected FAC mediated cell death.
Conclusion
Targeting a combination of DNA damage and cell cycle checkpoints enhances therapeutic efficacy, and is proposed as a novel strategy for high-risk MDS and AML. It also provides promising clinical relevance as a therapeutic candidate for MDS patients.
Keyword(s): Apoptosis, Cell cycle
Abstract: EP903
Type: E-Poster Presentation
Session title: Myelodysplastic syndromes - Biology & Translational Research
Background
Myelodysplastic syndrome (MDS) is the clonal myeloid neoplasm which is characterized by blood cytopenia and an increased risk of transformation to acute myelogenous leukemia (AML). About 30 to 40% of MDS patients develop AML and these patients are chemoresistance. Moreover, iron overload (IOL) often begins to develop in patients with MDS through repeated blood transfusion. Because the outcome of MDS is poor, alternative therapeutic strategies are required to improve the survival of MDS patients.
Aims
The inhibition of the DNA damage response pathway including Poly (ADP-ribose) polymerase 1 (PARP-1) in the treatment of cancer has recently approved in clinically. Wee1 is also an oncogenic nuclear kinase, which regulate the cell cycle as a crucial G2/M checkpoint. Because overexpression of Wee1 can be observed in various cancer types, Wee1 inhibitor could suppress MDS and AML cells in combination with PARP-1 inhibitor.
Methods
In this study, we investigated whether Wee1 was involved in MDS progress. We also investigated the efficacy of Wee1 inhibitor, MK-1775 and PARP1 inhibitor, talazoparib by using MDS and AML cell line, SKM-1, MDS-L, U937, THP-1, MV4;11 and osteoblastic cell line, MC3T3-E1.
Results
We first investigated the relationship between Wee1 expression and MDS patients by microarray gene expression data from the online Gene Expression Omnibus (GEO). Wee1 expression was increased in AML transformed cells and AML cells compared to refractory anemia with excess blasts cells (GSE14468). In contrast, PARP-1 expression was not changed. We next evaluated the effect of MK-1775 or talazoparib on proliferation of MDS and AML cell lines. 72 h treatment of MDS and AML cells were inhibited by MK-1775 or talazoparib in a dose dependent manner. Cellular cytotoxicity and caspase 3/7 activity was also increased. We next investigated the relationship between MK-1775 and talazoparib. We found that cotreatment with MK-1775 and talazoparib showed superior effect than single drug treatment. We found that cellular cytotoxicity, caspase 3/7 activity, intracellular reactive oxygen species (ROS) and beta-galactosidase staining positive cells were also increased after MK-1775 and talazoparib treatment. Cotreatment with MK-1775 and talazoparib reduced colony formation. Cell cycle analysis showed significant cell population in sub G1 and induced apoptosis. The study of gene expression profiling of cells revealed that the expression of anti-apoptotic genes such as BCL11A and BCL2 were reduced. We also found γ-H2AX was increased and BCL2 was reduced after MK-1775 and talazoparib treatment by immunoblot analysis. The change of mitochondrial transmembrane potential is one of the early intracellular events to occur following induction of apoptosis. Mitochondrial membrane potential was changed after MK-1775 and talazoparib treatment. Wee1 shRNA transfected cells reduced cellular proliferation. Cell cycle analysis showed G2/M arrest. We found that sensitivity of talazoparib was increased and induced apoptosis in Wee1 shRNA transfected cells. We next examined IOL by using osteoblastic cell line, MC3T3-E1. The cellular proliferation was reduced by ferric ammonium citrate (FAC) in a dose dependent manner. Intracellular ROS was increased. Talazoparib treatment protected FAC mediated cell death.
Conclusion
Targeting a combination of DNA damage and cell cycle checkpoints enhances therapeutic efficacy, and is proposed as a novel strategy for high-risk MDS and AML. It also provides promising clinical relevance as a therapeutic candidate for MDS patients.
Keyword(s): Apoptosis, Cell cycle