FUNCTIONAL RESTORATION OF MUTANT P53 AND CELL DEATH SENSITIZATION IN ACUTE LYMPHOBLASTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. demir S. 06/10/16; 133146; P158
Salih demir
Contributions
Contributions
Abstract
Abstract: P158
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
In acute lymphoblastic leukemia (ALL), TP53 alterations are rarely found at diagnosis but in about 12% of patients at relapse. TP53 mutations (TP53mut) have been described to be associated with poor response to therapy and inferior outcome in ALL. Compounds targeting mutated p53 such as APR-246 demonstrated activity in different types of TP53mut malignancies. In ALL however, targeting mutant TP53 has so far not been addressed.
Aims
To evaluate mutant TP53 as target for directed anti-leukemia therapy in ALL.
Methods
Patient-derived primograft samples established in our NOD/SCID/huALL mouse model were analyzed for TP53mut by denaturating high-performance liquid chromatography and validated by Sanger sequencing. Sensitivities of ALL cells in response to different drugs and combinations were assessed analyzing half maximal inhibitory concentrations (IC50) and combination indices (CI). Apoptosis was detected by caspase-3 activation and Annexin-V/propidium iodide positivity. Activation of p53 was analyzed by phosphorylation of p53 (p53pSer15, flowcytometry), expression of p53 downstream molecules and confirmation of phosphorylation was carried out by western blot analysis. p53 deficient cell lines were generated by lentiviral shRNA mediated knock-down.
Results
Of 62 primograft samples, 4 cases with TP53mut (6.5 %) were identified, corresponding to reported numbers in clinical studies. In parallel, we analyzed 6 BCP-ALL cell lines and identified 2 TP53mut and 4 wild type (TP53wt) lines.We analyzed the effects of the DNA damaging agent doxorubicin and the p53 targeting small molecule APR-246 (kindly provided by Aprea, Stockholm, Sweden) and observed an insensitivity to doxorubicin in TP53mut cell lines and primografts (mean IC50: 100 nM and >65 nM) compared to sensitive TP53wt cell lines and primografts (mean IC50: 9 nM and 7.4 nM). In contrast, TP53mut ALL cells were highly sensitive to APR-246 (mean IC50: 4.5 μM and 4.4 μM), compared to insensitivity of TP53wt cell lines and primografts (mean IC50: 58.3 μM and >90 μM). Increased Annexin-V/PI positivity and caspase-3 activity upon APR-246 exposure indicated apoptosis as mechanism of APR-246 mediated cell death.In order to investigate the specificity of APR-246 for p53, we investigated APR-246 mediated cell death induction in 2 genetically modified, p53 deficient ALL cell lines. No cell death induction could be detected upon p53 absence in contrast to high APR-246 sensitivity in the control transduced cell lines, clearly indicating dependency of APR-246 on the presence of mutant p53.Most importantly, a clear activation of p53 was identified upon APR-246 exposure in TP53mut but not TP53wt leukemias indicated by increased p53 phosphorylation and increased expression of the p53 target molecules NOXA and PUMA, pointing to restoration of p53 function and induction of downstream signaling in TP53mut ALL by APR-246.Moreover, we investigated the ability of APR-246 to re-sensitize TP53mut, DNA damage resistant ALL. Upon exposure of TP53mut ALL cell lines and primografts to combinations of APR-246 and doxorubicin, a strong synergism and re-sensitization to DNA-damage induced cell death was observed.
Conclusion
We provide evidence that APR-246 specifically targets TP53mut BCP-ALL leading to re-activation of p53, apoptosis induction, and re-sensitization of DNA-damage resistant TP53mut ALL cells. Thus, targeting mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Acute lymphoblastic leukemia, Mutation status, P53, Targeted therapy
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
In acute lymphoblastic leukemia (ALL), TP53 alterations are rarely found at diagnosis but in about 12% of patients at relapse. TP53 mutations (TP53mut) have been described to be associated with poor response to therapy and inferior outcome in ALL. Compounds targeting mutated p53 such as APR-246 demonstrated activity in different types of TP53mut malignancies. In ALL however, targeting mutant TP53 has so far not been addressed.
Aims
To evaluate mutant TP53 as target for directed anti-leukemia therapy in ALL.
Methods
Patient-derived primograft samples established in our NOD/SCID/huALL mouse model were analyzed for TP53mut by denaturating high-performance liquid chromatography and validated by Sanger sequencing. Sensitivities of ALL cells in response to different drugs and combinations were assessed analyzing half maximal inhibitory concentrations (IC50) and combination indices (CI). Apoptosis was detected by caspase-3 activation and Annexin-V/propidium iodide positivity. Activation of p53 was analyzed by phosphorylation of p53 (p53pSer15, flowcytometry), expression of p53 downstream molecules and confirmation of phosphorylation was carried out by western blot analysis. p53 deficient cell lines were generated by lentiviral shRNA mediated knock-down.
Results
Of 62 primograft samples, 4 cases with TP53mut (6.5 %) were identified, corresponding to reported numbers in clinical studies. In parallel, we analyzed 6 BCP-ALL cell lines and identified 2 TP53mut and 4 wild type (TP53wt) lines.We analyzed the effects of the DNA damaging agent doxorubicin and the p53 targeting small molecule APR-246 (kindly provided by Aprea, Stockholm, Sweden) and observed an insensitivity to doxorubicin in TP53mut cell lines and primografts (mean IC50: 100 nM and >65 nM) compared to sensitive TP53wt cell lines and primografts (mean IC50: 9 nM and 7.4 nM). In contrast, TP53mut ALL cells were highly sensitive to APR-246 (mean IC50: 4.5 μM and 4.4 μM), compared to insensitivity of TP53wt cell lines and primografts (mean IC50: 58.3 μM and >90 μM). Increased Annexin-V/PI positivity and caspase-3 activity upon APR-246 exposure indicated apoptosis as mechanism of APR-246 mediated cell death.In order to investigate the specificity of APR-246 for p53, we investigated APR-246 mediated cell death induction in 2 genetically modified, p53 deficient ALL cell lines. No cell death induction could be detected upon p53 absence in contrast to high APR-246 sensitivity in the control transduced cell lines, clearly indicating dependency of APR-246 on the presence of mutant p53.Most importantly, a clear activation of p53 was identified upon APR-246 exposure in TP53mut but not TP53wt leukemias indicated by increased p53 phosphorylation and increased expression of the p53 target molecules NOXA and PUMA, pointing to restoration of p53 function and induction of downstream signaling in TP53mut ALL by APR-246.Moreover, we investigated the ability of APR-246 to re-sensitize TP53mut, DNA damage resistant ALL. Upon exposure of TP53mut ALL cell lines and primografts to combinations of APR-246 and doxorubicin, a strong synergism and re-sensitization to DNA-damage induced cell death was observed.
Conclusion
We provide evidence that APR-246 specifically targets TP53mut BCP-ALL leading to re-activation of p53, apoptosis induction, and re-sensitization of DNA-damage resistant TP53mut ALL cells. Thus, targeting mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Acute lymphoblastic leukemia, Mutation status, P53, Targeted therapy
Abstract: P158
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
In acute lymphoblastic leukemia (ALL), TP53 alterations are rarely found at diagnosis but in about 12% of patients at relapse. TP53 mutations (TP53mut) have been described to be associated with poor response to therapy and inferior outcome in ALL. Compounds targeting mutated p53 such as APR-246 demonstrated activity in different types of TP53mut malignancies. In ALL however, targeting mutant TP53 has so far not been addressed.
Aims
To evaluate mutant TP53 as target for directed anti-leukemia therapy in ALL.
Methods
Patient-derived primograft samples established in our NOD/SCID/huALL mouse model were analyzed for TP53mut by denaturating high-performance liquid chromatography and validated by Sanger sequencing. Sensitivities of ALL cells in response to different drugs and combinations were assessed analyzing half maximal inhibitory concentrations (IC50) and combination indices (CI). Apoptosis was detected by caspase-3 activation and Annexin-V/propidium iodide positivity. Activation of p53 was analyzed by phosphorylation of p53 (p53pSer15, flowcytometry), expression of p53 downstream molecules and confirmation of phosphorylation was carried out by western blot analysis. p53 deficient cell lines were generated by lentiviral shRNA mediated knock-down.
Results
Of 62 primograft samples, 4 cases with TP53mut (6.5 %) were identified, corresponding to reported numbers in clinical studies. In parallel, we analyzed 6 BCP-ALL cell lines and identified 2 TP53mut and 4 wild type (TP53wt) lines.We analyzed the effects of the DNA damaging agent doxorubicin and the p53 targeting small molecule APR-246 (kindly provided by Aprea, Stockholm, Sweden) and observed an insensitivity to doxorubicin in TP53mut cell lines and primografts (mean IC50: 100 nM and >65 nM) compared to sensitive TP53wt cell lines and primografts (mean IC50: 9 nM and 7.4 nM). In contrast, TP53mut ALL cells were highly sensitive to APR-246 (mean IC50: 4.5 μM and 4.4 μM), compared to insensitivity of TP53wt cell lines and primografts (mean IC50: 58.3 μM and >90 μM). Increased Annexin-V/PI positivity and caspase-3 activity upon APR-246 exposure indicated apoptosis as mechanism of APR-246 mediated cell death.In order to investigate the specificity of APR-246 for p53, we investigated APR-246 mediated cell death induction in 2 genetically modified, p53 deficient ALL cell lines. No cell death induction could be detected upon p53 absence in contrast to high APR-246 sensitivity in the control transduced cell lines, clearly indicating dependency of APR-246 on the presence of mutant p53.Most importantly, a clear activation of p53 was identified upon APR-246 exposure in TP53mut but not TP53wt leukemias indicated by increased p53 phosphorylation and increased expression of the p53 target molecules NOXA and PUMA, pointing to restoration of p53 function and induction of downstream signaling in TP53mut ALL by APR-246.Moreover, we investigated the ability of APR-246 to re-sensitize TP53mut, DNA damage resistant ALL. Upon exposure of TP53mut ALL cell lines and primografts to combinations of APR-246 and doxorubicin, a strong synergism and re-sensitization to DNA-damage induced cell death was observed.
Conclusion
We provide evidence that APR-246 specifically targets TP53mut BCP-ALL leading to re-activation of p53, apoptosis induction, and re-sensitization of DNA-damage resistant TP53mut ALL cells. Thus, targeting mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Acute lymphoblastic leukemia, Mutation status, P53, Targeted therapy
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
In acute lymphoblastic leukemia (ALL), TP53 alterations are rarely found at diagnosis but in about 12% of patients at relapse. TP53 mutations (TP53mut) have been described to be associated with poor response to therapy and inferior outcome in ALL. Compounds targeting mutated p53 such as APR-246 demonstrated activity in different types of TP53mut malignancies. In ALL however, targeting mutant TP53 has so far not been addressed.
Aims
To evaluate mutant TP53 as target for directed anti-leukemia therapy in ALL.
Methods
Patient-derived primograft samples established in our NOD/SCID/huALL mouse model were analyzed for TP53mut by denaturating high-performance liquid chromatography and validated by Sanger sequencing. Sensitivities of ALL cells in response to different drugs and combinations were assessed analyzing half maximal inhibitory concentrations (IC50) and combination indices (CI). Apoptosis was detected by caspase-3 activation and Annexin-V/propidium iodide positivity. Activation of p53 was analyzed by phosphorylation of p53 (p53pSer15, flowcytometry), expression of p53 downstream molecules and confirmation of phosphorylation was carried out by western blot analysis. p53 deficient cell lines were generated by lentiviral shRNA mediated knock-down.
Results
Of 62 primograft samples, 4 cases with TP53mut (6.5 %) were identified, corresponding to reported numbers in clinical studies. In parallel, we analyzed 6 BCP-ALL cell lines and identified 2 TP53mut and 4 wild type (TP53wt) lines.We analyzed the effects of the DNA damaging agent doxorubicin and the p53 targeting small molecule APR-246 (kindly provided by Aprea, Stockholm, Sweden) and observed an insensitivity to doxorubicin in TP53mut cell lines and primografts (mean IC50: 100 nM and >65 nM) compared to sensitive TP53wt cell lines and primografts (mean IC50: 9 nM and 7.4 nM). In contrast, TP53mut ALL cells were highly sensitive to APR-246 (mean IC50: 4.5 μM and 4.4 μM), compared to insensitivity of TP53wt cell lines and primografts (mean IC50: 58.3 μM and >90 μM). Increased Annexin-V/PI positivity and caspase-3 activity upon APR-246 exposure indicated apoptosis as mechanism of APR-246 mediated cell death.In order to investigate the specificity of APR-246 for p53, we investigated APR-246 mediated cell death induction in 2 genetically modified, p53 deficient ALL cell lines. No cell death induction could be detected upon p53 absence in contrast to high APR-246 sensitivity in the control transduced cell lines, clearly indicating dependency of APR-246 on the presence of mutant p53.Most importantly, a clear activation of p53 was identified upon APR-246 exposure in TP53mut but not TP53wt leukemias indicated by increased p53 phosphorylation and increased expression of the p53 target molecules NOXA and PUMA, pointing to restoration of p53 function and induction of downstream signaling in TP53mut ALL by APR-246.Moreover, we investigated the ability of APR-246 to re-sensitize TP53mut, DNA damage resistant ALL. Upon exposure of TP53mut ALL cell lines and primografts to combinations of APR-246 and doxorubicin, a strong synergism and re-sensitization to DNA-damage induced cell death was observed.
Conclusion
We provide evidence that APR-246 specifically targets TP53mut BCP-ALL leading to re-activation of p53, apoptosis induction, and re-sensitization of DNA-damage resistant TP53mut ALL cells. Thus, targeting mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Acute lymphoblastic leukemia, Mutation status, P53, Targeted therapy
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