A NOVEL EPIGENETIC RESISTANCE MECHANISM TO THE FLT3 INHIBITOR PKC412 INDUCES CROSS-RESISTANCE TO STANDARD CHEMOTHERAPY IN ACUTE MYELOID LEUKEMIA (AML)
(Abstract release date: 05/19/16)
EHA Library. Göllner S. 06/11/16; 135216; S460
Dr. Stefanie Göllner
Contributions
Contributions
Abstract
Abstract: S460
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall C13
Background
In acute myeloid leukemia (AML) about 20-30% of patients possess a FLT3-internal tandem duplication (ITD) associated with a poor prognosis and a higher propensity for relapse after remission. Tyrosine kinase inhibitors (TKI) such as PKC412 have been developed for therapy of FLT3-ITD-positive AMLs. So far, resistance to tyrosine kinase inhibitors has been demonstrated to occur by point mutations in FLT3 whereas further mechanisms that render leukemic cells drug resistant remain incompletely understood.
Aims
We utilized a PKC412-resistant model of FLT3-ITD-positive MV4-11 cells to (1) identify novel mechanisms of drug resistance, (2) to analyze cross resistance to the standard chemotherapeutics cytarabine and daunorubicin and translate our findings to FLT3-ITD-negative AMLs and (3) to develop strategies to reverse chemoresistance in AML.
Methods
Drug resistance mechanisms were analyzed in vitro by immunohistochemistry, Western blot, shRNA-mediated mRNA knockdown, lentiviral overexpression, immunoprecipitation, label-free- and SILAC (Stable isotope labeling by amino acids in cell culture)- mass spectrometry, ChIP-seq and ex-vivo- culture of patient samples. For in vivo analysis xenograft mouse models were applied.
Results
We identified loss of the histone methyltransferase EZH2 with subsequent reduction of histone H3K27 trimethylation in PKC412- resistant MV4-11 cells which led to cross-resistance to cytarabine and daunorubicin. In AML patients, the absence of EZH2 protein expression was closely associated with poor overall survival (p=0.008), poor event-free (p=0.005) and poor relapse-free survival (p=0.047) as analyzed by tissue microarrays. The reduction of EZH2 protein levels via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown was sufficient to induce chemoresistance of Normal Karyotype (NK)- AML blasts and cell lines in vitro and in a xenograft mouse model. The loss of EZH2 in drug resistant MV4-11 cells was regulated by posttranslational mechanisms. Immunoprecipitation of EZH2 revealed a 2.6-fold increased phosphorylation of residue T487 in resistant cells (p= 0.014) and the cyclin-dependent kinase 1 (CDK1) was associated with EZH2 specifically in resistant MV4-11. Several ubiquitin-E3-ligases such as TRIM21 were increasingly bound to EZH2 in resistant compared to sensitive cells resulting in a strong ubiquitination of EZH2 and subsequent degradation. Pharmacological inhibition of CDK1 and treatment with the proteasome inhibitor bortezomib, respectively, increased EZH2 protein and restored drug sensitivity in resistant cells. Functionally, reduction of EZH2 directly induced upregulation of HOX genes and the drug transporter MRP1, suggesting a stem-cell-like signature to be associated with the resistance phenotype, which could be reverted by proteasome inhibitors. In primary AML samples sensitivity to cytarabine was specifically increased in samples with elevated EZH2 protein levels upon bortezomib exposure whereas no change was observed in patient samples without EZH2 increase upon bortezomib treatment (p=0.0079, Fisher´s exact test).
Conclusion
In summary, our findings identify a novel epigenetic resistance mechanism that can be induced by prolonged exposure to PKC412 or cytotoxic drugs. EZH2 protein restoration by proteasome inhibitors in combination with established therapy approaches may be a promising therapy concept to restore drug sensitivity in AML.
Session topic: AML Biology Mutant FLT
Keyword(s): Acute myeloid leukemia, Chemoresistance, Epigenetic, Proteasome inhibitor
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall C13
Background
In acute myeloid leukemia (AML) about 20-30% of patients possess a FLT3-internal tandem duplication (ITD) associated with a poor prognosis and a higher propensity for relapse after remission. Tyrosine kinase inhibitors (TKI) such as PKC412 have been developed for therapy of FLT3-ITD-positive AMLs. So far, resistance to tyrosine kinase inhibitors has been demonstrated to occur by point mutations in FLT3 whereas further mechanisms that render leukemic cells drug resistant remain incompletely understood.
Aims
We utilized a PKC412-resistant model of FLT3-ITD-positive MV4-11 cells to (1) identify novel mechanisms of drug resistance, (2) to analyze cross resistance to the standard chemotherapeutics cytarabine and daunorubicin and translate our findings to FLT3-ITD-negative AMLs and (3) to develop strategies to reverse chemoresistance in AML.
Methods
Drug resistance mechanisms were analyzed in vitro by immunohistochemistry, Western blot, shRNA-mediated mRNA knockdown, lentiviral overexpression, immunoprecipitation, label-free- and SILAC (Stable isotope labeling by amino acids in cell culture)- mass spectrometry, ChIP-seq and ex-vivo- culture of patient samples. For in vivo analysis xenograft mouse models were applied.
Results
We identified loss of the histone methyltransferase EZH2 with subsequent reduction of histone H3K27 trimethylation in PKC412- resistant MV4-11 cells which led to cross-resistance to cytarabine and daunorubicin. In AML patients, the absence of EZH2 protein expression was closely associated with poor overall survival (p=0.008), poor event-free (p=0.005) and poor relapse-free survival (p=0.047) as analyzed by tissue microarrays. The reduction of EZH2 protein levels via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown was sufficient to induce chemoresistance of Normal Karyotype (NK)- AML blasts and cell lines in vitro and in a xenograft mouse model. The loss of EZH2 in drug resistant MV4-11 cells was regulated by posttranslational mechanisms. Immunoprecipitation of EZH2 revealed a 2.6-fold increased phosphorylation of residue T487 in resistant cells (p= 0.014) and the cyclin-dependent kinase 1 (CDK1) was associated with EZH2 specifically in resistant MV4-11. Several ubiquitin-E3-ligases such as TRIM21 were increasingly bound to EZH2 in resistant compared to sensitive cells resulting in a strong ubiquitination of EZH2 and subsequent degradation. Pharmacological inhibition of CDK1 and treatment with the proteasome inhibitor bortezomib, respectively, increased EZH2 protein and restored drug sensitivity in resistant cells. Functionally, reduction of EZH2 directly induced upregulation of HOX genes and the drug transporter MRP1, suggesting a stem-cell-like signature to be associated with the resistance phenotype, which could be reverted by proteasome inhibitors. In primary AML samples sensitivity to cytarabine was specifically increased in samples with elevated EZH2 protein levels upon bortezomib exposure whereas no change was observed in patient samples without EZH2 increase upon bortezomib treatment (p=0.0079, Fisher´s exact test).
Conclusion
In summary, our findings identify a novel epigenetic resistance mechanism that can be induced by prolonged exposure to PKC412 or cytotoxic drugs. EZH2 protein restoration by proteasome inhibitors in combination with established therapy approaches may be a promising therapy concept to restore drug sensitivity in AML.
Session topic: AML Biology Mutant FLT
Keyword(s): Acute myeloid leukemia, Chemoresistance, Epigenetic, Proteasome inhibitor
Abstract: S460
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall C13
Background
In acute myeloid leukemia (AML) about 20-30% of patients possess a FLT3-internal tandem duplication (ITD) associated with a poor prognosis and a higher propensity for relapse after remission. Tyrosine kinase inhibitors (TKI) such as PKC412 have been developed for therapy of FLT3-ITD-positive AMLs. So far, resistance to tyrosine kinase inhibitors has been demonstrated to occur by point mutations in FLT3 whereas further mechanisms that render leukemic cells drug resistant remain incompletely understood.
Aims
We utilized a PKC412-resistant model of FLT3-ITD-positive MV4-11 cells to (1) identify novel mechanisms of drug resistance, (2) to analyze cross resistance to the standard chemotherapeutics cytarabine and daunorubicin and translate our findings to FLT3-ITD-negative AMLs and (3) to develop strategies to reverse chemoresistance in AML.
Methods
Drug resistance mechanisms were analyzed in vitro by immunohistochemistry, Western blot, shRNA-mediated mRNA knockdown, lentiviral overexpression, immunoprecipitation, label-free- and SILAC (Stable isotope labeling by amino acids in cell culture)- mass spectrometry, ChIP-seq and ex-vivo- culture of patient samples. For in vivo analysis xenograft mouse models were applied.
Results
We identified loss of the histone methyltransferase EZH2 with subsequent reduction of histone H3K27 trimethylation in PKC412- resistant MV4-11 cells which led to cross-resistance to cytarabine and daunorubicin. In AML patients, the absence of EZH2 protein expression was closely associated with poor overall survival (p=0.008), poor event-free (p=0.005) and poor relapse-free survival (p=0.047) as analyzed by tissue microarrays. The reduction of EZH2 protein levels via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown was sufficient to induce chemoresistance of Normal Karyotype (NK)- AML blasts and cell lines in vitro and in a xenograft mouse model. The loss of EZH2 in drug resistant MV4-11 cells was regulated by posttranslational mechanisms. Immunoprecipitation of EZH2 revealed a 2.6-fold increased phosphorylation of residue T487 in resistant cells (p= 0.014) and the cyclin-dependent kinase 1 (CDK1) was associated with EZH2 specifically in resistant MV4-11. Several ubiquitin-E3-ligases such as TRIM21 were increasingly bound to EZH2 in resistant compared to sensitive cells resulting in a strong ubiquitination of EZH2 and subsequent degradation. Pharmacological inhibition of CDK1 and treatment with the proteasome inhibitor bortezomib, respectively, increased EZH2 protein and restored drug sensitivity in resistant cells. Functionally, reduction of EZH2 directly induced upregulation of HOX genes and the drug transporter MRP1, suggesting a stem-cell-like signature to be associated with the resistance phenotype, which could be reverted by proteasome inhibitors. In primary AML samples sensitivity to cytarabine was specifically increased in samples with elevated EZH2 protein levels upon bortezomib exposure whereas no change was observed in patient samples without EZH2 increase upon bortezomib treatment (p=0.0079, Fisher´s exact test).
Conclusion
In summary, our findings identify a novel epigenetic resistance mechanism that can be induced by prolonged exposure to PKC412 or cytotoxic drugs. EZH2 protein restoration by proteasome inhibitors in combination with established therapy approaches may be a promising therapy concept to restore drug sensitivity in AML.
Session topic: AML Biology Mutant FLT
Keyword(s): Acute myeloid leukemia, Chemoresistance, Epigenetic, Proteasome inhibitor
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall C13
Background
In acute myeloid leukemia (AML) about 20-30% of patients possess a FLT3-internal tandem duplication (ITD) associated with a poor prognosis and a higher propensity for relapse after remission. Tyrosine kinase inhibitors (TKI) such as PKC412 have been developed for therapy of FLT3-ITD-positive AMLs. So far, resistance to tyrosine kinase inhibitors has been demonstrated to occur by point mutations in FLT3 whereas further mechanisms that render leukemic cells drug resistant remain incompletely understood.
Aims
We utilized a PKC412-resistant model of FLT3-ITD-positive MV4-11 cells to (1) identify novel mechanisms of drug resistance, (2) to analyze cross resistance to the standard chemotherapeutics cytarabine and daunorubicin and translate our findings to FLT3-ITD-negative AMLs and (3) to develop strategies to reverse chemoresistance in AML.
Methods
Drug resistance mechanisms were analyzed in vitro by immunohistochemistry, Western blot, shRNA-mediated mRNA knockdown, lentiviral overexpression, immunoprecipitation, label-free- and SILAC (Stable isotope labeling by amino acids in cell culture)- mass spectrometry, ChIP-seq and ex-vivo- culture of patient samples. For in vivo analysis xenograft mouse models were applied.
Results
We identified loss of the histone methyltransferase EZH2 with subsequent reduction of histone H3K27 trimethylation in PKC412- resistant MV4-11 cells which led to cross-resistance to cytarabine and daunorubicin. In AML patients, the absence of EZH2 protein expression was closely associated with poor overall survival (p=0.008), poor event-free (p=0.005) and poor relapse-free survival (p=0.047) as analyzed by tissue microarrays. The reduction of EZH2 protein levels via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown was sufficient to induce chemoresistance of Normal Karyotype (NK)- AML blasts and cell lines in vitro and in a xenograft mouse model. The loss of EZH2 in drug resistant MV4-11 cells was regulated by posttranslational mechanisms. Immunoprecipitation of EZH2 revealed a 2.6-fold increased phosphorylation of residue T487 in resistant cells (p= 0.014) and the cyclin-dependent kinase 1 (CDK1) was associated with EZH2 specifically in resistant MV4-11. Several ubiquitin-E3-ligases such as TRIM21 were increasingly bound to EZH2 in resistant compared to sensitive cells resulting in a strong ubiquitination of EZH2 and subsequent degradation. Pharmacological inhibition of CDK1 and treatment with the proteasome inhibitor bortezomib, respectively, increased EZH2 protein and restored drug sensitivity in resistant cells. Functionally, reduction of EZH2 directly induced upregulation of HOX genes and the drug transporter MRP1, suggesting a stem-cell-like signature to be associated with the resistance phenotype, which could be reverted by proteasome inhibitors. In primary AML samples sensitivity to cytarabine was specifically increased in samples with elevated EZH2 protein levels upon bortezomib exposure whereas no change was observed in patient samples without EZH2 increase upon bortezomib treatment (p=0.0079, Fisher´s exact test).
Conclusion
In summary, our findings identify a novel epigenetic resistance mechanism that can be induced by prolonged exposure to PKC412 or cytotoxic drugs. EZH2 protein restoration by proteasome inhibitors in combination with established therapy approaches may be a promising therapy concept to restore drug sensitivity in AML.
Session topic: AML Biology Mutant FLT
Keyword(s): Acute myeloid leukemia, Chemoresistance, Epigenetic, Proteasome inhibitor
{{ help_message }}
{{filter}}