A COMPARATIVE STUDY ON THE EFFECTS OF NUTLIN-3A IN B-CLL CHRONIC LYMPHOCYTIC LEUKEMIA CELLS WITH AND WITHOUT 17P DELETION
(Abstract release date: 05/19/16)
EHA Library. Shehata M. 06/09/16; 132597; E1048
Dr. Medhat Shehata
Contributions
Contributions
Abstract
Abstract: E1048
Type: Eposter Presentation
Background
The p53-MDM2 interaction is emerging as a promising therapeutic target in chronic lymphocytic leukemia (CLL). It is well known that MDM2 oncoprotein inhibits the function of wild-type p53 by its degradation and that MDM2 inhibitors are mainly effective in CLL cases with wild type p53. However, there is no substantial data on the effect of MDM2 inhibitors on CLL cells with p53 mutation or 17p deletion (17p-).
Aims
The aim of this work is to have a deeper insight into the effect MDM2 inhibitor Nutlin-3a on CLL cells obtained from patients with or without 17p deletion under standard suspension culture conditions and in a microenvironment model using co-cultures of CLL cells with primary bone marrow stromal cells (BMSC). This includes evaluating the effect of Nutli-3a on cell viability, induction of apoptosis and the regulation of key p53 downstream targets.
Methods
Peripheral blood mononuclear cells (PBMC) were obtained from 25 B-CLL patients with (n=7) and without (n=18) 17p deletion and exposed to Nutlin-3a under suspension or co-culture conditions for 2-3 days. Dose and time kinetics were also performed and cell viability was evaluated by MTT assay. Induction of apoptosis was assessed by AnnexinV/propidium iodide (PI) staining and FACS analysis. In parallel, a selective effect of Nutlin-3a on CLL cells, T-cells and monocytes was investigated using specific markers for B cells (CD19), T-cells (CD3) and monocytes (CD14) in combination with PI. In addition, the effect of Nutlin-3a on the key p53 downstream targets involved in the regulation of apoptosis (MDM2, Bax, PUMA, Noxa and Mcl-1), cell cycle regulation (p21) or DNA repair (PARP) was assessed by Western blotting and RT-PCR.
Results
In vitro treatment with Nutlin-3a induced cell killing in CLL cells from patients with and without 17p deletion as demonstrated by MTT assays. The effect of the inhibitor was relatively higher on the samples from patients with wild-type 17p at low concentrations (0,5µM to 1µM) compared to patients with 17p deletion. However, at higher concentrations (1-10µM), Nutlin-3a showed a remarkable effect on both groups. A similar pro-apoptotic response to Nutlin-3a was detected by FACS analysis experiments where Nutlin-3a induced 2,5 fold increase in apoptosis rate in patients without 17p deletion compared to 1,6 fold increase in apoptosis in patients with 17p deletion. The pro-apoptotic effect of Nutlin-3a was comparable under suspension and co-culture conditions and was higher in CLL (CD19+) cells compared to T-cells and monocytes. Consistent with the increase in apoptosis rates, Nutlin-3a treatment induced the expression of p53 downstream target p21 mRNA and this was associated with PARP cleavage in both patient groups.
Conclusion
These data confirm the beneficial effect of Nutlin-3a in CLL and further suggest that MDM2 inhibitors might be effective in 17p wild-type and 17p deleted patients depending on the concentration of Nutlin-3a. Therefore, the clinical relevance of these data deserves further investigations.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, P53
Type: Eposter Presentation
Background
The p53-MDM2 interaction is emerging as a promising therapeutic target in chronic lymphocytic leukemia (CLL). It is well known that MDM2 oncoprotein inhibits the function of wild-type p53 by its degradation and that MDM2 inhibitors are mainly effective in CLL cases with wild type p53. However, there is no substantial data on the effect of MDM2 inhibitors on CLL cells with p53 mutation or 17p deletion (17p-).
Aims
The aim of this work is to have a deeper insight into the effect MDM2 inhibitor Nutlin-3a on CLL cells obtained from patients with or without 17p deletion under standard suspension culture conditions and in a microenvironment model using co-cultures of CLL cells with primary bone marrow stromal cells (BMSC). This includes evaluating the effect of Nutli-3a on cell viability, induction of apoptosis and the regulation of key p53 downstream targets.
Methods
Peripheral blood mononuclear cells (PBMC) were obtained from 25 B-CLL patients with (n=7) and without (n=18) 17p deletion and exposed to Nutlin-3a under suspension or co-culture conditions for 2-3 days. Dose and time kinetics were also performed and cell viability was evaluated by MTT assay. Induction of apoptosis was assessed by AnnexinV/propidium iodide (PI) staining and FACS analysis. In parallel, a selective effect of Nutlin-3a on CLL cells, T-cells and monocytes was investigated using specific markers for B cells (CD19), T-cells (CD3) and monocytes (CD14) in combination with PI. In addition, the effect of Nutlin-3a on the key p53 downstream targets involved in the regulation of apoptosis (MDM2, Bax, PUMA, Noxa and Mcl-1), cell cycle regulation (p21) or DNA repair (PARP) was assessed by Western blotting and RT-PCR.
Results
In vitro treatment with Nutlin-3a induced cell killing in CLL cells from patients with and without 17p deletion as demonstrated by MTT assays. The effect of the inhibitor was relatively higher on the samples from patients with wild-type 17p at low concentrations (0,5µM to 1µM) compared to patients with 17p deletion. However, at higher concentrations (1-10µM), Nutlin-3a showed a remarkable effect on both groups. A similar pro-apoptotic response to Nutlin-3a was detected by FACS analysis experiments where Nutlin-3a induced 2,5 fold increase in apoptosis rate in patients without 17p deletion compared to 1,6 fold increase in apoptosis in patients with 17p deletion. The pro-apoptotic effect of Nutlin-3a was comparable under suspension and co-culture conditions and was higher in CLL (CD19+) cells compared to T-cells and monocytes. Consistent with the increase in apoptosis rates, Nutlin-3a treatment induced the expression of p53 downstream target p21 mRNA and this was associated with PARP cleavage in both patient groups.
Conclusion
These data confirm the beneficial effect of Nutlin-3a in CLL and further suggest that MDM2 inhibitors might be effective in 17p wild-type and 17p deleted patients depending on the concentration of Nutlin-3a. Therefore, the clinical relevance of these data deserves further investigations.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, P53
Abstract: E1048
Type: Eposter Presentation
Background
The p53-MDM2 interaction is emerging as a promising therapeutic target in chronic lymphocytic leukemia (CLL). It is well known that MDM2 oncoprotein inhibits the function of wild-type p53 by its degradation and that MDM2 inhibitors are mainly effective in CLL cases with wild type p53. However, there is no substantial data on the effect of MDM2 inhibitors on CLL cells with p53 mutation or 17p deletion (17p-).
Aims
The aim of this work is to have a deeper insight into the effect MDM2 inhibitor Nutlin-3a on CLL cells obtained from patients with or without 17p deletion under standard suspension culture conditions and in a microenvironment model using co-cultures of CLL cells with primary bone marrow stromal cells (BMSC). This includes evaluating the effect of Nutli-3a on cell viability, induction of apoptosis and the regulation of key p53 downstream targets.
Methods
Peripheral blood mononuclear cells (PBMC) were obtained from 25 B-CLL patients with (n=7) and without (n=18) 17p deletion and exposed to Nutlin-3a under suspension or co-culture conditions for 2-3 days. Dose and time kinetics were also performed and cell viability was evaluated by MTT assay. Induction of apoptosis was assessed by AnnexinV/propidium iodide (PI) staining and FACS analysis. In parallel, a selective effect of Nutlin-3a on CLL cells, T-cells and monocytes was investigated using specific markers for B cells (CD19), T-cells (CD3) and monocytes (CD14) in combination with PI. In addition, the effect of Nutlin-3a on the key p53 downstream targets involved in the regulation of apoptosis (MDM2, Bax, PUMA, Noxa and Mcl-1), cell cycle regulation (p21) or DNA repair (PARP) was assessed by Western blotting and RT-PCR.
Results
In vitro treatment with Nutlin-3a induced cell killing in CLL cells from patients with and without 17p deletion as demonstrated by MTT assays. The effect of the inhibitor was relatively higher on the samples from patients with wild-type 17p at low concentrations (0,5µM to 1µM) compared to patients with 17p deletion. However, at higher concentrations (1-10µM), Nutlin-3a showed a remarkable effect on both groups. A similar pro-apoptotic response to Nutlin-3a was detected by FACS analysis experiments where Nutlin-3a induced 2,5 fold increase in apoptosis rate in patients without 17p deletion compared to 1,6 fold increase in apoptosis in patients with 17p deletion. The pro-apoptotic effect of Nutlin-3a was comparable under suspension and co-culture conditions and was higher in CLL (CD19+) cells compared to T-cells and monocytes. Consistent with the increase in apoptosis rates, Nutlin-3a treatment induced the expression of p53 downstream target p21 mRNA and this was associated with PARP cleavage in both patient groups.
Conclusion
These data confirm the beneficial effect of Nutlin-3a in CLL and further suggest that MDM2 inhibitors might be effective in 17p wild-type and 17p deleted patients depending on the concentration of Nutlin-3a. Therefore, the clinical relevance of these data deserves further investigations.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, P53
Type: Eposter Presentation
Background
The p53-MDM2 interaction is emerging as a promising therapeutic target in chronic lymphocytic leukemia (CLL). It is well known that MDM2 oncoprotein inhibits the function of wild-type p53 by its degradation and that MDM2 inhibitors are mainly effective in CLL cases with wild type p53. However, there is no substantial data on the effect of MDM2 inhibitors on CLL cells with p53 mutation or 17p deletion (17p-).
Aims
The aim of this work is to have a deeper insight into the effect MDM2 inhibitor Nutlin-3a on CLL cells obtained from patients with or without 17p deletion under standard suspension culture conditions and in a microenvironment model using co-cultures of CLL cells with primary bone marrow stromal cells (BMSC). This includes evaluating the effect of Nutli-3a on cell viability, induction of apoptosis and the regulation of key p53 downstream targets.
Methods
Peripheral blood mononuclear cells (PBMC) were obtained from 25 B-CLL patients with (n=7) and without (n=18) 17p deletion and exposed to Nutlin-3a under suspension or co-culture conditions for 2-3 days. Dose and time kinetics were also performed and cell viability was evaluated by MTT assay. Induction of apoptosis was assessed by AnnexinV/propidium iodide (PI) staining and FACS analysis. In parallel, a selective effect of Nutlin-3a on CLL cells, T-cells and monocytes was investigated using specific markers for B cells (CD19), T-cells (CD3) and monocytes (CD14) in combination with PI. In addition, the effect of Nutlin-3a on the key p53 downstream targets involved in the regulation of apoptosis (MDM2, Bax, PUMA, Noxa and Mcl-1), cell cycle regulation (p21) or DNA repair (PARP) was assessed by Western blotting and RT-PCR.
Results
In vitro treatment with Nutlin-3a induced cell killing in CLL cells from patients with and without 17p deletion as demonstrated by MTT assays. The effect of the inhibitor was relatively higher on the samples from patients with wild-type 17p at low concentrations (0,5µM to 1µM) compared to patients with 17p deletion. However, at higher concentrations (1-10µM), Nutlin-3a showed a remarkable effect on both groups. A similar pro-apoptotic response to Nutlin-3a was detected by FACS analysis experiments where Nutlin-3a induced 2,5 fold increase in apoptosis rate in patients without 17p deletion compared to 1,6 fold increase in apoptosis in patients with 17p deletion. The pro-apoptotic effect of Nutlin-3a was comparable under suspension and co-culture conditions and was higher in CLL (CD19+) cells compared to T-cells and monocytes. Consistent with the increase in apoptosis rates, Nutlin-3a treatment induced the expression of p53 downstream target p21 mRNA and this was associated with PARP cleavage in both patient groups.
Conclusion
These data confirm the beneficial effect of Nutlin-3a in CLL and further suggest that MDM2 inhibitors might be effective in 17p wild-type and 17p deleted patients depending on the concentration of Nutlin-3a. Therefore, the clinical relevance of these data deserves further investigations.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, P53
{{ help_message }}
{{filter}}