CHECKPOINT KINASE 1 INHIBITION POTENTIATES APOPTOSIS AND INFLICTS MITOTIC FAILURE IN CLL-DERIVED MEC-1 CELL LINE
(Abstract release date: 05/19/16)
EHA Library. Zemanova J. 06/09/16; 132584; E1035
Ms. Jana Zemanova
Contributions
Contributions
Abstract
Abstract: E1035
Type: Eposter Presentation
Background
Treatment options for patients with TP53-mutated lymphoid tumors are very limited. In chronic lymphocytic leukemia (CLL), TP53 defects were not surmounted by chemoimmunotherapy regimens functioning in other patients, and also remain challenging for innovative small-molecule inhibitors of B-cell receptor signaling. Recently, the inhibition of ataxia-telangiectasia mutated and Rad-3 related (ATR) kinase operating in the DNA damage response (DDR) pathway has been identified as a potential alternative therapeutic strategy in CLL. ATR is a key molecule activating the DDR pathway through phosphorylation of checkpoint kinase 1 (Chk1) upon recognition of DNA damage.
Aims
(1) to test the Chk1 inhibition potential for sensitizing TP53-mutated CLL-derived MEC-1 and MEC-2 cell lines to nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine, and (2) to analyze appropriate cell death mechanisms in MEC-1 cells.
Methods
We used highly specific Chk1 inhibitor SCH900776 and measured (a) overall cell viability using a metabolic WST-1 assay, (b) DNA double strand breaks (DSBs) accumulation using pS139 detection on H2AX (γ-H2AX), and (c) apoptosis and cell cycle profile using annexin-V/PI and DNA content staining, respectively. In case of fludarabine, we also analyzed structure of mitotic chromosomes and employed a cell-live imaging for direct cell death monitoring.
Results
The MEC-1 cell line was significantly (P<0.01; two-way ANOVA) sensitized to all three NAs using 200 nM SCH900776, as evidenced by 72 h cell cultivation and concentration-dependent curves of viability. Interestingly, the serial sister cell line MEC-2 established from the same patient one year later completely lost the Chk1 inhibition-mediated sensitization to fludarabine. Analyzing further cell death mechanisms in MEC-1 cells, we observed that SCH900776 greatly facilitated γ-H2AX accumulation in the co-treatments with all three NAs and in all tested time intervals (4, 14, 24, and 48 h). Accordingly, apoptosis was clearly augmented in the co-treatments compared to single agent treatments with NAs. In the cell cycle analysis, an increase of sub-G1 (apoptotic) fraction in the co-treatments with Chk1 inhibitor resulted in all three NAs from diminished post-G1 populations (S and G2/M). The cytogenetic analysis for the co-treatment of SCH900776 with fludarabine disclosed a substantially reduced mitotic index (0.6% compared to 15.8% in control), and severe chromosome pulverization in cells undergoing mitosis (detected in 42% of analyzed mitotic cells). By contrast, this pulverization was only occasionally observed in single agent treatment with fludarabine (4%) and never present in control untreated MEC-1 cells. The cell-live imaging then disclosed several abnormal features during mitosis including (a) hardly recognizable metaphase plate, (b) inner cell mass asymmetric division between daughter cells, (c) accelerated time course of division process involving approximately 14 minutes (compared to 22 minutes in untreated cells), and (d) signs of apoptosis such as membrane blebbing.
Conclusion
The intrinsic resistance of TP53-mutated CLL cells to NAs can be significantly surmounted by Chk1 inhibition leading to cell cycle checkpoints abrogation and augmented DNA damage. Further testing of clinical candidate SCH900776 and other specific Chk1 inhibitors in CLL is justified and particularly appropriate among TP53 mutated patients. Supported by grant 15-33999A (MH CR), project MUNI/A/1028/2015, grant CZ.1.05/1.1.00/02.0123 (MEYS CR), and project CEITEC 2020
(LQ1601).
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, DNA damage, P53
Type: Eposter Presentation
Background
Treatment options for patients with TP53-mutated lymphoid tumors are very limited. In chronic lymphocytic leukemia (CLL), TP53 defects were not surmounted by chemoimmunotherapy regimens functioning in other patients, and also remain challenging for innovative small-molecule inhibitors of B-cell receptor signaling. Recently, the inhibition of ataxia-telangiectasia mutated and Rad-3 related (ATR) kinase operating in the DNA damage response (DDR) pathway has been identified as a potential alternative therapeutic strategy in CLL. ATR is a key molecule activating the DDR pathway through phosphorylation of checkpoint kinase 1 (Chk1) upon recognition of DNA damage.
Aims
(1) to test the Chk1 inhibition potential for sensitizing TP53-mutated CLL-derived MEC-1 and MEC-2 cell lines to nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine, and (2) to analyze appropriate cell death mechanisms in MEC-1 cells.
Methods
We used highly specific Chk1 inhibitor SCH900776 and measured (a) overall cell viability using a metabolic WST-1 assay, (b) DNA double strand breaks (DSBs) accumulation using pS139 detection on H2AX (γ-H2AX), and (c) apoptosis and cell cycle profile using annexin-V/PI and DNA content staining, respectively. In case of fludarabine, we also analyzed structure of mitotic chromosomes and employed a cell-live imaging for direct cell death monitoring.
Results
The MEC-1 cell line was significantly (P<0.01; two-way ANOVA) sensitized to all three NAs using 200 nM SCH900776, as evidenced by 72 h cell cultivation and concentration-dependent curves of viability. Interestingly, the serial sister cell line MEC-2 established from the same patient one year later completely lost the Chk1 inhibition-mediated sensitization to fludarabine. Analyzing further cell death mechanisms in MEC-1 cells, we observed that SCH900776 greatly facilitated γ-H2AX accumulation in the co-treatments with all three NAs and in all tested time intervals (4, 14, 24, and 48 h). Accordingly, apoptosis was clearly augmented in the co-treatments compared to single agent treatments with NAs. In the cell cycle analysis, an increase of sub-G1 (apoptotic) fraction in the co-treatments with Chk1 inhibitor resulted in all three NAs from diminished post-G1 populations (S and G2/M). The cytogenetic analysis for the co-treatment of SCH900776 with fludarabine disclosed a substantially reduced mitotic index (0.6% compared to 15.8% in control), and severe chromosome pulverization in cells undergoing mitosis (detected in 42% of analyzed mitotic cells). By contrast, this pulverization was only occasionally observed in single agent treatment with fludarabine (4%) and never present in control untreated MEC-1 cells. The cell-live imaging then disclosed several abnormal features during mitosis including (a) hardly recognizable metaphase plate, (b) inner cell mass asymmetric division between daughter cells, (c) accelerated time course of division process involving approximately 14 minutes (compared to 22 minutes in untreated cells), and (d) signs of apoptosis such as membrane blebbing.
Conclusion
The intrinsic resistance of TP53-mutated CLL cells to NAs can be significantly surmounted by Chk1 inhibition leading to cell cycle checkpoints abrogation and augmented DNA damage. Further testing of clinical candidate SCH900776 and other specific Chk1 inhibitors in CLL is justified and particularly appropriate among TP53 mutated patients. Supported by grant 15-33999A (MH CR), project MUNI/A/1028/2015, grant CZ.1.05/1.1.00/02.0123 (MEYS CR), and project CEITEC 2020
(LQ1601).
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, DNA damage, P53
Abstract: E1035
Type: Eposter Presentation
Background
Treatment options for patients with TP53-mutated lymphoid tumors are very limited. In chronic lymphocytic leukemia (CLL), TP53 defects were not surmounted by chemoimmunotherapy regimens functioning in other patients, and also remain challenging for innovative small-molecule inhibitors of B-cell receptor signaling. Recently, the inhibition of ataxia-telangiectasia mutated and Rad-3 related (ATR) kinase operating in the DNA damage response (DDR) pathway has been identified as a potential alternative therapeutic strategy in CLL. ATR is a key molecule activating the DDR pathway through phosphorylation of checkpoint kinase 1 (Chk1) upon recognition of DNA damage.
Aims
(1) to test the Chk1 inhibition potential for sensitizing TP53-mutated CLL-derived MEC-1 and MEC-2 cell lines to nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine, and (2) to analyze appropriate cell death mechanisms in MEC-1 cells.
Methods
We used highly specific Chk1 inhibitor SCH900776 and measured (a) overall cell viability using a metabolic WST-1 assay, (b) DNA double strand breaks (DSBs) accumulation using pS139 detection on H2AX (γ-H2AX), and (c) apoptosis and cell cycle profile using annexin-V/PI and DNA content staining, respectively. In case of fludarabine, we also analyzed structure of mitotic chromosomes and employed a cell-live imaging for direct cell death monitoring.
Results
The MEC-1 cell line was significantly (P<0.01; two-way ANOVA) sensitized to all three NAs using 200 nM SCH900776, as evidenced by 72 h cell cultivation and concentration-dependent curves of viability. Interestingly, the serial sister cell line MEC-2 established from the same patient one year later completely lost the Chk1 inhibition-mediated sensitization to fludarabine. Analyzing further cell death mechanisms in MEC-1 cells, we observed that SCH900776 greatly facilitated γ-H2AX accumulation in the co-treatments with all three NAs and in all tested time intervals (4, 14, 24, and 48 h). Accordingly, apoptosis was clearly augmented in the co-treatments compared to single agent treatments with NAs. In the cell cycle analysis, an increase of sub-G1 (apoptotic) fraction in the co-treatments with Chk1 inhibitor resulted in all three NAs from diminished post-G1 populations (S and G2/M). The cytogenetic analysis for the co-treatment of SCH900776 with fludarabine disclosed a substantially reduced mitotic index (0.6% compared to 15.8% in control), and severe chromosome pulverization in cells undergoing mitosis (detected in 42% of analyzed mitotic cells). By contrast, this pulverization was only occasionally observed in single agent treatment with fludarabine (4%) and never present in control untreated MEC-1 cells. The cell-live imaging then disclosed several abnormal features during mitosis including (a) hardly recognizable metaphase plate, (b) inner cell mass asymmetric division between daughter cells, (c) accelerated time course of division process involving approximately 14 minutes (compared to 22 minutes in untreated cells), and (d) signs of apoptosis such as membrane blebbing.
Conclusion
The intrinsic resistance of TP53-mutated CLL cells to NAs can be significantly surmounted by Chk1 inhibition leading to cell cycle checkpoints abrogation and augmented DNA damage. Further testing of clinical candidate SCH900776 and other specific Chk1 inhibitors in CLL is justified and particularly appropriate among TP53 mutated patients. Supported by grant 15-33999A (MH CR), project MUNI/A/1028/2015, grant CZ.1.05/1.1.00/02.0123 (MEYS CR), and project CEITEC 2020
(LQ1601).
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, DNA damage, P53
Type: Eposter Presentation
Background
Treatment options for patients with TP53-mutated lymphoid tumors are very limited. In chronic lymphocytic leukemia (CLL), TP53 defects were not surmounted by chemoimmunotherapy regimens functioning in other patients, and also remain challenging for innovative small-molecule inhibitors of B-cell receptor signaling. Recently, the inhibition of ataxia-telangiectasia mutated and Rad-3 related (ATR) kinase operating in the DNA damage response (DDR) pathway has been identified as a potential alternative therapeutic strategy in CLL. ATR is a key molecule activating the DDR pathway through phosphorylation of checkpoint kinase 1 (Chk1) upon recognition of DNA damage.
Aims
(1) to test the Chk1 inhibition potential for sensitizing TP53-mutated CLL-derived MEC-1 and MEC-2 cell lines to nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine, and (2) to analyze appropriate cell death mechanisms in MEC-1 cells.
Methods
We used highly specific Chk1 inhibitor SCH900776 and measured (a) overall cell viability using a metabolic WST-1 assay, (b) DNA double strand breaks (DSBs) accumulation using pS139 detection on H2AX (γ-H2AX), and (c) apoptosis and cell cycle profile using annexin-V/PI and DNA content staining, respectively. In case of fludarabine, we also analyzed structure of mitotic chromosomes and employed a cell-live imaging for direct cell death monitoring.
Results
The MEC-1 cell line was significantly (P<0.01; two-way ANOVA) sensitized to all three NAs using 200 nM SCH900776, as evidenced by 72 h cell cultivation and concentration-dependent curves of viability. Interestingly, the serial sister cell line MEC-2 established from the same patient one year later completely lost the Chk1 inhibition-mediated sensitization to fludarabine. Analyzing further cell death mechanisms in MEC-1 cells, we observed that SCH900776 greatly facilitated γ-H2AX accumulation in the co-treatments with all three NAs and in all tested time intervals (4, 14, 24, and 48 h). Accordingly, apoptosis was clearly augmented in the co-treatments compared to single agent treatments with NAs. In the cell cycle analysis, an increase of sub-G1 (apoptotic) fraction in the co-treatments with Chk1 inhibitor resulted in all three NAs from diminished post-G1 populations (S and G2/M). The cytogenetic analysis for the co-treatment of SCH900776 with fludarabine disclosed a substantially reduced mitotic index (0.6% compared to 15.8% in control), and severe chromosome pulverization in cells undergoing mitosis (detected in 42% of analyzed mitotic cells). By contrast, this pulverization was only occasionally observed in single agent treatment with fludarabine (4%) and never present in control untreated MEC-1 cells. The cell-live imaging then disclosed several abnormal features during mitosis including (a) hardly recognizable metaphase plate, (b) inner cell mass asymmetric division between daughter cells, (c) accelerated time course of division process involving approximately 14 minutes (compared to 22 minutes in untreated cells), and (d) signs of apoptosis such as membrane blebbing.
Conclusion
The intrinsic resistance of TP53-mutated CLL cells to NAs can be significantly surmounted by Chk1 inhibition leading to cell cycle checkpoints abrogation and augmented DNA damage. Further testing of clinical candidate SCH900776 and other specific Chk1 inhibitors in CLL is justified and particularly appropriate among TP53 mutated patients. Supported by grant 15-33999A (MH CR), project MUNI/A/1028/2015, grant CZ.1.05/1.1.00/02.0123 (MEYS CR), and project CEITEC 2020
(LQ1601).
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, DNA damage, P53
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