THE B-CELL RECEPTOR PATHWAY IS HIGHLY ACTIVED IN CANINE DLBCL CELL LINE CLBL-1
(Abstract release date: 05/21/15)
EHA Library. Zoellner A. 06/12/15; 102606; PB1939
Disclosure(s): LMU
Anna Zoellner
Contributions
Contributions
Abstract
Abstract: PB1939
Type: Publication Only
Background
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of canine lymphoma and shows significant similarities in its clinical and biological presentation to human DLBCL. Since the majority of canine DLBCL appears to have genetic parallels to the human Activated B-Cell (ABC) subtype of DLBCL, analysis of the biology and evaluation of possible treatment targets in canine DLBCL could supply valuable insights about the disease.
Aims
Here we demonstrate the expression of hallmark kinases of the B-cell receptor pathway in the canine DLBCL cell line CLBL-1. Further we investigated the treatment with B-cell receptor pathway interacting compounds such as BTK inhibitor Ibrutinib and PI3K inhibitor Idelalisib and substances lately known for their anti-lymphoid activity.
Methods
Established canine DLBCL (CLBL-1) and T-cell lymphoma (CL-1) cell lines were cultivated under standard conditions in RPMI and exposed to ascending doses of Idelalisib, Ibrutinib, Temsirolimus, BX-912, Ku-63764, Enzastaurin and Bortezomib. Cell proliferation was determined after 48 hours based on WST cell proliferation assay. Western blotting was performed after 24h. All experiments were performed at least in triplicates.
Results
In Western blot analysis kinases hallmarking the B-cell receptor- PI3K-AKT pathway (AKT, PDK, PI3K, mTOR) and their phosphorylated isoforms were detected in CLBL-1 cells. Furthermore untreated CLBL-1 cells expressed p42/44, p38, MEK, GSK alpha and GSK beta and their phosphorylated isoforms as well as the cyclins CDK2, CDK4, CDK7, CDK9 but no cyclin D1. Significantly, treatment with only 1,25nM Ibrutinib induced in WST analysis a growth reduction to 45%, with 1µM arresting growth thoroughly. The PI3K-delta inhibitor Idelalisib showed dose dependent effects: 0,6µM reduced cell growth to 41%, whereas 5µM reduced proliferation to 13%. The mTor inhibitor Temsirolimus showed high efficacy: 1,25nM Temsirolimus reduced cell proliferation to 38%, while the mtorc1/mtorc2 inhibitor Ku-63794 induced at the dose of 0,25µM a reduction to 49%. CLBL-1 was also sensitive towards other compounds with anti-lymphoid activity such as the PDK-1 inhibitor BX-912 (0,25µM; 20%), the PKC inhibitor Enzastaurin (1,25µM; 52%) and the proteasome inhibitor Bortezomib (5nM; 50%). To identify B-cell receptor pathway specific mechanisms the T-cell lymphoma cell line CL-1 was treated as control. Increasing doses of Ibrutinib, Idelalisib and Enzastaurin (up to 5µM) and Temsirolimus (up to 20nM) did not have significant effects.
Summary
The detected activated B-cell receptor pathway in CLBL-1 and the sensitivity towards small molecule inhibitors targeting this pathway indicates the similarity to human ABC DLBCL. This data strongly supports the relevance of canine DLBCL as model for its human counterpart.
Keyword(s): Canine
Type: Publication Only
Background
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of canine lymphoma and shows significant similarities in its clinical and biological presentation to human DLBCL. Since the majority of canine DLBCL appears to have genetic parallels to the human Activated B-Cell (ABC) subtype of DLBCL, analysis of the biology and evaluation of possible treatment targets in canine DLBCL could supply valuable insights about the disease.
Aims
Here we demonstrate the expression of hallmark kinases of the B-cell receptor pathway in the canine DLBCL cell line CLBL-1. Further we investigated the treatment with B-cell receptor pathway interacting compounds such as BTK inhibitor Ibrutinib and PI3K inhibitor Idelalisib and substances lately known for their anti-lymphoid activity.
Methods
Established canine DLBCL (CLBL-1) and T-cell lymphoma (CL-1) cell lines were cultivated under standard conditions in RPMI and exposed to ascending doses of Idelalisib, Ibrutinib, Temsirolimus, BX-912, Ku-63764, Enzastaurin and Bortezomib. Cell proliferation was determined after 48 hours based on WST cell proliferation assay. Western blotting was performed after 24h. All experiments were performed at least in triplicates.
Results
In Western blot analysis kinases hallmarking the B-cell receptor- PI3K-AKT pathway (AKT, PDK, PI3K, mTOR) and their phosphorylated isoforms were detected in CLBL-1 cells. Furthermore untreated CLBL-1 cells expressed p42/44, p38, MEK, GSK alpha and GSK beta and their phosphorylated isoforms as well as the cyclins CDK2, CDK4, CDK7, CDK9 but no cyclin D1. Significantly, treatment with only 1,25nM Ibrutinib induced in WST analysis a growth reduction to 45%, with 1µM arresting growth thoroughly. The PI3K-delta inhibitor Idelalisib showed dose dependent effects: 0,6µM reduced cell growth to 41%, whereas 5µM reduced proliferation to 13%. The mTor inhibitor Temsirolimus showed high efficacy: 1,25nM Temsirolimus reduced cell proliferation to 38%, while the mtorc1/mtorc2 inhibitor Ku-63794 induced at the dose of 0,25µM a reduction to 49%. CLBL-1 was also sensitive towards other compounds with anti-lymphoid activity such as the PDK-1 inhibitor BX-912 (0,25µM; 20%), the PKC inhibitor Enzastaurin (1,25µM; 52%) and the proteasome inhibitor Bortezomib (5nM; 50%). To identify B-cell receptor pathway specific mechanisms the T-cell lymphoma cell line CL-1 was treated as control. Increasing doses of Ibrutinib, Idelalisib and Enzastaurin (up to 5µM) and Temsirolimus (up to 20nM) did not have significant effects.
Summary
The detected activated B-cell receptor pathway in CLBL-1 and the sensitivity towards small molecule inhibitors targeting this pathway indicates the similarity to human ABC DLBCL. This data strongly supports the relevance of canine DLBCL as model for its human counterpart.
Keyword(s): Canine
Abstract: PB1939
Type: Publication Only
Background
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of canine lymphoma and shows significant similarities in its clinical and biological presentation to human DLBCL. Since the majority of canine DLBCL appears to have genetic parallels to the human Activated B-Cell (ABC) subtype of DLBCL, analysis of the biology and evaluation of possible treatment targets in canine DLBCL could supply valuable insights about the disease.
Aims
Here we demonstrate the expression of hallmark kinases of the B-cell receptor pathway in the canine DLBCL cell line CLBL-1. Further we investigated the treatment with B-cell receptor pathway interacting compounds such as BTK inhibitor Ibrutinib and PI3K inhibitor Idelalisib and substances lately known for their anti-lymphoid activity.
Methods
Established canine DLBCL (CLBL-1) and T-cell lymphoma (CL-1) cell lines were cultivated under standard conditions in RPMI and exposed to ascending doses of Idelalisib, Ibrutinib, Temsirolimus, BX-912, Ku-63764, Enzastaurin and Bortezomib. Cell proliferation was determined after 48 hours based on WST cell proliferation assay. Western blotting was performed after 24h. All experiments were performed at least in triplicates.
Results
In Western blot analysis kinases hallmarking the B-cell receptor- PI3K-AKT pathway (AKT, PDK, PI3K, mTOR) and their phosphorylated isoforms were detected in CLBL-1 cells. Furthermore untreated CLBL-1 cells expressed p42/44, p38, MEK, GSK alpha and GSK beta and their phosphorylated isoforms as well as the cyclins CDK2, CDK4, CDK7, CDK9 but no cyclin D1. Significantly, treatment with only 1,25nM Ibrutinib induced in WST analysis a growth reduction to 45%, with 1µM arresting growth thoroughly. The PI3K-delta inhibitor Idelalisib showed dose dependent effects: 0,6µM reduced cell growth to 41%, whereas 5µM reduced proliferation to 13%. The mTor inhibitor Temsirolimus showed high efficacy: 1,25nM Temsirolimus reduced cell proliferation to 38%, while the mtorc1/mtorc2 inhibitor Ku-63794 induced at the dose of 0,25µM a reduction to 49%. CLBL-1 was also sensitive towards other compounds with anti-lymphoid activity such as the PDK-1 inhibitor BX-912 (0,25µM; 20%), the PKC inhibitor Enzastaurin (1,25µM; 52%) and the proteasome inhibitor Bortezomib (5nM; 50%). To identify B-cell receptor pathway specific mechanisms the T-cell lymphoma cell line CL-1 was treated as control. Increasing doses of Ibrutinib, Idelalisib and Enzastaurin (up to 5µM) and Temsirolimus (up to 20nM) did not have significant effects.
Summary
The detected activated B-cell receptor pathway in CLBL-1 and the sensitivity towards small molecule inhibitors targeting this pathway indicates the similarity to human ABC DLBCL. This data strongly supports the relevance of canine DLBCL as model for its human counterpart.
Keyword(s): Canine
Type: Publication Only
Background
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of canine lymphoma and shows significant similarities in its clinical and biological presentation to human DLBCL. Since the majority of canine DLBCL appears to have genetic parallels to the human Activated B-Cell (ABC) subtype of DLBCL, analysis of the biology and evaluation of possible treatment targets in canine DLBCL could supply valuable insights about the disease.
Aims
Here we demonstrate the expression of hallmark kinases of the B-cell receptor pathway in the canine DLBCL cell line CLBL-1. Further we investigated the treatment with B-cell receptor pathway interacting compounds such as BTK inhibitor Ibrutinib and PI3K inhibitor Idelalisib and substances lately known for their anti-lymphoid activity.
Methods
Established canine DLBCL (CLBL-1) and T-cell lymphoma (CL-1) cell lines were cultivated under standard conditions in RPMI and exposed to ascending doses of Idelalisib, Ibrutinib, Temsirolimus, BX-912, Ku-63764, Enzastaurin and Bortezomib. Cell proliferation was determined after 48 hours based on WST cell proliferation assay. Western blotting was performed after 24h. All experiments were performed at least in triplicates.
Results
In Western blot analysis kinases hallmarking the B-cell receptor- PI3K-AKT pathway (AKT, PDK, PI3K, mTOR) and their phosphorylated isoforms were detected in CLBL-1 cells. Furthermore untreated CLBL-1 cells expressed p42/44, p38, MEK, GSK alpha and GSK beta and their phosphorylated isoforms as well as the cyclins CDK2, CDK4, CDK7, CDK9 but no cyclin D1. Significantly, treatment with only 1,25nM Ibrutinib induced in WST analysis a growth reduction to 45%, with 1µM arresting growth thoroughly. The PI3K-delta inhibitor Idelalisib showed dose dependent effects: 0,6µM reduced cell growth to 41%, whereas 5µM reduced proliferation to 13%. The mTor inhibitor Temsirolimus showed high efficacy: 1,25nM Temsirolimus reduced cell proliferation to 38%, while the mtorc1/mtorc2 inhibitor Ku-63794 induced at the dose of 0,25µM a reduction to 49%. CLBL-1 was also sensitive towards other compounds with anti-lymphoid activity such as the PDK-1 inhibitor BX-912 (0,25µM; 20%), the PKC inhibitor Enzastaurin (1,25µM; 52%) and the proteasome inhibitor Bortezomib (5nM; 50%). To identify B-cell receptor pathway specific mechanisms the T-cell lymphoma cell line CL-1 was treated as control. Increasing doses of Ibrutinib, Idelalisib and Enzastaurin (up to 5µM) and Temsirolimus (up to 20nM) did not have significant effects.
Summary
The detected activated B-cell receptor pathway in CLBL-1 and the sensitivity towards small molecule inhibitors targeting this pathway indicates the similarity to human ABC DLBCL. This data strongly supports the relevance of canine DLBCL as model for its human counterpart.
Keyword(s): Canine
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