CALCINEURIN COMPLEX ISOLATED FROM T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (T-ALL) CELLS IDENTIFIES NEW SIGNALING PATHWAYS WHOSE INHIBITION SYNERGIZE WITH CALCINEURIN INHIBITION TO PROMOTE T-ALL CELL DEATH
(Abstract release date: 05/19/16)
EHA Library. Piovan E. 06/09/16; 132397; E848
Dr. Erich Piovan
Contributions
Contributions
Abstract
Abstract: E848
Type: Eposter Presentation
Background
Calcineurin (Cn) is a calcium activated protein phosphatase, composed of a catalytic subunit (PPP3CA) and a regulatory subunit (PPP3CB). It is critically involved in calcium signaling in diverse cells and is involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined.
Aims
Available evidence shows that Nuclear Factor of Activated T cells (NFAT) transcription factors are mediators of Cn action in different cancers. However, it is possible that NFAT factors are not the only targets of Cn in leukemogenesis, as Cn can dephosphorylate other factors possibly relevant to its oncogenic properties. Aim of this study was to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL), evaluate if the interactors are enriched in cellular signaling pathways relevant for T-ALL pathogenesis and if novel drug combinations can be proposed to enhance the efficacy of chemotherapeutic drugs and/or reduce the side effects of clinically relevant immunosuppressive drugs targeting Cn.
Methods
In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-ALL we used tandem affinity chromatography, followed by mass spectrometry (MS) to purify novel Cn-interacting partners. Novel interactions were validated by immunoprecipitation following transient overexpression in 293T cells or as native interactions in T-ALL cells. Drug synergisms using relevant drug combinations were evaluated through the execution of cell viability assays (bioluminescent method and annexin V-PI staining). Effects of Cn activity modulation on cellular signaling pathways found to be enriched in our MS list were evaluated by western blotting and flow cytometry.
Results
We identified numerous PPP3CA interactors, including proteins implicated in leukemia pathobiology such as NPM1, BCL11B, GSK3b and KDM1. The identified Cn-interacting proteins were found to be part of diverse cellular signaling pathways including eIF2 signaling, cell cycle control of chromosomal replication, mammalian target of Rapamycin (mTOR) signaling and 14-3-3 mediated signaling. Interestingly, modulation of Cn activity (by inhibitors and activators) lead to alterations in the identified signaling pathways including PI3K-AKT-mTOR signaling and eIF2 signaling. In addition, jointly targeting pathways enriched in our Cn complex (such as PI3K-mTOR signaling, cell cycle control of chromosomal replication mediated by topoisomerase II) together with Cn unveiled novel synergistic pro-apoptotic drug combinations.
Conclusion
The signaling pathways reported here will contribute to identify novel drug combinations which could enhance the efficacy of chemotherapeutic drugs and/or reduce the side effects of clinically relevant immunosuppressive drugs targeting Cn.
Session topic: E-poster
Keyword(s): Calcium, MTOR, PI3-K/AKT, T-ALL
Type: Eposter Presentation
Background
Calcineurin (Cn) is a calcium activated protein phosphatase, composed of a catalytic subunit (PPP3CA) and a regulatory subunit (PPP3CB). It is critically involved in calcium signaling in diverse cells and is involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined.
Aims
Available evidence shows that Nuclear Factor of Activated T cells (NFAT) transcription factors are mediators of Cn action in different cancers. However, it is possible that NFAT factors are not the only targets of Cn in leukemogenesis, as Cn can dephosphorylate other factors possibly relevant to its oncogenic properties. Aim of this study was to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL), evaluate if the interactors are enriched in cellular signaling pathways relevant for T-ALL pathogenesis and if novel drug combinations can be proposed to enhance the efficacy of chemotherapeutic drugs and/or reduce the side effects of clinically relevant immunosuppressive drugs targeting Cn.
Methods
In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-ALL we used tandem affinity chromatography, followed by mass spectrometry (MS) to purify novel Cn-interacting partners. Novel interactions were validated by immunoprecipitation following transient overexpression in 293T cells or as native interactions in T-ALL cells. Drug synergisms using relevant drug combinations were evaluated through the execution of cell viability assays (bioluminescent method and annexin V-PI staining). Effects of Cn activity modulation on cellular signaling pathways found to be enriched in our MS list were evaluated by western blotting and flow cytometry.
Results
We identified numerous PPP3CA interactors, including proteins implicated in leukemia pathobiology such as NPM1, BCL11B, GSK3b and KDM1. The identified Cn-interacting proteins were found to be part of diverse cellular signaling pathways including eIF2 signaling, cell cycle control of chromosomal replication, mammalian target of Rapamycin (mTOR) signaling and 14-3-3 mediated signaling. Interestingly, modulation of Cn activity (by inhibitors and activators) lead to alterations in the identified signaling pathways including PI3K-AKT-mTOR signaling and eIF2 signaling. In addition, jointly targeting pathways enriched in our Cn complex (such as PI3K-mTOR signaling, cell cycle control of chromosomal replication mediated by topoisomerase II) together with Cn unveiled novel synergistic pro-apoptotic drug combinations.
Conclusion
The signaling pathways reported here will contribute to identify novel drug combinations which could enhance the efficacy of chemotherapeutic drugs and/or reduce the side effects of clinically relevant immunosuppressive drugs targeting Cn.
Session topic: E-poster
Keyword(s): Calcium, MTOR, PI3-K/AKT, T-ALL
Abstract: E848
Type: Eposter Presentation
Background
Calcineurin (Cn) is a calcium activated protein phosphatase, composed of a catalytic subunit (PPP3CA) and a regulatory subunit (PPP3CB). It is critically involved in calcium signaling in diverse cells and is involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined.
Aims
Available evidence shows that Nuclear Factor of Activated T cells (NFAT) transcription factors are mediators of Cn action in different cancers. However, it is possible that NFAT factors are not the only targets of Cn in leukemogenesis, as Cn can dephosphorylate other factors possibly relevant to its oncogenic properties. Aim of this study was to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL), evaluate if the interactors are enriched in cellular signaling pathways relevant for T-ALL pathogenesis and if novel drug combinations can be proposed to enhance the efficacy of chemotherapeutic drugs and/or reduce the side effects of clinically relevant immunosuppressive drugs targeting Cn.
Methods
In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-ALL we used tandem affinity chromatography, followed by mass spectrometry (MS) to purify novel Cn-interacting partners. Novel interactions were validated by immunoprecipitation following transient overexpression in 293T cells or as native interactions in T-ALL cells. Drug synergisms using relevant drug combinations were evaluated through the execution of cell viability assays (bioluminescent method and annexin V-PI staining). Effects of Cn activity modulation on cellular signaling pathways found to be enriched in our MS list were evaluated by western blotting and flow cytometry.
Results
We identified numerous PPP3CA interactors, including proteins implicated in leukemia pathobiology such as NPM1, BCL11B, GSK3b and KDM1. The identified Cn-interacting proteins were found to be part of diverse cellular signaling pathways including eIF2 signaling, cell cycle control of chromosomal replication, mammalian target of Rapamycin (mTOR) signaling and 14-3-3 mediated signaling. Interestingly, modulation of Cn activity (by inhibitors and activators) lead to alterations in the identified signaling pathways including PI3K-AKT-mTOR signaling and eIF2 signaling. In addition, jointly targeting pathways enriched in our Cn complex (such as PI3K-mTOR signaling, cell cycle control of chromosomal replication mediated by topoisomerase II) together with Cn unveiled novel synergistic pro-apoptotic drug combinations.
Conclusion
The signaling pathways reported here will contribute to identify novel drug combinations which could enhance the efficacy of chemotherapeutic drugs and/or reduce the side effects of clinically relevant immunosuppressive drugs targeting Cn.
Session topic: E-poster
Keyword(s): Calcium, MTOR, PI3-K/AKT, T-ALL
Type: Eposter Presentation
Background
Calcineurin (Cn) is a calcium activated protein phosphatase, composed of a catalytic subunit (PPP3CA) and a regulatory subunit (PPP3CB). It is critically involved in calcium signaling in diverse cells and is involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined.
Aims
Available evidence shows that Nuclear Factor of Activated T cells (NFAT) transcription factors are mediators of Cn action in different cancers. However, it is possible that NFAT factors are not the only targets of Cn in leukemogenesis, as Cn can dephosphorylate other factors possibly relevant to its oncogenic properties. Aim of this study was to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL), evaluate if the interactors are enriched in cellular signaling pathways relevant for T-ALL pathogenesis and if novel drug combinations can be proposed to enhance the efficacy of chemotherapeutic drugs and/or reduce the side effects of clinically relevant immunosuppressive drugs targeting Cn.
Methods
In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-ALL we used tandem affinity chromatography, followed by mass spectrometry (MS) to purify novel Cn-interacting partners. Novel interactions were validated by immunoprecipitation following transient overexpression in 293T cells or as native interactions in T-ALL cells. Drug synergisms using relevant drug combinations were evaluated through the execution of cell viability assays (bioluminescent method and annexin V-PI staining). Effects of Cn activity modulation on cellular signaling pathways found to be enriched in our MS list were evaluated by western blotting and flow cytometry.
Results
We identified numerous PPP3CA interactors, including proteins implicated in leukemia pathobiology such as NPM1, BCL11B, GSK3b and KDM1. The identified Cn-interacting proteins were found to be part of diverse cellular signaling pathways including eIF2 signaling, cell cycle control of chromosomal replication, mammalian target of Rapamycin (mTOR) signaling and 14-3-3 mediated signaling. Interestingly, modulation of Cn activity (by inhibitors and activators) lead to alterations in the identified signaling pathways including PI3K-AKT-mTOR signaling and eIF2 signaling. In addition, jointly targeting pathways enriched in our Cn complex (such as PI3K-mTOR signaling, cell cycle control of chromosomal replication mediated by topoisomerase II) together with Cn unveiled novel synergistic pro-apoptotic drug combinations.
Conclusion
The signaling pathways reported here will contribute to identify novel drug combinations which could enhance the efficacy of chemotherapeutic drugs and/or reduce the side effects of clinically relevant immunosuppressive drugs targeting Cn.
Session topic: E-poster
Keyword(s): Calcium, MTOR, PI3-K/AKT, T-ALL
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