DNA REPLICATION CONTROL MECHANISMS INVOLVED IN CHEMORESISTANCE OF CHRONIC LYMPHOCYTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Grgurevic S. 06/09/16; 132567; E1018
Ms. Srdana Grgurevic
Contributions
Contributions
Abstract
Abstract: E1018
Type: Eposter Presentation
Background
Chronic lymphocytic leukemia (CLL), the most common leukemia in the Western hemisphere, is characterized by clonal expansion of B lymphocytes co-expressing CD5 and CD19 that arise from the lymph nodes and accumulate in patient’s peripheral blood and bone marrow. Historically, standard first line treatment of CLL patients has been based on a DNA targeting agent, cyclophosphamide, nucleoside analogue, fludarabine and an anti-CD20 antibody, rituximab. Although the chemotherapeutic regime results in high rate of complete response, majority of CLL patients, nevertheless, relapse.
Aims
CLL genomes are characterized by, both, chromosome aberrations affecting the karyotypic integrity and elevated frequencies of nucleotide point mutations. Altered DNA replication, repair and recombination (3R) mechanisms may account for chromosomal instability and mutator phenotype that propels the development of chemoresistance towards genotoxic therapies. Here, we aimed to determine the expression of 3R genes and decipher their functional role in CLL.
Methods
For this purpose we assessed complete 3R gene expression profiles in 140 peripheral blood (PB) samples obtained from clinically annotated CLL patients and correlated the obtained data with patients’ clinical outcomes. Next, in order to investigate the DNA replisome of the cycling CLL cells, we used an in vitro approach based on interleukin-2 (IL-2) and CpG-oligonucleotide stimulation to obtain proliferative primary patient leukemic cells. To determine the chemotherapeutic implication of the 3R gene of interest (GOI), we performed drug sensitivity screens in presence of fludarabine on two CLL cell lines established successively from the same patient that differed in the expression of the 3R GOI. Also, we used a mouse embryonic fibroblast (MEF) cell line and its heterozygous and homozygous knock-outs for the 3R GOI to confirm the obtained results. We characterized chemoresistance profiles using cellular viability screens, cell cycle and DNA synthesis analysis. In order to decipher the molecular basis of chemoresistance driven by our 3R GOI, we used drugs that partially recapitulate the mechanism of action (MOA) of fludarabine, namely, hydroxyurea (HU), an inhibitor of the ribonucleotide reductase (RNR) and aphidicolin, an inhibitor of replicative DNA polymerases.
Results
Our qRT-PCR data revealed deregulated expression of several 3R genes among which, alternative DNA polymerase nu (POLN). Interestingly, we observed that relatively high expression of POLN correlates with a shorter progression free survival (PFS) in patients that received the chemotherapeutic treatment. In addition, in vitro proliferation assays showed that primary CLL lymphocytes that were stimulated to enter the cell cycle and divide, exclusively upregulated POLN. Drug screens in two independent cellular models showed that upregulation of POLN contributed to fludarabine chemoresistance in terms of cellular viability and sustained DNA synthesis. Our chemoresistance data demonstrated that, unlike POLN knock-out cells, POLN expressing cells are able to continue synthesizing DNA in presence of fludarabine. These results support observed clinical correlation between POLN expression and PFS. In terms of the molecular mechanism of POLN driven chemoresistance, differential DNA synthesis profiles obtained in presence of fludarabine were recapitulated when cells were treated with hydroxyurea but not aphidicolin.
Conclusion
Finally, our data reveal deregulated expression of 3R genes in CLL, among which that of a specialized DNA polymerase nu (POLN). We show that POLN overexpression correlates with worse clinical outcome in CLL patients upon receiving therapy. Moreover, our in vitro results clearly show that upregulation of POLN observed in cycling CLL cells can drive fludarabine chemoresistance. In conclusion, we propose that, on the molecular level, POLN contributes to chemoresistance by sustaining the DNA synthesis and preserving cellular viability in presence of a low dNTP pool imposed by fludarabine.
Session topic: E-poster
Keyword(s): Chemoresistance, Chronic lymphocytic leukemia, Fludarabine phosphate
Type: Eposter Presentation
Background
Chronic lymphocytic leukemia (CLL), the most common leukemia in the Western hemisphere, is characterized by clonal expansion of B lymphocytes co-expressing CD5 and CD19 that arise from the lymph nodes and accumulate in patient’s peripheral blood and bone marrow. Historically, standard first line treatment of CLL patients has been based on a DNA targeting agent, cyclophosphamide, nucleoside analogue, fludarabine and an anti-CD20 antibody, rituximab. Although the chemotherapeutic regime results in high rate of complete response, majority of CLL patients, nevertheless, relapse.
Aims
CLL genomes are characterized by, both, chromosome aberrations affecting the karyotypic integrity and elevated frequencies of nucleotide point mutations. Altered DNA replication, repair and recombination (3R) mechanisms may account for chromosomal instability and mutator phenotype that propels the development of chemoresistance towards genotoxic therapies. Here, we aimed to determine the expression of 3R genes and decipher their functional role in CLL.
Methods
For this purpose we assessed complete 3R gene expression profiles in 140 peripheral blood (PB) samples obtained from clinically annotated CLL patients and correlated the obtained data with patients’ clinical outcomes. Next, in order to investigate the DNA replisome of the cycling CLL cells, we used an in vitro approach based on interleukin-2 (IL-2) and CpG-oligonucleotide stimulation to obtain proliferative primary patient leukemic cells. To determine the chemotherapeutic implication of the 3R gene of interest (GOI), we performed drug sensitivity screens in presence of fludarabine on two CLL cell lines established successively from the same patient that differed in the expression of the 3R GOI. Also, we used a mouse embryonic fibroblast (MEF) cell line and its heterozygous and homozygous knock-outs for the 3R GOI to confirm the obtained results. We characterized chemoresistance profiles using cellular viability screens, cell cycle and DNA synthesis analysis. In order to decipher the molecular basis of chemoresistance driven by our 3R GOI, we used drugs that partially recapitulate the mechanism of action (MOA) of fludarabine, namely, hydroxyurea (HU), an inhibitor of the ribonucleotide reductase (RNR) and aphidicolin, an inhibitor of replicative DNA polymerases.
Results
Our qRT-PCR data revealed deregulated expression of several 3R genes among which, alternative DNA polymerase nu (POLN). Interestingly, we observed that relatively high expression of POLN correlates with a shorter progression free survival (PFS) in patients that received the chemotherapeutic treatment. In addition, in vitro proliferation assays showed that primary CLL lymphocytes that were stimulated to enter the cell cycle and divide, exclusively upregulated POLN. Drug screens in two independent cellular models showed that upregulation of POLN contributed to fludarabine chemoresistance in terms of cellular viability and sustained DNA synthesis. Our chemoresistance data demonstrated that, unlike POLN knock-out cells, POLN expressing cells are able to continue synthesizing DNA in presence of fludarabine. These results support observed clinical correlation between POLN expression and PFS. In terms of the molecular mechanism of POLN driven chemoresistance, differential DNA synthesis profiles obtained in presence of fludarabine were recapitulated when cells were treated with hydroxyurea but not aphidicolin.
Conclusion
Finally, our data reveal deregulated expression of 3R genes in CLL, among which that of a specialized DNA polymerase nu (POLN). We show that POLN overexpression correlates with worse clinical outcome in CLL patients upon receiving therapy. Moreover, our in vitro results clearly show that upregulation of POLN observed in cycling CLL cells can drive fludarabine chemoresistance. In conclusion, we propose that, on the molecular level, POLN contributes to chemoresistance by sustaining the DNA synthesis and preserving cellular viability in presence of a low dNTP pool imposed by fludarabine.
Session topic: E-poster
Keyword(s): Chemoresistance, Chronic lymphocytic leukemia, Fludarabine phosphate
Abstract: E1018
Type: Eposter Presentation
Background
Chronic lymphocytic leukemia (CLL), the most common leukemia in the Western hemisphere, is characterized by clonal expansion of B lymphocytes co-expressing CD5 and CD19 that arise from the lymph nodes and accumulate in patient’s peripheral blood and bone marrow. Historically, standard first line treatment of CLL patients has been based on a DNA targeting agent, cyclophosphamide, nucleoside analogue, fludarabine and an anti-CD20 antibody, rituximab. Although the chemotherapeutic regime results in high rate of complete response, majority of CLL patients, nevertheless, relapse.
Aims
CLL genomes are characterized by, both, chromosome aberrations affecting the karyotypic integrity and elevated frequencies of nucleotide point mutations. Altered DNA replication, repair and recombination (3R) mechanisms may account for chromosomal instability and mutator phenotype that propels the development of chemoresistance towards genotoxic therapies. Here, we aimed to determine the expression of 3R genes and decipher their functional role in CLL.
Methods
For this purpose we assessed complete 3R gene expression profiles in 140 peripheral blood (PB) samples obtained from clinically annotated CLL patients and correlated the obtained data with patients’ clinical outcomes. Next, in order to investigate the DNA replisome of the cycling CLL cells, we used an in vitro approach based on interleukin-2 (IL-2) and CpG-oligonucleotide stimulation to obtain proliferative primary patient leukemic cells. To determine the chemotherapeutic implication of the 3R gene of interest (GOI), we performed drug sensitivity screens in presence of fludarabine on two CLL cell lines established successively from the same patient that differed in the expression of the 3R GOI. Also, we used a mouse embryonic fibroblast (MEF) cell line and its heterozygous and homozygous knock-outs for the 3R GOI to confirm the obtained results. We characterized chemoresistance profiles using cellular viability screens, cell cycle and DNA synthesis analysis. In order to decipher the molecular basis of chemoresistance driven by our 3R GOI, we used drugs that partially recapitulate the mechanism of action (MOA) of fludarabine, namely, hydroxyurea (HU), an inhibitor of the ribonucleotide reductase (RNR) and aphidicolin, an inhibitor of replicative DNA polymerases.
Results
Our qRT-PCR data revealed deregulated expression of several 3R genes among which, alternative DNA polymerase nu (POLN). Interestingly, we observed that relatively high expression of POLN correlates with a shorter progression free survival (PFS) in patients that received the chemotherapeutic treatment. In addition, in vitro proliferation assays showed that primary CLL lymphocytes that were stimulated to enter the cell cycle and divide, exclusively upregulated POLN. Drug screens in two independent cellular models showed that upregulation of POLN contributed to fludarabine chemoresistance in terms of cellular viability and sustained DNA synthesis. Our chemoresistance data demonstrated that, unlike POLN knock-out cells, POLN expressing cells are able to continue synthesizing DNA in presence of fludarabine. These results support observed clinical correlation between POLN expression and PFS. In terms of the molecular mechanism of POLN driven chemoresistance, differential DNA synthesis profiles obtained in presence of fludarabine were recapitulated when cells were treated with hydroxyurea but not aphidicolin.
Conclusion
Finally, our data reveal deregulated expression of 3R genes in CLL, among which that of a specialized DNA polymerase nu (POLN). We show that POLN overexpression correlates with worse clinical outcome in CLL patients upon receiving therapy. Moreover, our in vitro results clearly show that upregulation of POLN observed in cycling CLL cells can drive fludarabine chemoresistance. In conclusion, we propose that, on the molecular level, POLN contributes to chemoresistance by sustaining the DNA synthesis and preserving cellular viability in presence of a low dNTP pool imposed by fludarabine.
Session topic: E-poster
Keyword(s): Chemoresistance, Chronic lymphocytic leukemia, Fludarabine phosphate
Type: Eposter Presentation
Background
Chronic lymphocytic leukemia (CLL), the most common leukemia in the Western hemisphere, is characterized by clonal expansion of B lymphocytes co-expressing CD5 and CD19 that arise from the lymph nodes and accumulate in patient’s peripheral blood and bone marrow. Historically, standard first line treatment of CLL patients has been based on a DNA targeting agent, cyclophosphamide, nucleoside analogue, fludarabine and an anti-CD20 antibody, rituximab. Although the chemotherapeutic regime results in high rate of complete response, majority of CLL patients, nevertheless, relapse.
Aims
CLL genomes are characterized by, both, chromosome aberrations affecting the karyotypic integrity and elevated frequencies of nucleotide point mutations. Altered DNA replication, repair and recombination (3R) mechanisms may account for chromosomal instability and mutator phenotype that propels the development of chemoresistance towards genotoxic therapies. Here, we aimed to determine the expression of 3R genes and decipher their functional role in CLL.
Methods
For this purpose we assessed complete 3R gene expression profiles in 140 peripheral blood (PB) samples obtained from clinically annotated CLL patients and correlated the obtained data with patients’ clinical outcomes. Next, in order to investigate the DNA replisome of the cycling CLL cells, we used an in vitro approach based on interleukin-2 (IL-2) and CpG-oligonucleotide stimulation to obtain proliferative primary patient leukemic cells. To determine the chemotherapeutic implication of the 3R gene of interest (GOI), we performed drug sensitivity screens in presence of fludarabine on two CLL cell lines established successively from the same patient that differed in the expression of the 3R GOI. Also, we used a mouse embryonic fibroblast (MEF) cell line and its heterozygous and homozygous knock-outs for the 3R GOI to confirm the obtained results. We characterized chemoresistance profiles using cellular viability screens, cell cycle and DNA synthesis analysis. In order to decipher the molecular basis of chemoresistance driven by our 3R GOI, we used drugs that partially recapitulate the mechanism of action (MOA) of fludarabine, namely, hydroxyurea (HU), an inhibitor of the ribonucleotide reductase (RNR) and aphidicolin, an inhibitor of replicative DNA polymerases.
Results
Our qRT-PCR data revealed deregulated expression of several 3R genes among which, alternative DNA polymerase nu (POLN). Interestingly, we observed that relatively high expression of POLN correlates with a shorter progression free survival (PFS) in patients that received the chemotherapeutic treatment. In addition, in vitro proliferation assays showed that primary CLL lymphocytes that were stimulated to enter the cell cycle and divide, exclusively upregulated POLN. Drug screens in two independent cellular models showed that upregulation of POLN contributed to fludarabine chemoresistance in terms of cellular viability and sustained DNA synthesis. Our chemoresistance data demonstrated that, unlike POLN knock-out cells, POLN expressing cells are able to continue synthesizing DNA in presence of fludarabine. These results support observed clinical correlation between POLN expression and PFS. In terms of the molecular mechanism of POLN driven chemoresistance, differential DNA synthesis profiles obtained in presence of fludarabine were recapitulated when cells were treated with hydroxyurea but not aphidicolin.
Conclusion
Finally, our data reveal deregulated expression of 3R genes in CLL, among which that of a specialized DNA polymerase nu (POLN). We show that POLN overexpression correlates with worse clinical outcome in CLL patients upon receiving therapy. Moreover, our in vitro results clearly show that upregulation of POLN observed in cycling CLL cells can drive fludarabine chemoresistance. In conclusion, we propose that, on the molecular level, POLN contributes to chemoresistance by sustaining the DNA synthesis and preserving cellular viability in presence of a low dNTP pool imposed by fludarabine.
Session topic: E-poster
Keyword(s): Chemoresistance, Chronic lymphocytic leukemia, Fludarabine phosphate
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