THE ADVERSE EFFECTS OF CANCEROUS INHIBITOR OF PROTEIN PHOSPHATASE 2A, ARE LINKED WITH REDUCTION OF FUNCTIONAL B56GAMMA, A REGULATORY SUBUNIT OF PROTEIN PHOSPHATASE 2A, IN CHRONIC MYELOID LEUKAEMIA
(Abstract release date: 05/19/16)
EHA Library. Austin J. 06/11/16; 135250; S494
Dr. James Austin
Contributions
Contributions
Abstract
Abstract: S494
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Hall C11
Background
Protein phosphatase 2A (PP2A) is an important phosphatase which opposes the deregulated kinase activity that typifies many malignancies. It comprises a scaffold (A) subunit linked to a catalytic (C) and regulatory (B) subunit; the latter dictates activity, substrate specificity and intracellular localisation and has 26 isoforms. Of these, B56γ confers a PP2A nuclear localisation signal, and mutations interfering with its PP2A binding are reported in solid tumours. We have shown that a high diagnostic level of CIP2A, a key PP2A inhibitor, is a predictive biomarker of poor outcome in both acute and chronic myeloid leukaemia (CML), and ~30 studies in non-haematological tumours all also show a similar association with poor outcome. PP2A is known to dephosphorylate MYC at serine 62, thus destabilising it and shortening its half-life. PP2A also regulates mTORC1, MAPK/ERK, AKT, P53 and NfKB pathways which are associated with malignancy, but whether CIP2A involvement is direct or indirect is not understood; furthermore the detailed molecular and biochemical consequences of high CIP2A levels in malignant cells remain elusive.
Aims
We aim to test whether CIP2A binding to PP2A is dependent on which regulatory subunits are attached in the PP2A heterotrimeric complex. This will help to explain how CIP2A can mediate some but not all of PP2A’s specific functions on pathways involved in cancer progression.
Methods
The CML cell line K562 expresses high levels of CIP2A. Its CIP2A levels and those of MYC, S62 MYC, and PP2A components were assessed by flow cytometry. Primary CML cells, both mononuclear and CD34+, with either low or high levels of CIP2A were also studied, after obtaining informed consent. CML cells and cell-lines were used in fractionation (to seperate into distinct cellular compartments), western blotting, co-immunoprecipitation and qPCR.
Results
CIP2A co-immunoprecipitates not only with PR65, the heat repeat scaffold (A) subunit of PP2A, but also with B56γ, one of the B regulatory subunits of PP2A, though not with the alternative B subunits PPP2R4, B56α, B56β, PPP2R5E or B72/130. In primary CML cells with high CIP2A levels, B56γ mRNA expression is lower than in either control or CML samples with low CIP2A levels, to suggest it has an inhibitory effect on B56y protein levels. In high CIP2A K562 cells, CIP2A is predominantly cytoplasmic, as is B56γ. In addition, S62 MYC is almost entirely cytoplasmic. CIP2A knockdown by transient transfection of siRNA will increase the level of B56γ and vice versa, suggesting that expression is negatively correlated.
Conclusion
These data suggest that CIP2A reduces the active PP2A-B56γ associated complex and is compatible with the following model: In normal cells (which have very little CIP2A present) and malignant cells with low CIP2A, the PP2A scaffold plus C unit complex moves freely around the cell with full activity, getting access to the nucleus by the nuclear localisation signal obtained on binding B56γ. CIP2A is at too low a level to interfere with this. Cytoplasmic PP2A is also fully active and can thus regulate important cytoplasmic pathways such as MAPK/Jun which can promote proliferation. If CIP2A is high (exclusively a feature of some malignant cells), it binds B56γ as part of the PP2A complex. This results in the specific loss of function performed by the PP2A-B56γ attached complex. This could be achieved by CIP2A in a number of ways, including interfering with PP2A- B56γ nuclear transport, by restricting access of the nuclear localisation signal (NLS) or a direct inhibitory effect on function (i.e. blocking of the active site). Concurrently, a reduction in B56γ mRNA is observed. Overall, these effects reduce the amount of PP2A- B56γ phosphatase function, leading to deregulation of known pathways that require functional B56γ; these include the P53 tumour suppressor function and MAPK/ERK in uncontrolled cell proliferation. The effect on S62 MYC may also be affected by reducing total nuclear levels of PP2A or causing a misbalance of other active B regulatory subunits that compete to bind PP2A. Thus, high CIP2A results in a specific deregulation of PP2A function likely to promote clonal progression, with potential overlap in other haematological malignancies.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, MYC, PP2A, Protein-protein interaction
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Hall C11
Background
Protein phosphatase 2A (PP2A) is an important phosphatase which opposes the deregulated kinase activity that typifies many malignancies. It comprises a scaffold (A) subunit linked to a catalytic (C) and regulatory (B) subunit; the latter dictates activity, substrate specificity and intracellular localisation and has 26 isoforms. Of these, B56γ confers a PP2A nuclear localisation signal, and mutations interfering with its PP2A binding are reported in solid tumours. We have shown that a high diagnostic level of CIP2A, a key PP2A inhibitor, is a predictive biomarker of poor outcome in both acute and chronic myeloid leukaemia (CML), and ~30 studies in non-haematological tumours all also show a similar association with poor outcome. PP2A is known to dephosphorylate MYC at serine 62, thus destabilising it and shortening its half-life. PP2A also regulates mTORC1, MAPK/ERK, AKT, P53 and NfKB pathways which are associated with malignancy, but whether CIP2A involvement is direct or indirect is not understood; furthermore the detailed molecular and biochemical consequences of high CIP2A levels in malignant cells remain elusive.
Aims
We aim to test whether CIP2A binding to PP2A is dependent on which regulatory subunits are attached in the PP2A heterotrimeric complex. This will help to explain how CIP2A can mediate some but not all of PP2A’s specific functions on pathways involved in cancer progression.
Methods
The CML cell line K562 expresses high levels of CIP2A. Its CIP2A levels and those of MYC, S62 MYC, and PP2A components were assessed by flow cytometry. Primary CML cells, both mononuclear and CD34+, with either low or high levels of CIP2A were also studied, after obtaining informed consent. CML cells and cell-lines were used in fractionation (to seperate into distinct cellular compartments), western blotting, co-immunoprecipitation and qPCR.
Results
CIP2A co-immunoprecipitates not only with PR65, the heat repeat scaffold (A) subunit of PP2A, but also with B56γ, one of the B regulatory subunits of PP2A, though not with the alternative B subunits PPP2R4, B56α, B56β, PPP2R5E or B72/130. In primary CML cells with high CIP2A levels, B56γ mRNA expression is lower than in either control or CML samples with low CIP2A levels, to suggest it has an inhibitory effect on B56y protein levels. In high CIP2A K562 cells, CIP2A is predominantly cytoplasmic, as is B56γ. In addition, S62 MYC is almost entirely cytoplasmic. CIP2A knockdown by transient transfection of siRNA will increase the level of B56γ and vice versa, suggesting that expression is negatively correlated.
Conclusion
These data suggest that CIP2A reduces the active PP2A-B56γ associated complex and is compatible with the following model: In normal cells (which have very little CIP2A present) and malignant cells with low CIP2A, the PP2A scaffold plus C unit complex moves freely around the cell with full activity, getting access to the nucleus by the nuclear localisation signal obtained on binding B56γ. CIP2A is at too low a level to interfere with this. Cytoplasmic PP2A is also fully active and can thus regulate important cytoplasmic pathways such as MAPK/Jun which can promote proliferation. If CIP2A is high (exclusively a feature of some malignant cells), it binds B56γ as part of the PP2A complex. This results in the specific loss of function performed by the PP2A-B56γ attached complex. This could be achieved by CIP2A in a number of ways, including interfering with PP2A- B56γ nuclear transport, by restricting access of the nuclear localisation signal (NLS) or a direct inhibitory effect on function (i.e. blocking of the active site). Concurrently, a reduction in B56γ mRNA is observed. Overall, these effects reduce the amount of PP2A- B56γ phosphatase function, leading to deregulation of known pathways that require functional B56γ; these include the P53 tumour suppressor function and MAPK/ERK in uncontrolled cell proliferation. The effect on S62 MYC may also be affected by reducing total nuclear levels of PP2A or causing a misbalance of other active B regulatory subunits that compete to bind PP2A. Thus, high CIP2A results in a specific deregulation of PP2A function likely to promote clonal progression, with potential overlap in other haematological malignancies.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, MYC, PP2A, Protein-protein interaction
Abstract: S494
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Hall C11
Background
Protein phosphatase 2A (PP2A) is an important phosphatase which opposes the deregulated kinase activity that typifies many malignancies. It comprises a scaffold (A) subunit linked to a catalytic (C) and regulatory (B) subunit; the latter dictates activity, substrate specificity and intracellular localisation and has 26 isoforms. Of these, B56γ confers a PP2A nuclear localisation signal, and mutations interfering with its PP2A binding are reported in solid tumours. We have shown that a high diagnostic level of CIP2A, a key PP2A inhibitor, is a predictive biomarker of poor outcome in both acute and chronic myeloid leukaemia (CML), and ~30 studies in non-haematological tumours all also show a similar association with poor outcome. PP2A is known to dephosphorylate MYC at serine 62, thus destabilising it and shortening its half-life. PP2A also regulates mTORC1, MAPK/ERK, AKT, P53 and NfKB pathways which are associated with malignancy, but whether CIP2A involvement is direct or indirect is not understood; furthermore the detailed molecular and biochemical consequences of high CIP2A levels in malignant cells remain elusive.
Aims
We aim to test whether CIP2A binding to PP2A is dependent on which regulatory subunits are attached in the PP2A heterotrimeric complex. This will help to explain how CIP2A can mediate some but not all of PP2A’s specific functions on pathways involved in cancer progression.
Methods
The CML cell line K562 expresses high levels of CIP2A. Its CIP2A levels and those of MYC, S62 MYC, and PP2A components were assessed by flow cytometry. Primary CML cells, both mononuclear and CD34+, with either low or high levels of CIP2A were also studied, after obtaining informed consent. CML cells and cell-lines were used in fractionation (to seperate into distinct cellular compartments), western blotting, co-immunoprecipitation and qPCR.
Results
CIP2A co-immunoprecipitates not only with PR65, the heat repeat scaffold (A) subunit of PP2A, but also with B56γ, one of the B regulatory subunits of PP2A, though not with the alternative B subunits PPP2R4, B56α, B56β, PPP2R5E or B72/130. In primary CML cells with high CIP2A levels, B56γ mRNA expression is lower than in either control or CML samples with low CIP2A levels, to suggest it has an inhibitory effect on B56y protein levels. In high CIP2A K562 cells, CIP2A is predominantly cytoplasmic, as is B56γ. In addition, S62 MYC is almost entirely cytoplasmic. CIP2A knockdown by transient transfection of siRNA will increase the level of B56γ and vice versa, suggesting that expression is negatively correlated.
Conclusion
These data suggest that CIP2A reduces the active PP2A-B56γ associated complex and is compatible with the following model: In normal cells (which have very little CIP2A present) and malignant cells with low CIP2A, the PP2A scaffold plus C unit complex moves freely around the cell with full activity, getting access to the nucleus by the nuclear localisation signal obtained on binding B56γ. CIP2A is at too low a level to interfere with this. Cytoplasmic PP2A is also fully active and can thus regulate important cytoplasmic pathways such as MAPK/Jun which can promote proliferation. If CIP2A is high (exclusively a feature of some malignant cells), it binds B56γ as part of the PP2A complex. This results in the specific loss of function performed by the PP2A-B56γ attached complex. This could be achieved by CIP2A in a number of ways, including interfering with PP2A- B56γ nuclear transport, by restricting access of the nuclear localisation signal (NLS) or a direct inhibitory effect on function (i.e. blocking of the active site). Concurrently, a reduction in B56γ mRNA is observed. Overall, these effects reduce the amount of PP2A- B56γ phosphatase function, leading to deregulation of known pathways that require functional B56γ; these include the P53 tumour suppressor function and MAPK/ERK in uncontrolled cell proliferation. The effect on S62 MYC may also be affected by reducing total nuclear levels of PP2A or causing a misbalance of other active B regulatory subunits that compete to bind PP2A. Thus, high CIP2A results in a specific deregulation of PP2A function likely to promote clonal progression, with potential overlap in other haematological malignancies.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, MYC, PP2A, Protein-protein interaction
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Hall C11
Background
Protein phosphatase 2A (PP2A) is an important phosphatase which opposes the deregulated kinase activity that typifies many malignancies. It comprises a scaffold (A) subunit linked to a catalytic (C) and regulatory (B) subunit; the latter dictates activity, substrate specificity and intracellular localisation and has 26 isoforms. Of these, B56γ confers a PP2A nuclear localisation signal, and mutations interfering with its PP2A binding are reported in solid tumours. We have shown that a high diagnostic level of CIP2A, a key PP2A inhibitor, is a predictive biomarker of poor outcome in both acute and chronic myeloid leukaemia (CML), and ~30 studies in non-haematological tumours all also show a similar association with poor outcome. PP2A is known to dephosphorylate MYC at serine 62, thus destabilising it and shortening its half-life. PP2A also regulates mTORC1, MAPK/ERK, AKT, P53 and NfKB pathways which are associated with malignancy, but whether CIP2A involvement is direct or indirect is not understood; furthermore the detailed molecular and biochemical consequences of high CIP2A levels in malignant cells remain elusive.
Aims
We aim to test whether CIP2A binding to PP2A is dependent on which regulatory subunits are attached in the PP2A heterotrimeric complex. This will help to explain how CIP2A can mediate some but not all of PP2A’s specific functions on pathways involved in cancer progression.
Methods
The CML cell line K562 expresses high levels of CIP2A. Its CIP2A levels and those of MYC, S62 MYC, and PP2A components were assessed by flow cytometry. Primary CML cells, both mononuclear and CD34+, with either low or high levels of CIP2A were also studied, after obtaining informed consent. CML cells and cell-lines were used in fractionation (to seperate into distinct cellular compartments), western blotting, co-immunoprecipitation and qPCR.
Results
CIP2A co-immunoprecipitates not only with PR65, the heat repeat scaffold (A) subunit of PP2A, but also with B56γ, one of the B regulatory subunits of PP2A, though not with the alternative B subunits PPP2R4, B56α, B56β, PPP2R5E or B72/130. In primary CML cells with high CIP2A levels, B56γ mRNA expression is lower than in either control or CML samples with low CIP2A levels, to suggest it has an inhibitory effect on B56y protein levels. In high CIP2A K562 cells, CIP2A is predominantly cytoplasmic, as is B56γ. In addition, S62 MYC is almost entirely cytoplasmic. CIP2A knockdown by transient transfection of siRNA will increase the level of B56γ and vice versa, suggesting that expression is negatively correlated.
Conclusion
These data suggest that CIP2A reduces the active PP2A-B56γ associated complex and is compatible with the following model: In normal cells (which have very little CIP2A present) and malignant cells with low CIP2A, the PP2A scaffold plus C unit complex moves freely around the cell with full activity, getting access to the nucleus by the nuclear localisation signal obtained on binding B56γ. CIP2A is at too low a level to interfere with this. Cytoplasmic PP2A is also fully active and can thus regulate important cytoplasmic pathways such as MAPK/Jun which can promote proliferation. If CIP2A is high (exclusively a feature of some malignant cells), it binds B56γ as part of the PP2A complex. This results in the specific loss of function performed by the PP2A-B56γ attached complex. This could be achieved by CIP2A in a number of ways, including interfering with PP2A- B56γ nuclear transport, by restricting access of the nuclear localisation signal (NLS) or a direct inhibitory effect on function (i.e. blocking of the active site). Concurrently, a reduction in B56γ mRNA is observed. Overall, these effects reduce the amount of PP2A- B56γ phosphatase function, leading to deregulation of known pathways that require functional B56γ; these include the P53 tumour suppressor function and MAPK/ERK in uncontrolled cell proliferation. The effect on S62 MYC may also be affected by reducing total nuclear levels of PP2A or causing a misbalance of other active B regulatory subunits that compete to bind PP2A. Thus, high CIP2A results in a specific deregulation of PP2A function likely to promote clonal progression, with potential overlap in other haematological malignancies.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, MYC, PP2A, Protein-protein interaction
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