FYN INHIBITOR INDUCES APOPTOSIS IN NK LEUKEMIA CELLS
(Abstract release date: 05/19/16)
EHA Library. Laurenzana I. 06/09/16; 132590; E1041
Dr. Ilaria Laurenzana
Contributions
Contributions
Abstract
Abstract: E1041
Type: Eposter Presentation
Background
Fyn protein, a member of Src-family kinase, has different biological functions including growth factor/cytokine receptor, T and B-cell receptor and integrin-mediated signaling, cell-cell adhesion, mitosis, differentiation of natural killer (NK) cells. Some studies show its involvement in hematologic malignancies. However, the tissue-specific pattern of Fyn mRNA expression indicate that it is more expressed in NK and T cells respect to other human tissues. NK cell neoplasms are a rare and heterogeneous group of disorders characterized by increased proliferation of cytotoxic NK cells. They include aggressive NK leukemia (ANKL) and chronic lymphoproliferative disorder of NK cells (NK-CLPD). ANKL has a fulminant clinical course frequently resulting in death within two months; NK-CLPD is typically indolent. Since no standard therapies for immature NK cell neoplasms have been established so far, new therapeutic options are needed. Tintori et al. found a new Fyn inhibitor by a structure-based drug design protocol followed by hit-to-lead optimization. The 4c pyrazolo[3,4-d]pyrimidine compound showed antiproliferative activity against different cancer cell lines.
Aims
The aim of this study was to determine the effect of 4c compound in NK leukemic cells.
Methods
Fyn mRNA level was evaluated in peripheral blood mononuclear cells (PBMC) from 10 healthy controls (HC) and 8 NK-CLPD patients by qRT-PCR; protein level was evaluated in PBMC from 3 HC and 3 NK-CLPD and in 2 ANKL cell lines (KHYG1 and NK92) by western blotting (WB). The 4c compound was tested in a time course MTS assay at increasing concentrations on HC-PBMC, KHYG1 and NK92. KHYG1 were treated with 4c at 4µM for 24h to evaluate apoptosis and cell cycle by cytometric analysis, as well as gene expression profile by Illumina HiScanSQ. Fyn immunoprecipitation and proteome profile arrays of apoptosis and phospho-kinase were also performed. Expression of procaspase3, active caspase3, P70 and Akt was validated by WB. 4c compound was tested also in 3 NK-CLPD e 1 ANKL patients. Cell viability was assessed by trypan blue dye count method and apoptosis was verified by cytometric analysis.
Results
qRT-PCR data showed a significant overexpression of Fyn level in PBMC of NK-CLPD patients compared to HC. Protein level was highest in NK-CLPD patients (p<0.001) and NK cell lines (p<0.0001) respect to HC. To verify the effect of 4c compound, we treated KHYG1 and NK92 with different concentrations, showing a reduction of cell viability (EC50= 4µM and 14µM respectively at 24h); on the contrary, there was no effect on HC-PBMC. Moreover, this treatment induced a significant caspase3 dependent apoptosis (p<0.01) and G2/M cell cycle arrest. Gene expression analysis of 4c treated or not KHYG1 identified 455 down-regulated and 326 up-regulated genes, most of which involved in cell death function, thus confirming the apoptosis data. To corroborate Fyn inhibition by 4c treatment, we detected a reduction of its phosphorylation and we also observed a decrease of phospho-Akt and phospho-P70, two proteins involved in apoptotic process and cell proliferation. Interestingly, 4c treatment reduced cell viability and activated caspase3 dependent apoptosis (p<0.05) in 3 NK-CLPD and 1 ANKL patients.
Conclusion
In this study we demonstrated a higher Fyn expression in NK leukemia patients compared to healthy controls. We also demonstrated an apoptotic effect of pyrazolo[3,4-d]pyrimidine 4c compound on NK leukemia cells and we provided in vitro preliminary evidences that Fyn is its possible cellular target.
Session topic: E-poster
Keyword(s): Kinase inhibitor, NK cell, Src kinase
Type: Eposter Presentation
Background
Fyn protein, a member of Src-family kinase, has different biological functions including growth factor/cytokine receptor, T and B-cell receptor and integrin-mediated signaling, cell-cell adhesion, mitosis, differentiation of natural killer (NK) cells. Some studies show its involvement in hematologic malignancies. However, the tissue-specific pattern of Fyn mRNA expression indicate that it is more expressed in NK and T cells respect to other human tissues. NK cell neoplasms are a rare and heterogeneous group of disorders characterized by increased proliferation of cytotoxic NK cells. They include aggressive NK leukemia (ANKL) and chronic lymphoproliferative disorder of NK cells (NK-CLPD). ANKL has a fulminant clinical course frequently resulting in death within two months; NK-CLPD is typically indolent. Since no standard therapies for immature NK cell neoplasms have been established so far, new therapeutic options are needed. Tintori et al. found a new Fyn inhibitor by a structure-based drug design protocol followed by hit-to-lead optimization. The 4c pyrazolo[3,4-d]pyrimidine compound showed antiproliferative activity against different cancer cell lines.
Aims
The aim of this study was to determine the effect of 4c compound in NK leukemic cells.
Methods
Fyn mRNA level was evaluated in peripheral blood mononuclear cells (PBMC) from 10 healthy controls (HC) and 8 NK-CLPD patients by qRT-PCR; protein level was evaluated in PBMC from 3 HC and 3 NK-CLPD and in 2 ANKL cell lines (KHYG1 and NK92) by western blotting (WB). The 4c compound was tested in a time course MTS assay at increasing concentrations on HC-PBMC, KHYG1 and NK92. KHYG1 were treated with 4c at 4µM for 24h to evaluate apoptosis and cell cycle by cytometric analysis, as well as gene expression profile by Illumina HiScanSQ. Fyn immunoprecipitation and proteome profile arrays of apoptosis and phospho-kinase were also performed. Expression of procaspase3, active caspase3, P70 and Akt was validated by WB. 4c compound was tested also in 3 NK-CLPD e 1 ANKL patients. Cell viability was assessed by trypan blue dye count method and apoptosis was verified by cytometric analysis.
Results
qRT-PCR data showed a significant overexpression of Fyn level in PBMC of NK-CLPD patients compared to HC. Protein level was highest in NK-CLPD patients (p<0.001) and NK cell lines (p<0.0001) respect to HC. To verify the effect of 4c compound, we treated KHYG1 and NK92 with different concentrations, showing a reduction of cell viability (EC50= 4µM and 14µM respectively at 24h); on the contrary, there was no effect on HC-PBMC. Moreover, this treatment induced a significant caspase3 dependent apoptosis (p<0.01) and G2/M cell cycle arrest. Gene expression analysis of 4c treated or not KHYG1 identified 455 down-regulated and 326 up-regulated genes, most of which involved in cell death function, thus confirming the apoptosis data. To corroborate Fyn inhibition by 4c treatment, we detected a reduction of its phosphorylation and we also observed a decrease of phospho-Akt and phospho-P70, two proteins involved in apoptotic process and cell proliferation. Interestingly, 4c treatment reduced cell viability and activated caspase3 dependent apoptosis (p<0.05) in 3 NK-CLPD and 1 ANKL patients.
Conclusion
In this study we demonstrated a higher Fyn expression in NK leukemia patients compared to healthy controls. We also demonstrated an apoptotic effect of pyrazolo[3,4-d]pyrimidine 4c compound on NK leukemia cells and we provided in vitro preliminary evidences that Fyn is its possible cellular target.
Session topic: E-poster
Keyword(s): Kinase inhibitor, NK cell, Src kinase
Abstract: E1041
Type: Eposter Presentation
Background
Fyn protein, a member of Src-family kinase, has different biological functions including growth factor/cytokine receptor, T and B-cell receptor and integrin-mediated signaling, cell-cell adhesion, mitosis, differentiation of natural killer (NK) cells. Some studies show its involvement in hematologic malignancies. However, the tissue-specific pattern of Fyn mRNA expression indicate that it is more expressed in NK and T cells respect to other human tissues. NK cell neoplasms are a rare and heterogeneous group of disorders characterized by increased proliferation of cytotoxic NK cells. They include aggressive NK leukemia (ANKL) and chronic lymphoproliferative disorder of NK cells (NK-CLPD). ANKL has a fulminant clinical course frequently resulting in death within two months; NK-CLPD is typically indolent. Since no standard therapies for immature NK cell neoplasms have been established so far, new therapeutic options are needed. Tintori et al. found a new Fyn inhibitor by a structure-based drug design protocol followed by hit-to-lead optimization. The 4c pyrazolo[3,4-d]pyrimidine compound showed antiproliferative activity against different cancer cell lines.
Aims
The aim of this study was to determine the effect of 4c compound in NK leukemic cells.
Methods
Fyn mRNA level was evaluated in peripheral blood mononuclear cells (PBMC) from 10 healthy controls (HC) and 8 NK-CLPD patients by qRT-PCR; protein level was evaluated in PBMC from 3 HC and 3 NK-CLPD and in 2 ANKL cell lines (KHYG1 and NK92) by western blotting (WB). The 4c compound was tested in a time course MTS assay at increasing concentrations on HC-PBMC, KHYG1 and NK92. KHYG1 were treated with 4c at 4µM for 24h to evaluate apoptosis and cell cycle by cytometric analysis, as well as gene expression profile by Illumina HiScanSQ. Fyn immunoprecipitation and proteome profile arrays of apoptosis and phospho-kinase were also performed. Expression of procaspase3, active caspase3, P70 and Akt was validated by WB. 4c compound was tested also in 3 NK-CLPD e 1 ANKL patients. Cell viability was assessed by trypan blue dye count method and apoptosis was verified by cytometric analysis.
Results
qRT-PCR data showed a significant overexpression of Fyn level in PBMC of NK-CLPD patients compared to HC. Protein level was highest in NK-CLPD patients (p<0.001) and NK cell lines (p<0.0001) respect to HC. To verify the effect of 4c compound, we treated KHYG1 and NK92 with different concentrations, showing a reduction of cell viability (EC50= 4µM and 14µM respectively at 24h); on the contrary, there was no effect on HC-PBMC. Moreover, this treatment induced a significant caspase3 dependent apoptosis (p<0.01) and G2/M cell cycle arrest. Gene expression analysis of 4c treated or not KHYG1 identified 455 down-regulated and 326 up-regulated genes, most of which involved in cell death function, thus confirming the apoptosis data. To corroborate Fyn inhibition by 4c treatment, we detected a reduction of its phosphorylation and we also observed a decrease of phospho-Akt and phospho-P70, two proteins involved in apoptotic process and cell proliferation. Interestingly, 4c treatment reduced cell viability and activated caspase3 dependent apoptosis (p<0.05) in 3 NK-CLPD and 1 ANKL patients.
Conclusion
In this study we demonstrated a higher Fyn expression in NK leukemia patients compared to healthy controls. We also demonstrated an apoptotic effect of pyrazolo[3,4-d]pyrimidine 4c compound on NK leukemia cells and we provided in vitro preliminary evidences that Fyn is its possible cellular target.
Session topic: E-poster
Keyword(s): Kinase inhibitor, NK cell, Src kinase
Type: Eposter Presentation
Background
Fyn protein, a member of Src-family kinase, has different biological functions including growth factor/cytokine receptor, T and B-cell receptor and integrin-mediated signaling, cell-cell adhesion, mitosis, differentiation of natural killer (NK) cells. Some studies show its involvement in hematologic malignancies. However, the tissue-specific pattern of Fyn mRNA expression indicate that it is more expressed in NK and T cells respect to other human tissues. NK cell neoplasms are a rare and heterogeneous group of disorders characterized by increased proliferation of cytotoxic NK cells. They include aggressive NK leukemia (ANKL) and chronic lymphoproliferative disorder of NK cells (NK-CLPD). ANKL has a fulminant clinical course frequently resulting in death within two months; NK-CLPD is typically indolent. Since no standard therapies for immature NK cell neoplasms have been established so far, new therapeutic options are needed. Tintori et al. found a new Fyn inhibitor by a structure-based drug design protocol followed by hit-to-lead optimization. The 4c pyrazolo[3,4-d]pyrimidine compound showed antiproliferative activity against different cancer cell lines.
Aims
The aim of this study was to determine the effect of 4c compound in NK leukemic cells.
Methods
Fyn mRNA level was evaluated in peripheral blood mononuclear cells (PBMC) from 10 healthy controls (HC) and 8 NK-CLPD patients by qRT-PCR; protein level was evaluated in PBMC from 3 HC and 3 NK-CLPD and in 2 ANKL cell lines (KHYG1 and NK92) by western blotting (WB). The 4c compound was tested in a time course MTS assay at increasing concentrations on HC-PBMC, KHYG1 and NK92. KHYG1 were treated with 4c at 4µM for 24h to evaluate apoptosis and cell cycle by cytometric analysis, as well as gene expression profile by Illumina HiScanSQ. Fyn immunoprecipitation and proteome profile arrays of apoptosis and phospho-kinase were also performed. Expression of procaspase3, active caspase3, P70 and Akt was validated by WB. 4c compound was tested also in 3 NK-CLPD e 1 ANKL patients. Cell viability was assessed by trypan blue dye count method and apoptosis was verified by cytometric analysis.
Results
qRT-PCR data showed a significant overexpression of Fyn level in PBMC of NK-CLPD patients compared to HC. Protein level was highest in NK-CLPD patients (p<0.001) and NK cell lines (p<0.0001) respect to HC. To verify the effect of 4c compound, we treated KHYG1 and NK92 with different concentrations, showing a reduction of cell viability (EC50= 4µM and 14µM respectively at 24h); on the contrary, there was no effect on HC-PBMC. Moreover, this treatment induced a significant caspase3 dependent apoptosis (p<0.01) and G2/M cell cycle arrest. Gene expression analysis of 4c treated or not KHYG1 identified 455 down-regulated and 326 up-regulated genes, most of which involved in cell death function, thus confirming the apoptosis data. To corroborate Fyn inhibition by 4c treatment, we detected a reduction of its phosphorylation and we also observed a decrease of phospho-Akt and phospho-P70, two proteins involved in apoptotic process and cell proliferation. Interestingly, 4c treatment reduced cell viability and activated caspase3 dependent apoptosis (p<0.05) in 3 NK-CLPD and 1 ANKL patients.
Conclusion
In this study we demonstrated a higher Fyn expression in NK leukemia patients compared to healthy controls. We also demonstrated an apoptotic effect of pyrazolo[3,4-d]pyrimidine 4c compound on NK leukemia cells and we provided in vitro preliminary evidences that Fyn is its possible cellular target.
Session topic: E-poster
Keyword(s): Kinase inhibitor, NK cell, Src kinase
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