INTRINSIC AND SYNERGISTIC ACTIVITY OF ABT-199 AND OVERCOMING ABT-199 RESISTANCE BY THE CDK-INHIBITOR DINACICLIB IN B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Seyfried F. 06/10/16; 133149; P161
Dr. Felix Seyfried
Contributions
Contributions
Abstract
Abstract: P161
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Therapy resistance and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are closely associated with deficient cell death pathways. Anti-apoptotic Bcl-2 family proteins are central apoptosis regulators, thereby serving as promising targets for novel, directed therapies.ABT-199 binds selectively to Bcl-2, inhibits its anti-apoptotic function and releases pro-death Bcl-2 family molecules leading to apoptosis induction. ABT-199 demonstrated activity in different hematological malignancies, pre-clinically and in early clinical trials. However, resistance for ABT-199 highlights the need for predictive markers and effective combination treatment strategies.
Aims
We addressed the effectivity of ABT-199 on BCP-ALL including pre-clinical in vivo evaluation and addressed resistance mechanisms and strategies to overcome ABT-199 insensitivity.
Methods
The effects of different drugs or combinations on BCP-ALL cell lines (n=6) and patient-derived BCP-ALL primografts (n=16) established by transplantation of primary patient ALL cells onto NOD/SCID mice were investigated analyzing half maximal inhibitory concentrations (IC50) and combination indices (CI). Anti-leukemic activity of ABT-199 was evaluated pre-clinically in vivo. Expression of apoptosis regulators was detected by western blot analysis. Mcl-1 deficient cell lines were generated by CRISPR/Cas gene engineering.
Results
A high sensitivity for ABT-199 was identified in 4 of 6 BCP-ALL cell lines with nanomolar IC50 values (mean 212 nM) in contrast to ABT-199 insensitivity in two other lines (IC50 > 1µM). Similarly, a high sensitivity for ABT-199 was identified in the majority of ALL primografts (14 of 16, IC50 2 to 156 nM) with two insensitive cases (IC50 >1 µM). Of note, PBMCs from healthy donors also showed ABT-199 insensitivity.We assessed the expression of the apoptosis regulating molecules Bcl-2 and Mcl-1 and found high Bcl-2 levels in ABT-199 sensitive compared to low Bcl-2 levels in resistant ALL. In contrast, ABT-199 resistant leukemias showed high Mcl-1 expression and low levels were found in ABT-199 sensitive cases. Interestingly, CRISPR/Cas9-mediated knockout of Mcl-1 clearly sensitized ABT-199 insensitive cell lines for ABT-199 mediated cell death, indicating the role of Mcl-1 as mediator but also marker for ABT-199 resistance. Most interestingly, the CDK inhibitor dinaciclib induced rapid down-regulation of Mcl-1 at low nanomolar concentrations leading to sensitization for ABT-199 in resistant cell lines and primograft ALL.We also combined ABT-199 with chemotherapeutic agents used for remission induction in current therapy regimens. Co-exposure of ALL cells to vincristine, dexamethasone and asparaginase combined with ABT-199 showed a clear synergistic effect, both in cell lines and primograft ALL.Moreover, in a preclinical setting, recipient animals bearing a high-risk, t(11;19) leukemia were treated with ABT-199 or solvent. Importantly, a clear reduction of leukemia load was observed upon ABT-199 in vivo therapy compared to vehicle treated recipients (p<0.01).
Conclusion
Taken together, our data show apoptosis induction and high efficacy of ABT-199 as single compound, both ex vivo and pre-clinically in vivo, and synergy upon combination with conventional induction chemotherapy. Importantly, Mcl-1 was identified as mediator and indicator for ABT-199 resistance. Most importantly, the CDK-inhibitor dinaciclib was found to down-regulate Mcl-1 leading to sensitization for ABT-199 in previously resistant, Mcl-1 high expressing leukemia cells.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Apoptosis, BCL2, Leukemia, Pediatric
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Therapy resistance and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are closely associated with deficient cell death pathways. Anti-apoptotic Bcl-2 family proteins are central apoptosis regulators, thereby serving as promising targets for novel, directed therapies.ABT-199 binds selectively to Bcl-2, inhibits its anti-apoptotic function and releases pro-death Bcl-2 family molecules leading to apoptosis induction. ABT-199 demonstrated activity in different hematological malignancies, pre-clinically and in early clinical trials. However, resistance for ABT-199 highlights the need for predictive markers and effective combination treatment strategies.
Aims
We addressed the effectivity of ABT-199 on BCP-ALL including pre-clinical in vivo evaluation and addressed resistance mechanisms and strategies to overcome ABT-199 insensitivity.
Methods
The effects of different drugs or combinations on BCP-ALL cell lines (n=6) and patient-derived BCP-ALL primografts (n=16) established by transplantation of primary patient ALL cells onto NOD/SCID mice were investigated analyzing half maximal inhibitory concentrations (IC50) and combination indices (CI). Anti-leukemic activity of ABT-199 was evaluated pre-clinically in vivo. Expression of apoptosis regulators was detected by western blot analysis. Mcl-1 deficient cell lines were generated by CRISPR/Cas gene engineering.
Results
A high sensitivity for ABT-199 was identified in 4 of 6 BCP-ALL cell lines with nanomolar IC50 values (mean 212 nM) in contrast to ABT-199 insensitivity in two other lines (IC50 > 1µM). Similarly, a high sensitivity for ABT-199 was identified in the majority of ALL primografts (14 of 16, IC50 2 to 156 nM) with two insensitive cases (IC50 >1 µM). Of note, PBMCs from healthy donors also showed ABT-199 insensitivity.We assessed the expression of the apoptosis regulating molecules Bcl-2 and Mcl-1 and found high Bcl-2 levels in ABT-199 sensitive compared to low Bcl-2 levels in resistant ALL. In contrast, ABT-199 resistant leukemias showed high Mcl-1 expression and low levels were found in ABT-199 sensitive cases. Interestingly, CRISPR/Cas9-mediated knockout of Mcl-1 clearly sensitized ABT-199 insensitive cell lines for ABT-199 mediated cell death, indicating the role of Mcl-1 as mediator but also marker for ABT-199 resistance. Most interestingly, the CDK inhibitor dinaciclib induced rapid down-regulation of Mcl-1 at low nanomolar concentrations leading to sensitization for ABT-199 in resistant cell lines and primograft ALL.We also combined ABT-199 with chemotherapeutic agents used for remission induction in current therapy regimens. Co-exposure of ALL cells to vincristine, dexamethasone and asparaginase combined with ABT-199 showed a clear synergistic effect, both in cell lines and primograft ALL.Moreover, in a preclinical setting, recipient animals bearing a high-risk, t(11;19) leukemia were treated with ABT-199 or solvent. Importantly, a clear reduction of leukemia load was observed upon ABT-199 in vivo therapy compared to vehicle treated recipients (p<0.01).
Conclusion
Taken together, our data show apoptosis induction and high efficacy of ABT-199 as single compound, both ex vivo and pre-clinically in vivo, and synergy upon combination with conventional induction chemotherapy. Importantly, Mcl-1 was identified as mediator and indicator for ABT-199 resistance. Most importantly, the CDK-inhibitor dinaciclib was found to down-regulate Mcl-1 leading to sensitization for ABT-199 in previously resistant, Mcl-1 high expressing leukemia cells.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Apoptosis, BCL2, Leukemia, Pediatric
Abstract: P161
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Therapy resistance and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are closely associated with deficient cell death pathways. Anti-apoptotic Bcl-2 family proteins are central apoptosis regulators, thereby serving as promising targets for novel, directed therapies.ABT-199 binds selectively to Bcl-2, inhibits its anti-apoptotic function and releases pro-death Bcl-2 family molecules leading to apoptosis induction. ABT-199 demonstrated activity in different hematological malignancies, pre-clinically and in early clinical trials. However, resistance for ABT-199 highlights the need for predictive markers and effective combination treatment strategies.
Aims
We addressed the effectivity of ABT-199 on BCP-ALL including pre-clinical in vivo evaluation and addressed resistance mechanisms and strategies to overcome ABT-199 insensitivity.
Methods
The effects of different drugs or combinations on BCP-ALL cell lines (n=6) and patient-derived BCP-ALL primografts (n=16) established by transplantation of primary patient ALL cells onto NOD/SCID mice were investigated analyzing half maximal inhibitory concentrations (IC50) and combination indices (CI). Anti-leukemic activity of ABT-199 was evaluated pre-clinically in vivo. Expression of apoptosis regulators was detected by western blot analysis. Mcl-1 deficient cell lines were generated by CRISPR/Cas gene engineering.
Results
A high sensitivity for ABT-199 was identified in 4 of 6 BCP-ALL cell lines with nanomolar IC50 values (mean 212 nM) in contrast to ABT-199 insensitivity in two other lines (IC50 > 1µM). Similarly, a high sensitivity for ABT-199 was identified in the majority of ALL primografts (14 of 16, IC50 2 to 156 nM) with two insensitive cases (IC50 >1 µM). Of note, PBMCs from healthy donors also showed ABT-199 insensitivity.We assessed the expression of the apoptosis regulating molecules Bcl-2 and Mcl-1 and found high Bcl-2 levels in ABT-199 sensitive compared to low Bcl-2 levels in resistant ALL. In contrast, ABT-199 resistant leukemias showed high Mcl-1 expression and low levels were found in ABT-199 sensitive cases. Interestingly, CRISPR/Cas9-mediated knockout of Mcl-1 clearly sensitized ABT-199 insensitive cell lines for ABT-199 mediated cell death, indicating the role of Mcl-1 as mediator but also marker for ABT-199 resistance. Most interestingly, the CDK inhibitor dinaciclib induced rapid down-regulation of Mcl-1 at low nanomolar concentrations leading to sensitization for ABT-199 in resistant cell lines and primograft ALL.We also combined ABT-199 with chemotherapeutic agents used for remission induction in current therapy regimens. Co-exposure of ALL cells to vincristine, dexamethasone and asparaginase combined with ABT-199 showed a clear synergistic effect, both in cell lines and primograft ALL.Moreover, in a preclinical setting, recipient animals bearing a high-risk, t(11;19) leukemia were treated with ABT-199 or solvent. Importantly, a clear reduction of leukemia load was observed upon ABT-199 in vivo therapy compared to vehicle treated recipients (p<0.01).
Conclusion
Taken together, our data show apoptosis induction and high efficacy of ABT-199 as single compound, both ex vivo and pre-clinically in vivo, and synergy upon combination with conventional induction chemotherapy. Importantly, Mcl-1 was identified as mediator and indicator for ABT-199 resistance. Most importantly, the CDK-inhibitor dinaciclib was found to down-regulate Mcl-1 leading to sensitization for ABT-199 in previously resistant, Mcl-1 high expressing leukemia cells.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Apoptosis, BCL2, Leukemia, Pediatric
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Therapy resistance and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are closely associated with deficient cell death pathways. Anti-apoptotic Bcl-2 family proteins are central apoptosis regulators, thereby serving as promising targets for novel, directed therapies.ABT-199 binds selectively to Bcl-2, inhibits its anti-apoptotic function and releases pro-death Bcl-2 family molecules leading to apoptosis induction. ABT-199 demonstrated activity in different hematological malignancies, pre-clinically and in early clinical trials. However, resistance for ABT-199 highlights the need for predictive markers and effective combination treatment strategies.
Aims
We addressed the effectivity of ABT-199 on BCP-ALL including pre-clinical in vivo evaluation and addressed resistance mechanisms and strategies to overcome ABT-199 insensitivity.
Methods
The effects of different drugs or combinations on BCP-ALL cell lines (n=6) and patient-derived BCP-ALL primografts (n=16) established by transplantation of primary patient ALL cells onto NOD/SCID mice were investigated analyzing half maximal inhibitory concentrations (IC50) and combination indices (CI). Anti-leukemic activity of ABT-199 was evaluated pre-clinically in vivo. Expression of apoptosis regulators was detected by western blot analysis. Mcl-1 deficient cell lines were generated by CRISPR/Cas gene engineering.
Results
A high sensitivity for ABT-199 was identified in 4 of 6 BCP-ALL cell lines with nanomolar IC50 values (mean 212 nM) in contrast to ABT-199 insensitivity in two other lines (IC50 > 1µM). Similarly, a high sensitivity for ABT-199 was identified in the majority of ALL primografts (14 of 16, IC50 2 to 156 nM) with two insensitive cases (IC50 >1 µM). Of note, PBMCs from healthy donors also showed ABT-199 insensitivity.We assessed the expression of the apoptosis regulating molecules Bcl-2 and Mcl-1 and found high Bcl-2 levels in ABT-199 sensitive compared to low Bcl-2 levels in resistant ALL. In contrast, ABT-199 resistant leukemias showed high Mcl-1 expression and low levels were found in ABT-199 sensitive cases. Interestingly, CRISPR/Cas9-mediated knockout of Mcl-1 clearly sensitized ABT-199 insensitive cell lines for ABT-199 mediated cell death, indicating the role of Mcl-1 as mediator but also marker for ABT-199 resistance. Most interestingly, the CDK inhibitor dinaciclib induced rapid down-regulation of Mcl-1 at low nanomolar concentrations leading to sensitization for ABT-199 in resistant cell lines and primograft ALL.We also combined ABT-199 with chemotherapeutic agents used for remission induction in current therapy regimens. Co-exposure of ALL cells to vincristine, dexamethasone and asparaginase combined with ABT-199 showed a clear synergistic effect, both in cell lines and primograft ALL.Moreover, in a preclinical setting, recipient animals bearing a high-risk, t(11;19) leukemia were treated with ABT-199 or solvent. Importantly, a clear reduction of leukemia load was observed upon ABT-199 in vivo therapy compared to vehicle treated recipients (p<0.01).
Conclusion
Taken together, our data show apoptosis induction and high efficacy of ABT-199 as single compound, both ex vivo and pre-clinically in vivo, and synergy upon combination with conventional induction chemotherapy. Importantly, Mcl-1 was identified as mediator and indicator for ABT-199 resistance. Most importantly, the CDK-inhibitor dinaciclib was found to down-regulate Mcl-1 leading to sensitization for ABT-199 in previously resistant, Mcl-1 high expressing leukemia cells.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Apoptosis, BCL2, Leukemia, Pediatric
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