Contributions
Abstract: PB1625
Type: Publication Only
Background
Acute Lymphoblastic Leukaemia (ALL) is a malignant disease of hematopoietic system in which early lymphoid precursors proliferate and replace the normal hematopoietic cells, affecting both linages, B and T cells (B-ALL and T-ALL). NOTCH pathway is an evolutionary conserved signalling pathway that plays a significant role in cell fate decision during development, stem cell self-renewal and differentiation in haematopoiesis. Deregulation of NOTCH signalling was already reported in haematological disorders and various solid tumours, namely in T-ALL, were activating NOTCH1 mutations are present in more than half patients, playing a significant role in its pathogenesis. Therefore, modulation of NOTCH signalling pathway, for example with gamma-secretase inhibitors, might provide a potential novel therapeutic approach in ALL.
Aims
In this context, the aim of this study was to evaluate the NOTCH pathway as a therapeutic target in ALL, using a ƴ-secretase inhibitor in two in vitro models of ALL.
Methods
For this purpose, we used two different ALL cell lines, CEM a T-ALL and KOPN-8 a B-ALL cell line. These cells were incubated in the absence and presence of increasing concentrations of the ƴ-secretase inhibitor, GSI-XXI (20µM - 50µM). Cell viability and proliferation were assessed by trypan blue exclusion assay. Cell death was evaluated by optical microscopy and flow cytometry (FC) using annexin V/propidium iodide double staining and JC-1 probe to assess mitochondrial membrane potential. Apoptotic protein levels (BAX and BCL-2) and cell cycle distribution were also evaluated by FC. The expression levels of CCND1, CCNB1, CCNE1 and NF-ĸB genes were determined by RT-PCR. Results were considered statistically significant when p<0,05.
Results
Our results suggest that GSI-XXI reduced cell proliferation and viability in a dose- and cell type dependent manner with an IC50 at 24h of approximately 40µM for CEM and 30µM for KOPN-8 cells. This compound induced cell death mainly by apoptosis in both cell lines confirmed by morphological analysis, mediated by an increase in BAX/BCL-2 ratio and a decrease in mitochondrial membrane potential. The analysis of cell cycle also revealed a significant arrest in G0/G1 phase in CEM cells. This analysis also showed a sub-G1 peak in KOPN-8 treated cells, which correspond to DNA fragmentation a typical feature of apoptosis. Finally, GSI-XXI did not induce significant changes in the expression levels of CCND1, CCNB1, CCNE1 and NF-ĸB genes.
Conclusion
In conclusion, if these results can be translated to clinical practice, they suggest that NOTCH pathway and ƴ-secretase inhibitors, like GSI-XXI, might be a good therapeutic approach in acute lymphoblastic leukaemia patients.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Acute lymphoblastic leukemia, Notch signaling
Abstract: PB1625
Type: Publication Only
Background
Acute Lymphoblastic Leukaemia (ALL) is a malignant disease of hematopoietic system in which early lymphoid precursors proliferate and replace the normal hematopoietic cells, affecting both linages, B and T cells (B-ALL and T-ALL). NOTCH pathway is an evolutionary conserved signalling pathway that plays a significant role in cell fate decision during development, stem cell self-renewal and differentiation in haematopoiesis. Deregulation of NOTCH signalling was already reported in haematological disorders and various solid tumours, namely in T-ALL, were activating NOTCH1 mutations are present in more than half patients, playing a significant role in its pathogenesis. Therefore, modulation of NOTCH signalling pathway, for example with gamma-secretase inhibitors, might provide a potential novel therapeutic approach in ALL.
Aims
In this context, the aim of this study was to evaluate the NOTCH pathway as a therapeutic target in ALL, using a ƴ-secretase inhibitor in two in vitro models of ALL.
Methods
For this purpose, we used two different ALL cell lines, CEM a T-ALL and KOPN-8 a B-ALL cell line. These cells were incubated in the absence and presence of increasing concentrations of the ƴ-secretase inhibitor, GSI-XXI (20µM - 50µM). Cell viability and proliferation were assessed by trypan blue exclusion assay. Cell death was evaluated by optical microscopy and flow cytometry (FC) using annexin V/propidium iodide double staining and JC-1 probe to assess mitochondrial membrane potential. Apoptotic protein levels (BAX and BCL-2) and cell cycle distribution were also evaluated by FC. The expression levels of CCND1, CCNB1, CCNE1 and NF-ĸB genes were determined by RT-PCR. Results were considered statistically significant when p<0,05.
Results
Our results suggest that GSI-XXI reduced cell proliferation and viability in a dose- and cell type dependent manner with an IC50 at 24h of approximately 40µM for CEM and 30µM for KOPN-8 cells. This compound induced cell death mainly by apoptosis in both cell lines confirmed by morphological analysis, mediated by an increase in BAX/BCL-2 ratio and a decrease in mitochondrial membrane potential. The analysis of cell cycle also revealed a significant arrest in G0/G1 phase in CEM cells. This analysis also showed a sub-G1 peak in KOPN-8 treated cells, which correspond to DNA fragmentation a typical feature of apoptosis. Finally, GSI-XXI did not induce significant changes in the expression levels of CCND1, CCNB1, CCNE1 and NF-ĸB genes.
Conclusion
In conclusion, if these results can be translated to clinical practice, they suggest that NOTCH pathway and ƴ-secretase inhibitors, like GSI-XXI, might be a good therapeutic approach in acute lymphoblastic leukaemia patients.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Acute lymphoblastic leukemia, Notch signaling