THE WEE1 INHIBITION DEEPLY SENSITIZES ACUTE LYMPHOBLASTIC LEUKEMIA CELL LINES AND PRIMARY CELLS TO THE CYTOTOXIC EFFECT OF DIFFERENT ANTINEOPLASTIC COMPOUNDS
(Abstract release date: 05/19/16)
EHA Library. Ghelli Luserna Di Rora A. 06/09/16; 132390; E841
Mr. Andrea Ghelli Luserna Di Rora
Contributions
Contributions
Abstract
Abstract: E841
Type: Eposter Presentation
Background
Although the efficacy of Checkpoint kinase inhibitors have been established in different kind of tumors, only few studies have been performed in order to evaluate their effectiveness on hematological disorders. The Wee1, ATR/Chk1 and ATM/Chk2 pathways are involved in cell cycle regulation, DNA damages response and DNA repair. Wee1 is a checkpoint kinase, involved mainly in the regulation of G2/M transition through the phosphorylation of both Cyclin-dependent kinase 1 (CDK1) and 2 (CDK2).
Aims
In this study we evaluated the in vitro/ex vivo efficacy of a Wee1 inhibitor, MK-1775, in single agent and in combination on Acute Lymphoblastic Leukemia (ALL) cell lines and primary samples.
Methods
A panel B-/T-ALL cell lines and different primary cells isolated from adult ALL patients were treated in single agent or in combination with different compound (nucleoside analogue, Clofarabine; tyrosine kinase inhibitor, Bosutinib; Chk1/Chk2 inhibitor, PF-00477736) and then was evaluated the reduction of the cell viability (MTS reagent), the reduction of the proliferation(Trypan blue exclusion dye assay), cell cycle modification (Propidium iodide staining), induction of apoptosis (Annexin V/Pi staining), protein modification (Western blot) and protein expression modification (Prime PCR plate).
Results
MK-1775 deeply reduced the cell viability in a dose and time-dependent manner in all the treated cell lines. The reduction of both cell viability and proliferation were associated with the increment of apoptosis and the activation of different DNA damage markers (gH2AX and Parp-1), confirming the cytotoxicity of MK-1775 in single agent. We hypothesized that targeting Chk1, a kinase upstream, of Wee1, would be more effective in reducing cell proliferation. Indeed, the concomitant inhibition of Chk1 and Wee1 kinases, using the PF-0477736 in combination with MK-1775, synergized in the reduction of the cell viability, inhibition of the proliferation index and induction of apoptosis. We undertook further studies to understand the chemo-sensitizer activity of the compound, thus MK-1775 was combined with different drugs (Clofarabine, Bosutinib Authentic, Bos, and a particular isomer of Bosutinib, Bos-I).The combination between MK-1775 and clofarabine showed an additive effect in terms of reduction of the cell viability and induction of apoptosis. Finally the Wee1 inhibitor was combined with the tyrosine kinase inhibitors Bos and Bos-I. Both isomers in combination with MK-1775 showed an additive effect in term of reduction of the cell viability and induction of apoptosis. The cytotoxic effect of Bos-I was stronger on the Philadelphia-negative cell lines in comparison to the positive counterpart. Western blot analysis highlighted that this compound, but not the Bos, interfered with the Chk1/Chk2 and Wee1 pathway. The results found on the different cell lines were confirmed also on primary leukemic cells.
Conclusion
In our opinion the results of this study identify the Wee1 kinase as a promising target for the treatment of ALL. As monotherapy the inhibition of Wee1 increases the genetic instability of leukemic cells, promoting cell death caused by progressive addition of DNA damages. As chemo-sensitazer agent, the MK-1775 inhibits the DNA damage response pathway, increasing the cytotoxicity of different compounds.Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.Martinelli G. & YenT.J. equal contribution
Session topic: E-poster
Keyword(s): Cell cycle, Chemosensitivity, DNA repair, Ex vivo
Type: Eposter Presentation
Background
Although the efficacy of Checkpoint kinase inhibitors have been established in different kind of tumors, only few studies have been performed in order to evaluate their effectiveness on hematological disorders. The Wee1, ATR/Chk1 and ATM/Chk2 pathways are involved in cell cycle regulation, DNA damages response and DNA repair. Wee1 is a checkpoint kinase, involved mainly in the regulation of G2/M transition through the phosphorylation of both Cyclin-dependent kinase 1 (CDK1) and 2 (CDK2).
Aims
In this study we evaluated the in vitro/ex vivo efficacy of a Wee1 inhibitor, MK-1775, in single agent and in combination on Acute Lymphoblastic Leukemia (ALL) cell lines and primary samples.
Methods
A panel B-/T-ALL cell lines and different primary cells isolated from adult ALL patients were treated in single agent or in combination with different compound (nucleoside analogue, Clofarabine; tyrosine kinase inhibitor, Bosutinib; Chk1/Chk2 inhibitor, PF-00477736) and then was evaluated the reduction of the cell viability (MTS reagent), the reduction of the proliferation(Trypan blue exclusion dye assay), cell cycle modification (Propidium iodide staining), induction of apoptosis (Annexin V/Pi staining), protein modification (Western blot) and protein expression modification (Prime PCR plate).
Results
MK-1775 deeply reduced the cell viability in a dose and time-dependent manner in all the treated cell lines. The reduction of both cell viability and proliferation were associated with the increment of apoptosis and the activation of different DNA damage markers (gH2AX and Parp-1), confirming the cytotoxicity of MK-1775 in single agent. We hypothesized that targeting Chk1, a kinase upstream, of Wee1, would be more effective in reducing cell proliferation. Indeed, the concomitant inhibition of Chk1 and Wee1 kinases, using the PF-0477736 in combination with MK-1775, synergized in the reduction of the cell viability, inhibition of the proliferation index and induction of apoptosis. We undertook further studies to understand the chemo-sensitizer activity of the compound, thus MK-1775 was combined with different drugs (Clofarabine, Bosutinib Authentic, Bos, and a particular isomer of Bosutinib, Bos-I).The combination between MK-1775 and clofarabine showed an additive effect in terms of reduction of the cell viability and induction of apoptosis. Finally the Wee1 inhibitor was combined with the tyrosine kinase inhibitors Bos and Bos-I. Both isomers in combination with MK-1775 showed an additive effect in term of reduction of the cell viability and induction of apoptosis. The cytotoxic effect of Bos-I was stronger on the Philadelphia-negative cell lines in comparison to the positive counterpart. Western blot analysis highlighted that this compound, but not the Bos, interfered with the Chk1/Chk2 and Wee1 pathway. The results found on the different cell lines were confirmed also on primary leukemic cells.
Conclusion
In our opinion the results of this study identify the Wee1 kinase as a promising target for the treatment of ALL. As monotherapy the inhibition of Wee1 increases the genetic instability of leukemic cells, promoting cell death caused by progressive addition of DNA damages. As chemo-sensitazer agent, the MK-1775 inhibits the DNA damage response pathway, increasing the cytotoxicity of different compounds.Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.Martinelli G. & YenT.J. equal contribution
Session topic: E-poster
Keyword(s): Cell cycle, Chemosensitivity, DNA repair, Ex vivo
Abstract: E841
Type: Eposter Presentation
Background
Although the efficacy of Checkpoint kinase inhibitors have been established in different kind of tumors, only few studies have been performed in order to evaluate their effectiveness on hematological disorders. The Wee1, ATR/Chk1 and ATM/Chk2 pathways are involved in cell cycle regulation, DNA damages response and DNA repair. Wee1 is a checkpoint kinase, involved mainly in the regulation of G2/M transition through the phosphorylation of both Cyclin-dependent kinase 1 (CDK1) and 2 (CDK2).
Aims
In this study we evaluated the in vitro/ex vivo efficacy of a Wee1 inhibitor, MK-1775, in single agent and in combination on Acute Lymphoblastic Leukemia (ALL) cell lines and primary samples.
Methods
A panel B-/T-ALL cell lines and different primary cells isolated from adult ALL patients were treated in single agent or in combination with different compound (nucleoside analogue, Clofarabine; tyrosine kinase inhibitor, Bosutinib; Chk1/Chk2 inhibitor, PF-00477736) and then was evaluated the reduction of the cell viability (MTS reagent), the reduction of the proliferation(Trypan blue exclusion dye assay), cell cycle modification (Propidium iodide staining), induction of apoptosis (Annexin V/Pi staining), protein modification (Western blot) and protein expression modification (Prime PCR plate).
Results
MK-1775 deeply reduced the cell viability in a dose and time-dependent manner in all the treated cell lines. The reduction of both cell viability and proliferation were associated with the increment of apoptosis and the activation of different DNA damage markers (gH2AX and Parp-1), confirming the cytotoxicity of MK-1775 in single agent. We hypothesized that targeting Chk1, a kinase upstream, of Wee1, would be more effective in reducing cell proliferation. Indeed, the concomitant inhibition of Chk1 and Wee1 kinases, using the PF-0477736 in combination with MK-1775, synergized in the reduction of the cell viability, inhibition of the proliferation index and induction of apoptosis. We undertook further studies to understand the chemo-sensitizer activity of the compound, thus MK-1775 was combined with different drugs (Clofarabine, Bosutinib Authentic, Bos, and a particular isomer of Bosutinib, Bos-I).The combination between MK-1775 and clofarabine showed an additive effect in terms of reduction of the cell viability and induction of apoptosis. Finally the Wee1 inhibitor was combined with the tyrosine kinase inhibitors Bos and Bos-I. Both isomers in combination with MK-1775 showed an additive effect in term of reduction of the cell viability and induction of apoptosis. The cytotoxic effect of Bos-I was stronger on the Philadelphia-negative cell lines in comparison to the positive counterpart. Western blot analysis highlighted that this compound, but not the Bos, interfered with the Chk1/Chk2 and Wee1 pathway. The results found on the different cell lines were confirmed also on primary leukemic cells.
Conclusion
In our opinion the results of this study identify the Wee1 kinase as a promising target for the treatment of ALL. As monotherapy the inhibition of Wee1 increases the genetic instability of leukemic cells, promoting cell death caused by progressive addition of DNA damages. As chemo-sensitazer agent, the MK-1775 inhibits the DNA damage response pathway, increasing the cytotoxicity of different compounds.Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.Martinelli G. & YenT.J. equal contribution
Session topic: E-poster
Keyword(s): Cell cycle, Chemosensitivity, DNA repair, Ex vivo
Type: Eposter Presentation
Background
Although the efficacy of Checkpoint kinase inhibitors have been established in different kind of tumors, only few studies have been performed in order to evaluate their effectiveness on hematological disorders. The Wee1, ATR/Chk1 and ATM/Chk2 pathways are involved in cell cycle regulation, DNA damages response and DNA repair. Wee1 is a checkpoint kinase, involved mainly in the regulation of G2/M transition through the phosphorylation of both Cyclin-dependent kinase 1 (CDK1) and 2 (CDK2).
Aims
In this study we evaluated the in vitro/ex vivo efficacy of a Wee1 inhibitor, MK-1775, in single agent and in combination on Acute Lymphoblastic Leukemia (ALL) cell lines and primary samples.
Methods
A panel B-/T-ALL cell lines and different primary cells isolated from adult ALL patients were treated in single agent or in combination with different compound (nucleoside analogue, Clofarabine; tyrosine kinase inhibitor, Bosutinib; Chk1/Chk2 inhibitor, PF-00477736) and then was evaluated the reduction of the cell viability (MTS reagent), the reduction of the proliferation(Trypan blue exclusion dye assay), cell cycle modification (Propidium iodide staining), induction of apoptosis (Annexin V/Pi staining), protein modification (Western blot) and protein expression modification (Prime PCR plate).
Results
MK-1775 deeply reduced the cell viability in a dose and time-dependent manner in all the treated cell lines. The reduction of both cell viability and proliferation were associated with the increment of apoptosis and the activation of different DNA damage markers (gH2AX and Parp-1), confirming the cytotoxicity of MK-1775 in single agent. We hypothesized that targeting Chk1, a kinase upstream, of Wee1, would be more effective in reducing cell proliferation. Indeed, the concomitant inhibition of Chk1 and Wee1 kinases, using the PF-0477736 in combination with MK-1775, synergized in the reduction of the cell viability, inhibition of the proliferation index and induction of apoptosis. We undertook further studies to understand the chemo-sensitizer activity of the compound, thus MK-1775 was combined with different drugs (Clofarabine, Bosutinib Authentic, Bos, and a particular isomer of Bosutinib, Bos-I).The combination between MK-1775 and clofarabine showed an additive effect in terms of reduction of the cell viability and induction of apoptosis. Finally the Wee1 inhibitor was combined with the tyrosine kinase inhibitors Bos and Bos-I. Both isomers in combination with MK-1775 showed an additive effect in term of reduction of the cell viability and induction of apoptosis. The cytotoxic effect of Bos-I was stronger on the Philadelphia-negative cell lines in comparison to the positive counterpart. Western blot analysis highlighted that this compound, but not the Bos, interfered with the Chk1/Chk2 and Wee1 pathway. The results found on the different cell lines were confirmed also on primary leukemic cells.
Conclusion
In our opinion the results of this study identify the Wee1 kinase as a promising target for the treatment of ALL. As monotherapy the inhibition of Wee1 increases the genetic instability of leukemic cells, promoting cell death caused by progressive addition of DNA damages. As chemo-sensitazer agent, the MK-1775 inhibits the DNA damage response pathway, increasing the cytotoxicity of different compounds.Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.Martinelli G. & YenT.J. equal contribution
Session topic: E-poster
Keyword(s): Cell cycle, Chemosensitivity, DNA repair, Ex vivo
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