ACTIVATION OF MTORC2 COMPLEX CONTRIBUTES TO CELL CYCLE PROGRESSION AND SURVIVAL IN ALK+ ANAPLASTIC LARGE CELL LYMPHOMA
(Abstract release date: 05/19/16)
EHA Library. Rassidakis G. 06/09/16; 132923; E1374
Assoc. Prof. George Rassidakis
Contributions
Contributions
Abstract
Abstract: E1374
Type: Eposter Presentation
Background
ALK+ anaplastic large cell lymphoma (ALK+ ALCL) is characterized by chromosomal translocations of ALK gene locus, the most frequent being the t(2;5)(p23;q35) resulting in aberrrant expression and activation of the NPM-ALK oncoprotein. The NPM-ALK activates multiple oncogenic pathways including the mTOR-Raptor (mTORC1) pathway, however, the potential role of mTOR-Rictor (mTORC2) complex in ALCL pathogenesis is yet unknown.
Aims
To investigate the potential role of Sin1, a critical component of the mTORC2 complex required for its integrity and activation, in ALK+ ALCL pathogenesis.
Methods
Expression, phosphorylation, and localization of Sin1 protein were assessed by Western blot after subcellular fractionation, immunofluorescence and confocal microscopy in 3 ALK+ and 2 ALK- ALCL cell lines. Physical interaction between various proteins was assessed by co-immunoprecipitation assays. Real-time RT PCR was used to assess Sin1 gene products at the RNA level. Transfection experiments were performed in BaF3 cells and in ALK+ and ALK- ALCL cells using NPM-ALK and Sin1 expressing plasmids and Sin1-shRNA constructs (gene silencing). Standard cell viability, proliferation and colony formation assays as well as flow cytometry were utilized to assess cell growth and survival following forced expression or gene silencing of Sin1 gene, as well as after treatment with ALK and STAT3 inhibitors. In a cohort of 32 previously untreated patients with ALK+ ALCL, Sin1 protein expression was assessed by immunohistochemistry performed on a tissue microarray.
Results
Sin1.1 and Sin1.5 were the main isoforms detected in immunoblots and they were differentially expressed among ALK+ and ALK- ALCL cell lines. Sin1 protein was co-localized with activated (Ser473-phosphorylated) AKT kinase in ALCL cells. Treatment of ALK+ ALCL cells with Crizotinib led to reduced expression and de-phosphorylation of Sin1 protein. Forced expression of Sin1.1 and Sin1.5 isoforms resulted in increased cell proliferation in ALK+ ALCL cells with downregulation of the CDK inhibitor p21. Inversely, knocking down Sin1 gene by two specific Sin1-shRNA constructs resulted in dramatic decrease of cell viability and colony formation (by 80%) of ALK+ ALCL cells, which was associated with AKT de-activation and downregulation of the anti-apoptotic BCL-XL and MCL-1 proteins. Using our in vitro system and ex vivo (xenografts) mouse model, Sin1 gene expression was, at least in part, regulated by STAT3 transcription factor. In the cohort of ALK+ ALCL patients, Sin1 protein was expressed in all 32 tumors studied with cytoplasmic and nuclear pattern.
Conclusion
Taken together, these novel findings suggest that mTORC2 complex, through Sin1-dependent activation of AKT and possibly other AKT-independent functions, may contribute to cell cycle and apoptosis deregulation and oncogenesis in ALCL. Thus, modulation of mTORC2 activity might represent a novel target for investigational therapy in patients with ALCL.
Session topic: E-poster
Keyword(s): Akt, ALK/ALCL, MTOR, STAT3
Type: Eposter Presentation
Background
ALK+ anaplastic large cell lymphoma (ALK+ ALCL) is characterized by chromosomal translocations of ALK gene locus, the most frequent being the t(2;5)(p23;q35) resulting in aberrrant expression and activation of the NPM-ALK oncoprotein. The NPM-ALK activates multiple oncogenic pathways including the mTOR-Raptor (mTORC1) pathway, however, the potential role of mTOR-Rictor (mTORC2) complex in ALCL pathogenesis is yet unknown.
Aims
To investigate the potential role of Sin1, a critical component of the mTORC2 complex required for its integrity and activation, in ALK+ ALCL pathogenesis.
Methods
Expression, phosphorylation, and localization of Sin1 protein were assessed by Western blot after subcellular fractionation, immunofluorescence and confocal microscopy in 3 ALK+ and 2 ALK- ALCL cell lines. Physical interaction between various proteins was assessed by co-immunoprecipitation assays. Real-time RT PCR was used to assess Sin1 gene products at the RNA level. Transfection experiments were performed in BaF3 cells and in ALK+ and ALK- ALCL cells using NPM-ALK and Sin1 expressing plasmids and Sin1-shRNA constructs (gene silencing). Standard cell viability, proliferation and colony formation assays as well as flow cytometry were utilized to assess cell growth and survival following forced expression or gene silencing of Sin1 gene, as well as after treatment with ALK and STAT3 inhibitors. In a cohort of 32 previously untreated patients with ALK+ ALCL, Sin1 protein expression was assessed by immunohistochemistry performed on a tissue microarray.
Results
Sin1.1 and Sin1.5 were the main isoforms detected in immunoblots and they were differentially expressed among ALK+ and ALK- ALCL cell lines. Sin1 protein was co-localized with activated (Ser473-phosphorylated) AKT kinase in ALCL cells. Treatment of ALK+ ALCL cells with Crizotinib led to reduced expression and de-phosphorylation of Sin1 protein. Forced expression of Sin1.1 and Sin1.5 isoforms resulted in increased cell proliferation in ALK+ ALCL cells with downregulation of the CDK inhibitor p21. Inversely, knocking down Sin1 gene by two specific Sin1-shRNA constructs resulted in dramatic decrease of cell viability and colony formation (by 80%) of ALK+ ALCL cells, which was associated with AKT de-activation and downregulation of the anti-apoptotic BCL-XL and MCL-1 proteins. Using our in vitro system and ex vivo (xenografts) mouse model, Sin1 gene expression was, at least in part, regulated by STAT3 transcription factor. In the cohort of ALK+ ALCL patients, Sin1 protein was expressed in all 32 tumors studied with cytoplasmic and nuclear pattern.
Conclusion
Taken together, these novel findings suggest that mTORC2 complex, through Sin1-dependent activation of AKT and possibly other AKT-independent functions, may contribute to cell cycle and apoptosis deregulation and oncogenesis in ALCL. Thus, modulation of mTORC2 activity might represent a novel target for investigational therapy in patients with ALCL.
Session topic: E-poster
Keyword(s): Akt, ALK/ALCL, MTOR, STAT3
Abstract: E1374
Type: Eposter Presentation
Background
ALK+ anaplastic large cell lymphoma (ALK+ ALCL) is characterized by chromosomal translocations of ALK gene locus, the most frequent being the t(2;5)(p23;q35) resulting in aberrrant expression and activation of the NPM-ALK oncoprotein. The NPM-ALK activates multiple oncogenic pathways including the mTOR-Raptor (mTORC1) pathway, however, the potential role of mTOR-Rictor (mTORC2) complex in ALCL pathogenesis is yet unknown.
Aims
To investigate the potential role of Sin1, a critical component of the mTORC2 complex required for its integrity and activation, in ALK+ ALCL pathogenesis.
Methods
Expression, phosphorylation, and localization of Sin1 protein were assessed by Western blot after subcellular fractionation, immunofluorescence and confocal microscopy in 3 ALK+ and 2 ALK- ALCL cell lines. Physical interaction between various proteins was assessed by co-immunoprecipitation assays. Real-time RT PCR was used to assess Sin1 gene products at the RNA level. Transfection experiments were performed in BaF3 cells and in ALK+ and ALK- ALCL cells using NPM-ALK and Sin1 expressing plasmids and Sin1-shRNA constructs (gene silencing). Standard cell viability, proliferation and colony formation assays as well as flow cytometry were utilized to assess cell growth and survival following forced expression or gene silencing of Sin1 gene, as well as after treatment with ALK and STAT3 inhibitors. In a cohort of 32 previously untreated patients with ALK+ ALCL, Sin1 protein expression was assessed by immunohistochemistry performed on a tissue microarray.
Results
Sin1.1 and Sin1.5 were the main isoforms detected in immunoblots and they were differentially expressed among ALK+ and ALK- ALCL cell lines. Sin1 protein was co-localized with activated (Ser473-phosphorylated) AKT kinase in ALCL cells. Treatment of ALK+ ALCL cells with Crizotinib led to reduced expression and de-phosphorylation of Sin1 protein. Forced expression of Sin1.1 and Sin1.5 isoforms resulted in increased cell proliferation in ALK+ ALCL cells with downregulation of the CDK inhibitor p21. Inversely, knocking down Sin1 gene by two specific Sin1-shRNA constructs resulted in dramatic decrease of cell viability and colony formation (by 80%) of ALK+ ALCL cells, which was associated with AKT de-activation and downregulation of the anti-apoptotic BCL-XL and MCL-1 proteins. Using our in vitro system and ex vivo (xenografts) mouse model, Sin1 gene expression was, at least in part, regulated by STAT3 transcription factor. In the cohort of ALK+ ALCL patients, Sin1 protein was expressed in all 32 tumors studied with cytoplasmic and nuclear pattern.
Conclusion
Taken together, these novel findings suggest that mTORC2 complex, through Sin1-dependent activation of AKT and possibly other AKT-independent functions, may contribute to cell cycle and apoptosis deregulation and oncogenesis in ALCL. Thus, modulation of mTORC2 activity might represent a novel target for investigational therapy in patients with ALCL.
Session topic: E-poster
Keyword(s): Akt, ALK/ALCL, MTOR, STAT3
Type: Eposter Presentation
Background
ALK+ anaplastic large cell lymphoma (ALK+ ALCL) is characterized by chromosomal translocations of ALK gene locus, the most frequent being the t(2;5)(p23;q35) resulting in aberrrant expression and activation of the NPM-ALK oncoprotein. The NPM-ALK activates multiple oncogenic pathways including the mTOR-Raptor (mTORC1) pathway, however, the potential role of mTOR-Rictor (mTORC2) complex in ALCL pathogenesis is yet unknown.
Aims
To investigate the potential role of Sin1, a critical component of the mTORC2 complex required for its integrity and activation, in ALK+ ALCL pathogenesis.
Methods
Expression, phosphorylation, and localization of Sin1 protein were assessed by Western blot after subcellular fractionation, immunofluorescence and confocal microscopy in 3 ALK+ and 2 ALK- ALCL cell lines. Physical interaction between various proteins was assessed by co-immunoprecipitation assays. Real-time RT PCR was used to assess Sin1 gene products at the RNA level. Transfection experiments were performed in BaF3 cells and in ALK+ and ALK- ALCL cells using NPM-ALK and Sin1 expressing plasmids and Sin1-shRNA constructs (gene silencing). Standard cell viability, proliferation and colony formation assays as well as flow cytometry were utilized to assess cell growth and survival following forced expression or gene silencing of Sin1 gene, as well as after treatment with ALK and STAT3 inhibitors. In a cohort of 32 previously untreated patients with ALK+ ALCL, Sin1 protein expression was assessed by immunohistochemistry performed on a tissue microarray.
Results
Sin1.1 and Sin1.5 were the main isoforms detected in immunoblots and they were differentially expressed among ALK+ and ALK- ALCL cell lines. Sin1 protein was co-localized with activated (Ser473-phosphorylated) AKT kinase in ALCL cells. Treatment of ALK+ ALCL cells with Crizotinib led to reduced expression and de-phosphorylation of Sin1 protein. Forced expression of Sin1.1 and Sin1.5 isoforms resulted in increased cell proliferation in ALK+ ALCL cells with downregulation of the CDK inhibitor p21. Inversely, knocking down Sin1 gene by two specific Sin1-shRNA constructs resulted in dramatic decrease of cell viability and colony formation (by 80%) of ALK+ ALCL cells, which was associated with AKT de-activation and downregulation of the anti-apoptotic BCL-XL and MCL-1 proteins. Using our in vitro system and ex vivo (xenografts) mouse model, Sin1 gene expression was, at least in part, regulated by STAT3 transcription factor. In the cohort of ALK+ ALCL patients, Sin1 protein was expressed in all 32 tumors studied with cytoplasmic and nuclear pattern.
Conclusion
Taken together, these novel findings suggest that mTORC2 complex, through Sin1-dependent activation of AKT and possibly other AKT-independent functions, may contribute to cell cycle and apoptosis deregulation and oncogenesis in ALCL. Thus, modulation of mTORC2 activity might represent a novel target for investigational therapy in patients with ALCL.
Session topic: E-poster
Keyword(s): Akt, ALK/ALCL, MTOR, STAT3
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